Stressed Wild-type Mice Research Articles

Molecular signature of excessive female aggression: study of stressed mice with genetic inactivation of neuronal serotonin synthesis

Aggression is a complex social behavior, critically involving brain serotonin (5-HT) function. The neurobiology of female aggression remains elusive, while the incidence of its manifestations has been increasing. Yet, animal models of female aggression are scarce. We previously proposed a paradigm of female aggression in the context of gene x environment interaction where mice with partial genetic inactivation of tryptophan hydroxylase-2 (Tph2+/− mice), a key enzyme of neuronal 5-HT synthesis, are subjected to predation stress resulting in pathological aggression. Using deep sequencing and the EBSeq method, we studied the transcriptomic signature of excessive aggression in the prefrontal cortex of female Tph2+/− mice subjected to rat exposure stress and food deprivation. Challenged mutants, but not other groups, displayed marked aggressive behaviors. We found 26 genes with altered expression in the opposite direction between stressed groups of both Tph2 genotypes. We identified several molecular markers, including Dgkh, Arfgef3, Kcnh7, Grin2a, Tenm1 and Epha6, implicated in neurodevelopmental deficits and psychiatric conditions featuring impaired cognition and emotional dysregulation. Moreover, while 17 regulons, including several relevant to neural plasticity and function, were significantly altered in stressed mutants, no alteration in regulons was detected in stressed wildtype mice. An interplay of the uncovered pathways likely mediates partial Tph2 inactivation in interaction with severe stress experience, thus resulting in excessive female aggression.

Thelper1 and T helper2 cytokine production patterns in stressed beta2-adrenergic receptor-deficient mice during chlamydia genital infection.

Murine CD4+ T cells have two distinct cytokine secretion patterns to play different functions. We have demonstrated that cold-induced stress (CIS) suppresses the immune system leading to increased intensity of Chlamydia muridaum in mice. We have reported that beta2-adrenergic receptor (b2-AR) knockout (KO) mice resist chlamydia genital infection. However, the cytokine profile of CD4+ T cells is not well explored. This study aimed to determine the cytokine production of Th1 and Th2 types in stressed and non-stressed b2-AR KO mice. We investigated the cytokine production levels of stressed and non-stressed mice during C. muridarum genital infection. Significantly increased production of cytokines was observed in plates pre-coated with anti-CD3/CD28 or in the presence of Con A or LPS. A decrease in the production of IFN-g and IL-2, whereas an increase in the secretion of IL-10, IL-13, and IL-23 in CD4+ T cells of stressed wild-type mice was obtained; however, the secretion of IFN-g and IL-2 was restored in T cells of b2-AR KO mice. Moreover, in vitro proliferation of CD4 T cells in the presence of b2-AR antagonists, ICI 118, 551 stimulated the production of Th1 cytokines, whereas b2-AR agonist, Fenoterol, decreased the production of Th1-type cytokines. Overall, Th1 cytokine responses are reduced in stressed mice suggesting that the cytokine status was polarized toward a Th2 immune response that can be restored by removing b2-AR from immune cells.

Adipose Triglyceride Lipase Deficiency Aggravates Angiotensin II–Induced Atrial Fibrillation by Reducing Peroxisome Proliferator–Activated Receptor α Activation in Mice

Atrial fibrillation (AF) is a main risk factor for cerebrovascular diseases but lacks precision therapy. Adipose triglyceride lipase (ATGL) is a key enzyme involved in the intracellular degradation of triacylglycerol and plays an important role in lipid and energy metabolism. However, the role of ATGL in the regulation of AF remains unclear. In this study, AF was induced by infusion of angiotensin II (Ang II, 2000 ng/kg/min) for 3 weeks in male ATGL knockout (KO) mice and age-matched C57BL/6 wild-type mice. The atrial volume was measured by echocardiography. Atrial fibrosis, inflammatory cells, and superoxide production were detected by histologic examinations. The results showed that ATGL expression was significantly downregulated in the atrial tissue of the Ang II–infused mice. Moreover, Ang II–induced increase in the inducibility and duration of AF, atrial dilation, fibrosis, inflammation, and oxidative stress in wild-type mice were markedly accelerated in ATGL KO mice; however, these effects were dramatically reversed in the ATGL KO mice administered with peroxisome proliferator–activated receptor (PPAR)-α agonist clofibric acid. Mechanistically, Ang II downregulated ATGL expression and inhibited PPAR-α activity, activated multiple signaling pathways (inhibiting kappa B kinase α/β-nuclear factor-κB, nicotinamide adenine dinucleotide phosphate oxidase, and transforming growth factor-β1/SMAD2/3) and reducing Kv1.5, Cx40, and Cx43 expression, thereby contributing to atrial structural and electrical remodeling and subsequent AF. In summary, our results indicate that ATGL KO enhances AF inducibility, possibly through inhibiting PPAR-α activation and suggest that activating ATGL might be a new therapeutic option for treating hypertensive AF.

SIRT3 PREVENTS DOXORUBICIN INDUCED DILATED CARDIOMYOPATHY VIA REGULATION OF MITOCHONDRIAL PROTEIN ACETYLATION: INVESTIGATING METABOLIC DYSFUNCTION AS A RESULT OF ALTERATIONS TO CARDIAC LIPIDS

Doxorubicin (DOX) is a chemotherapeutic used in the treatment cancer, however it has dose-dependent cardiotoxic side effects such as the development of dilated cardiomyopathy. Previously, we showed that DOX treatment decreases the expression of mitochondrial lysine deacetylase, sirtuin 3 (SIRT3) and alters mitochondrial protein acetylation of enzymes involved in cardiac energy production and oxidative stress in wild-type mice. Cardiac expression of full length mitochondrial localized M1-SIRT3 prevented cardiac remodelling and dysfunction characteristic of dilated cardiomyopathy in female mice.

Abstract P3023: The Regulatory Role Of Mir-21 In Heart Failure With Preserved Ejection Fraction (hfpef)

Introduction: Coronary microvascular dysfunction (CMD) is characterized by impaired endothelial-dependent vasodilation. These impairments are seen in diabetic cardiomyopathy, ischemia with no obstructive coronary artery disease, and heart failure with preserved ejection fraction (HFpEF), but detailed mechanisms have yet to be elucidated. Moreover, how microRNA (miR-21) regulates CMD in HFpEF is not entirely understood. Methods: miR-21 knockout and wild-type (WT) mice were fed a diet high in fat and sugar (HFHS) for 6 months and blood lipid and glucose were measured. Echocardiography and treadmill exercise exertion tests were performed to assess cardiac function. Myocardial blood flow (MBF) under stress was measured by contrast echocardiography or doppler after the treatment with different dosages of norepinephrine and mean arterial blood pressure was measured simultaneously by femoral pressure catheter. Cardiac fibrosis was detected using trichrome staining and molecular pathways were elucidated via gene and protein analysis. Results: Our preliminary data show decreased MBF during stress in WT mice fed a HFHS diet. While ejection fraction was not changed, cardiac index, stroke volume, and running distance were decreased. Furthermore, E/E’ ratio was increased in WT mice fed HFHS diet compared to the WT mice fed a chow diet, suggesting heart failure and impaired diastolic function with normal systolic function. Moreover, perivascular fibrosis was increased in the WT mice fed a HFHS diet compared to WT mice fed a chow diet. However, the ablation of miR-21 reversed all these changes in the mice fed a HFHS diet. Conclusions: miR-21 regulates CMD and ameliorated HFpEF. Further investigation will elucidate the pathways and mechanisms converging with miR-21 to regulate HFpEF.

Profile of CD8+ T-cells in a Stressed beta2- Adrenergic Receptor Knockout Mouse Model during Chlamydia muridarum Genital Infection.


 Chlamydia trachomatis is the most common bacterial sexually transmitted disease. A negative correlation between the immune system and stress has been reported. We have demonstrated that cold-induced stress (CIS) increases the intensity of Chlamydia muridarum genital infection in a mouse model, but the status of CD8+ T cells is still unknown. This study profiles CD8+ T cell’s interaction with stress hormone; norepinephrine (NE) and the beta2-adrenergic receptor (β2-AR) in the presence of agonists and antagonists; and examines cytokines produced in the β2-AR knockout (KO) stress mouse model during C. muridarum genital infection. We hypothesize that CD8+ T cells produce high-level IFN-g and low-level TNF-a in stressed β2-AR KO mice than C57BL/6J wildtype (WT). Two groups of mice were cold-water stressed for 21 days, infected with C. muridarum, and then sacrificed for CD8+ T cell isolation. Viability was tested. The proliferation of CD8+ T cells was carried out for 72 h and culture supernatant was collected for cytokine production using ELISA. To confirm genital tract infection, Chlamydia muridarum isolation is underway. Our data show that the viability of CD8+ T cells treated with NE was significantly reduced compared to cells treated with LPS or Con A, indicating the suppressive effect of NE on immune cells. Stressed β2-AR KO mice displayed increased production of protective cytokines compared to stressed WT mice, indicating the absence of β2-AR leads to the functional restoration of protective immune cells.

Negative Allosteric Modulation of Gamma-Aminobutyric Acid A Receptors at α5 Subunit–Containing Benzodiazepine Sites Reverses Stress-Induced Anhedonia and Weakened Synaptic Function in Mice

BackgroundAbnormal reward processing, typically anhedonia, is a hallmark of human depression and is accompanied by altered functional connectivity in reward circuits. Negative allosteric modulators of GABAA (gamma-aminobutyric acid A) receptors (GABA-NAMs) have rapid antidepressant-like properties in rodents and exert few adverse effects, but molecular targets underlying their behavioral and synaptic effects remain undetermined. We hypothesized that GABA-NAMs act at the benzodiazepine site of GABAA receptors containing α5 subunits to increase gamma oscillatory activity, strengthen synapses in reward circuits, and reverse anhedonia. MethodsAnhedonia was induced by chronic stress in male mice and assayed by preferences for sucrose and female urine (n = 5–7 mice/group). Hippocampal slices were then prepared for electrophysiological recording (n = 1–6 slices/mouse, 4–6 mice/group). Electroencephalography power was quantified in response to GABA-NAM and ketamine administration (n = 7–9 mice/group). ResultsChronic stress reduced sucrose and female urine preferences and hippocampal temporoammonic-CA1 synaptic strength. A peripheral injection of the GABA-NAM MRK-016 restored hedonic behavior and AMPA-to-NMDA ratios in wild-type mice. These actions were prevented by pretreatment with the benzodiazepine site antagonist flumazenil. MRK-016 administration increased gamma power over the prefrontal cortex in wild-type mice but not α5 knockout mice, whereas ketamine promoted gamma power in both genotypes. Hedonic behavior and AMPA-to-NMDA ratios were only restored by MRK-016 in stressed wild-type mice but not α5 knockout mice. Conclusionsα5-Selective GABA-NAMs exert rapid anti-anhedonic actions and restore the strength of synapses in reward regions by acting at the benzodiazepine site of α5-containing GABAA receptors. These results encourage human studies using GABA-NAMs to treat depression by providing readily translatable measures of target engagement.

Ginsenoside Re Protects against Serotonergic Behaviors Evoked by 2,5-Dimethoxy-4-iodo-amphetamine in Mice via Inhibition of PKCδ-Mediated Mitochondrial Dysfunction.

It has been recognized that serotonin 2A receptor (5-HT2A) agonist 2,5-dimethoxy-4-iodo-amphetamine (DOI) impairs serotonergic homeostasis. However, the mechanism of DOI-induced serotonergic behaviors remains to be explored. Moreover, little is known about therapeutic interventions against serotonin syndrome, although evidence suggests that ginseng might possess modulating effects on the serotonin system. As ginsenoside Re (GRe) is well-known as a novel antioxidant in the nervous system, we investigated whether GRe modulates 5-HT2A receptor agonist DOI-induced serotonin impairments. We proposed that protein kinase Cδ (PKCδ) mediates serotonergic impairments. Treatment with GRe or 5-HT2A receptor antagonist MDL11939 significantly attenuated DOI-induced serotonergic behaviors (i.e., overall serotonergic syndrome behaviors, head twitch response, hyperthermia) by inhibiting mitochondrial translocation of PKCδ, reducing mitochondrial glutathione peroxidase activity, mitochondrial dysfunction, and mitochondrial oxidative stress in wild-type mice. These attenuations were in line with those observed upon PKCδ inhibition (i.e., pharmacologic inhibitor rottlerin or PKCδ knockout mice). Furthermore, GRe was not further implicated in attenuation mediated by PKCδ knockout in mice. Our results suggest that PKCδ is a therapeutic target for GRe against serotonergic behaviors induced by DOI.

Decreased Chlamydia muridarum genital infection and improved immune response in a β2-Adrenergic Receptor Knockout Mouse Stress Model

Chlamydia genital infection caused by Chlamydia trachomatis shows a high prevalence in low socioeconomic populations; however, whether stress is associated with the high prevalence of infection is not known. During chronic stress conditions, the interaction of beta2-adrenergic receptor (β2-AR) with norepinephrine (NE) is known to suppress the immune system in humans and animal models. However, the mechanism of suppression that leads to chlamydia infection is not well known. This study aims to measure the susceptibility of mice and cytokine production of a β2-AR knockout (KO) during Chlamydia muridarum genital infection. Stressed and non-stressed C57BL/6J wildtype (WT) and β2-AR gene KO mice will be infected with C. muridarum intravaginally post 21-day stressing period. Swabbing of mice at the 3-day interval for 42 days will be performed to determine C. muridarum counts and isolation. Moreover, splenic CD4+ T cells, bone marrow-derived dendritic cells at day 7 post-infection will be performed for immune response analysis by ELISA. We hypothesize that the deficiency of β2-AR leads to significantly less C. muridarum shedding from the genital tract of stressed KO and leads to an increase and decrease in the production of protective and suppressive cytokines, respectively. We anticipate results obtained will align with our central hypothesis that stressed WT mice have increased C. muridarum shedding of genital tracts and increased production of protective cytokines in stressed β2-AR KO, suggesting the importance of stress in β2-AR stimulation.

OR04-04 Identification of a Novel Transcriptional Regulator of Metabolic Disease in Circulating and Central Myeloid Cells

Derangement in systemic metabolic homeostasis is tightly associated with widespread activation of resident and circulating immune cells, a phenomenon known as ‘metaflammation’. Numerous studies have explored the role of tissue resident and circulating macrophages in contributing to metaflammation, obesity, and their sequelae; however, there is a dearth of information regarding targetable transcriptional regulators of the genesis and persistence of metabolic disease. Here, we identify myeloid Krüppel-like factor 2 (KLF2) as a novel regulator of metabolic disease. Previous reports demonstrate that KLF2 serves as a critical regulator of myeloid cell quiescence and is downregulated in numerous acute and chronic inflammatory states. Specifically in the context of chronic metaflammation, we note that KLF2 expression is decreased in circulating immune cells of obese patients and in adipose tissue macrophages of high fat diet (HFD) fed mice, which is consistent with the hypothesis that KLF2 regulates metaflammation. To explore this further, we utilized mice with myeloid cell-specific deletion of KLF2 (K2KO) which exhibit accelerated obesity and insulin resistance. K2KO mice have widespread central (i.e. CNS) and peripheral metaflammation both in the basal and HFD-stimulated states. To discern whether the effect of myeloid deletion of KLF2 on metabolism is due to deletion in microglia in the feeding centers of the hypothalamus or in peripheral immune cells, bone marrow chimeras with head shielding were created. 50% reconstitution of circulating immune cells with K2KO cells in wildtype (WT) mice was sufficient to maintain the metabolic disease phenotype, while mice with K2KO microglia + WT circulating cells had only slightly improved outcomes compared to K2KO mice. Conversely, ablation of microglia in K2KO mice using PLX5622 formulated in HFD also successfully attenuated the aberrant feeding behavior, weight gain, and glucose dyshomeostasis seen in K2KO mice. Together, these data demonstrate a role for loss of KLF2 in hematopoietic and CNS resident cells in causing metabolic disease. Given that myeloid KLF2 expression decreases under metabolic stress in WT mice and humans, we sought to explore whether maintenance of KLF2 expression in these cells would be protective against diet-induced metabolic disease. Indeed, mice with myeloid-specific overexpression of KLF2 demonstrated a markedly improved metabolic phenotype when challenged with HFD, providing evidence that targeting KLF2 expression in myeloid cells may prove to be a therapeutic option against metaflammation.

Translocator protein-mediated fast-onset antidepressant-like and memory-enhancing effects in chronically stressed mice.

Fast-acting and cognitive-enhancing antidepressants are desperately needed. Activation of translocator protein (18 kDa, TSPO) is a novel strategy for developing potential antidepressants, but there are no data available on the onset time of TSPO ligands. This study aimed to investigate the fast-onset antidepressant actions of AC-5216, a selective TSPO ligand, in TSPO knock-out (KO) mice. TSPO wild-type (WT) and KO mice were subjected to a six-week chronic unpredicted stress (CUS) paradigm. Then, the mice were treated with AC-5216 and tested with depressive and cognitive behaviours. A single dose of AC-5216 (0.3 mg/kg) exerted anxiolytic- and antidepressant-like actions in TSPO WT mice. Moreover, in chronically stressed WT mice, two to four days of AC-5216 treatment (0.3 mg/kg, once per day) produced fast-onset antidepressant-like effects in the novelty-suppressed feeding and sucrose preference tests, as well as memory-enhancing effects in the novel object recognition test. In addition, a rapid (with five days of treatment) restoration of serum corticosterone levels and prefrontal cortex (PFC) allopregnanolone levels was found. Further studies showed that in these stress-exposed WT mice, AC-5216 significantly increased the levels of mTOR signalling-related proteins (mBDNF, p-mTOR, PSD-95, synapsin-1, GluR1), as well as the total dendritic length and branching points of pyramidal neurons in the PFC. These results suggest that TSPO mediates the fast-onset antidepressant-like and memory-enhancing effects of AC-5216, possibly through the rapid activation of mTOR signalling and restoration of dendritic complexity in the PFC.

Macrophage Hypercellularity Accompanies Erythroid Hyperplasia in Sickle Cell Mice and during Recovery from Blood Loss in Wild Type Mice

Red blood cell (RBC) homeostasis tightly balances production of new erythroid cells and clearance of damaged or senescent RBCs. Macrophages play a central role in this process in the specialized niches termed erythroblastic island. Macrophages expressing CD169 (Sialoadhesin or Siglec-1), F4/80, CD11b, VCAM-1, ER-HR3 and Ly-6G play important roles in physiological and pathological erythropoiesis. Sickle cell disease (SCD) is characterized by chronic stress erythropoiesis as a compensatory mechanism for anemia. Recent publications have detected macrophage hypercellularity correlated with erythroid hyperplasia in genetic models of erythrocytosis and monocyte-derived macrophage proliferation in the spleen. Because macrophages are integral part of the erythropoietic niche, our objective was to assess the macrophage compartment in transgenic sickle cell mice (SS) at steady state compared to AA controls. Furthermore, we used acute blood loss in C57BL/6J wild type (WT) to confirm if macrophage hypercellularity accompanies erythroid hyperplasia in a mouse model without erythrocytosis or sickling. Using flow cytometry, macrophages were identified with F4/80, CD45, CD11b and CD169, while erythroid progenitor cells were identified with Ter119 and CD71 in the BM and spleen of SS mice and age-matched control (AA) mice. Each subset of macrophages is 3.8 to 6.8-fold higher in SS mice bone marrow compared to AA at steady state (p≤0.05, Fig 1a). Findings were similar for other CD169 subsets in the spleens of SS mice. This macrophage hypercellularity was accompanied by expected erythroid hyperplasia, with 12-fold higher immature erythroid progenitor cells (CD71hiTer119hi) in SS mice than AA controls (p≤0.001). To confirm the increase in macrophage numbers that accompanied erythroid hyperplasia in SS mice as a response to erythroid stress, we induce stress with acute blood loss in WT mice by phlebotomy (once). Interestingly, we found macrophage hypercellularity accompanies erythroid hyperplasia in WT mice during recovery from acute blood loss. WT mice showed about 47-61% increases in CD169 macrophages subsets in the marrow and 47-83% increase in the spleen during recovery from acute blood loss (5-7 days) compared to untreated mice (Fig. 1b). This macrophage hypercellularity was accompanied with 88% increase in immature erythroid progenitor cell. Similarly, we investigated if there was age dependence in macrophage hypercellularity that accompanies erythroid hyperplasia in SS mice. In SS mice, there was no significant differences in macrophage numbers in the BM juvenile mice (5 weeks old) compared to young adults (14 weeks old), presumably because the higher demand for erythroid production to compensate for anemia had already started in the juvenile animals. However, there was a significantly higher macrophage hypercellularity in the spleen at 14 weeks compared to juvenile animals (p≤0.001). There was also a reduction in hematocrit (p≤0.05) and hemoglobin (p≤0.01) from 5 to 14 weeks in SS mice, although this does not correlate to either macrophage hypercellularity or erythroid hyperplasia. We also quantified mRNA expression of heme oxygenase 1 (Hmox1), a critical regulator of erythroid stress response, in the BM of untreated SS mice, and untreated and stressed WT mice. As expected, BM cells from SS mice at steady state expressed significantly higher Hmox1 expression than in WT mice (p≤0.001). Acute blood loss induced-stress raised the Hmox1 expression in WT mice to comparable levels to that of SS mice at steady state. Our investigation is the first to our knowledge to detect BM macrophage hypercellularity is associated with erythroid hyperplasia in response to acute blood loss and in congenital hemolytic anemia. Our data of macrophage hypercellularity in SCD opens up further research opportunity to elucidate the role of these cells in multiorgan complication in SCD. Figure 1: Macrophage hypercellularity accompanies erythroid hyperplasia in sickle cell disease and during recovery from acute blood loss. (a) BM macrophage quantification (per femur) in untreated sickle cell mice compared to AA controls (n = 5). Student's Ttest * SS mice compared to AA mice. (b) Acute blood loss induced macrophage hypercellularity in the BM of WT mice (n = 4 - 5). Student's Ttest * Untreated compared to stressed WT mice. Figure 1 Disclosures Ofori-Acquah: Shire Human Genetic Therapies Inc: Other: Financial Relationship. Kato:Novartis, Global Blood Therapeutics: Consultancy, Research Funding; Bayer: Research Funding.

Loss of CD36 protects against diet-induced obesity but results in impaired muscle stem cell function, delayed muscle regeneration and hepatic steatosis.

The prevalence of obesity is a major risk factor for cardiovascular and metabolic diseases including impaired skeletal muscle regeneration. Since skeletal muscle regenerative capacity is regulated by satellite cells, we aimed to investigate whether a high-fat diet impairs satellite cell function and whether this is linked to fatty acid uptake via CD36. We also aimed to determine whether loss of CD36 impacts on muscle redox homeostasis and skeletal muscle regenerative capacity. We studied the impact of a high-fat diet and CD36 deficiency on murine skeletal muscle morphology, redox homeostasis, satellite cell function, bioenergetics and lipid accumulation in the liver. We also determined the effect of CD36 deficiency on skeletal muscle regeneration. High-fat diet increased body weight, intramuscular lipid accumulation and oxidative stress in wild-type mice that were significantly mitigated in CD36-deficient mice. High-fat diet and CD36 deficiency independently attenuated satellite cell function on single fibres and myogenic capacity on primary satellite cells. CD36deficiency resulted in delayed skeletal muscle regeneration following acute injury with cardiotoxin. CD36-deficient and wild-type primary satellite cells had distinct bioenergetic profiles in response to palmitate. High-fat diet induced hepatic steatosis in both genotypes that was more pronounced in the CD36-deficient mice. This study demonstrates that CD36 deficiency protects against diet-induced obesity, intramuscular lipid deposition and oxidative stress but results in impaired muscle satellite cell function, delayed muscle regeneration and hepatic steatosis. CD36 is a key mediator of fatty acid uptake in skeletal muscle, linking obesity with satellite cell function and muscle regeneration.

A Central Extended Amygdala Circuit That Modulates Anxiety.

Both the amygdala and the bed nucleus of the stria terminalis (BNST) have been implicated in maladaptive anxiety characteristics of anxiety disorders. However, the underlying circuit and cellular mechanisms have remained elusive. Here we show that mice with Erbb4 gene deficiency in somatostatin-expressing (SOM+) neurons exhibit heightened anxiety as measured in the elevated plus maze test and the open field test, two assays commonly used to assess anxiety-related behaviors in rodents. Using a combination of electrophysiological, molecular, genetic, and pharmacological techniques, we demonstrate that the abnormal anxiety in the mutant mice is caused by enhanced excitatory synaptic inputs onto SOM+ neurons in the central amygdala (CeA), and the resulting reduction in inhibition onto downstream SOM+ neurons in the BNST. Notably, our results indicate that an increase in dynorphin signaling in SOM+ CeA neurons mediates the paradoxical reduction in inhibition onto SOM+ BNST neurons, and that the consequent enhanced activity of SOM+ BNST neurons is both necessary for and sufficient to drive the elevated anxiety. Finally, we show that the elevated anxiety and the associated synaptic dysfunctions and increased dynorphin signaling in the CeA-BNST circuit of the Erbb4 mutant mice can be recapitulated by stress in wild-type mice. Together, our results unravel previously unknown circuit and cellular processes in the central extended amygdala that can cause maladaptive anxiety.SIGNIFICANCE STATEMENT The central extended amygdala has been implicated in anxiety-related behaviors, but the underlying mechanisms are unclear. Here we found that somatostatin-expressing neurons in the central amygdala (CeA) controls anxiety through modulation of the stria terminalis, a process that is mediated by an increase in dynorphin signaling in the CeA. Our results reveal circuit and cellular dysfunctions that may account for maladaptive anxiety.

Neuregulin 1 Deficiency Modulates Adolescent Stress-Induced Dendritic Spine Loss in a Brain Region-Specific Manner and Increases Complement 4 Expression in the Hippocampus.

One neuropathological feature of schizophrenia is a diminished number of dendritic spines in the prefrontal cortex and hippocampus. The neuregulin 1 (Nrg1) system is involved in the plasticity of dendritic spines, and chronic stress decreases dendritic spine densities in the prefrontal cortex and hippocampus. Here, we aimed to assess whether Nrg1 deficiency confers vulnerability to the effects of adolescent stress on dendritic spine plasticity. We also assessed other schizophrenia-relevant neurobiological changes such as microglial cell activation, loss of parvalbumin (PV) interneurons, and induction of complement factor 4 (C4). Adolescent male wild-type (WT) and Nrg1 heterozygous mice were subjected to chronic restraint stress before their brains underwent Golgi impregnation or immunofluorescent staining of PV interneurons, microglial cells, and C4. Stress in WT mice promoted dendritic spine loss and microglial cell activation in the prefrontal cortex and the hippocampus. However, Nrg1 deficiency rendered mice resilient to stress-induced dendritic spine loss in the infralimbic cortex and the CA3 region of the hippocampus without affecting stress-induced microglial cell activation in these brain regions. Nrg1 deficiency and adolescent stress combined to trigger increased dendritic spine densities in the prelimbic cortex. In the hippocampal CA1 region, Nrg1 deficiency accentuated stress-induced dendritic spine loss. Nrg1 deficiency increased C4 protein and decreased C4 mRNA expression in the hippocampus, and the number of PV interneurons in the basolateral amygdala. This study demonstrates that Nrg1 modulates the impact of stress on the adolescent brain in a region-specific manner. It also provides first evidence of a link between Nrg1 and C4 systems in the hippocampus.

Role of endoplasmic reticulum stress in 12/15-lipoxygenase-induced retinal microvascular dysfunction in a mouse model of diabetic retinopathy.

Our earlier studies have established the role of 12/15-lipoxygenase (LO) in mediating the inflammatory reaction in diabetic retinopathy. However, the exact mechanism is still unclear. The goal of the current study was to identify the potential role of endoplasmic reticulum (ER) stress as a major cellular stress response in the 12/15-LO-induced retinal changes in diabetic retinopathy. We used in vivo and in vitro approaches. For in vivo studies, experimental diabetes was induced in wild-type (WT) mice and 12/15-Lo (also known as Alox15) knockout mice (12/15-Lo-/-); ER stress was then evaluated after 12-14weeks of diabetes. We also tested the effect of intravitreal injection of 12-hydroxyeicosatetraenoic acid (HETE) on retinal ER stress in WT mice and in mice lacking the catalytic subunit of NADPH oxidase, encoded by Nox2 (also known as Cybb) (Nox2-/- mice). In vitro studies were performed using human retinal endothelial cells (HRECs) treated with 15-HETE (0.1μmol/l) or vehicle, with or without ER stress or NADPH oxidase inhibitors. This was followed by evaluation of ER stress response, NADPH oxidase expression/activity and the levels of phosphorylated vascular endothelial growth factor receptor-2 (p-VEGFR2) by western blotting and immunoprecipitation assays. Moreover, real-time imaging of intracellular calcium (Ca2+) release in HRECs treated with or without 15-HETE was performed using confocal microscopy. Deletion of 12/15-Lo significantly attenuated diabetes-induced ER stress in mouse retina. In vitro, 15-HETE upregulated ER stress markers such as phosphorylated RNA-dependent protein kinase-like ER-regulated kinase (p-PERK), activating transcription factor 6 (ATF6) and protein disulfide isomerase (PDI) in HRECs. Inhibition of ER stress reduced 15-HETE-induced-leucocyte adhesion, VEGFR2 phosphorylation and NADPH oxidase expression/activity. However, inhibition of NADPH oxidase or deletion of Nox2 had no effect on ER stress induced by the 12/15-LO-derived metabolites both in vitro and in vivo. We also found that 15-HETE increases the intracellular calcium in HRECs. ER stress contributes to 12/15-LO-induced retinal inflammation in diabetic retinopathy via activation of NADPH oxidase and VEGFR2. Perturbation of calcium homeostasis in the retina might also play a role in linking 12/15-LO to retinal ER stress and subsequent microvascular dysfunction in diabetic retinopathy.

P.6.b.007 - An fMRI study on the effect of cue reactivity on striatal reward anticipation in alcohol dependent patients

Schizophrenia is a neurodevelopmental disorder characterized by abnormal processing of information and attentional deficits. Schizophrenia has a high genetic component but is precipitated by environmental factors, as proposed by the ‘two-hit’ theory of schizophrenia. Here we compared latent inhibition as a measure of learning and attention, in CHL1-deficient mice, an animal model of schizophrenia, and their wild-type littermates, under no-stress and chronic mild stress conditions. All unstressed mice as well as the stressed wild-type mice showed latent inhibition. In contrast, CHL1-deficient mice did not show latent inhibition after exposure to chronic stress. Differences in neuronal activation (c-Fos-positive cell counts) were noted in brain regions associated with latent inhibition: Neuronal activation in the prelimbic/infralimbic cortices and the nucleus accumbens shell was affected solely by stress. Neuronal activation in basolateral amygdala and ventral hippocampus was affected independently by stress and genotype. Most importantly, neural activation in nucleus accumbens core was affected by the interaction between stress and genotype. These results provide strong support for a ‘two-hit’ (genes x environment) effect on latent inhibition in CHL1-deficient mice, and identify CHL1-deficient mice as a model of schizophrenia-like learning and attention impairments.

Role of Dopamine D2 Receptor in Stress-Induced Myelin Loss

Dopaminergic systems play a major role in reward-related behavior and dysregulation of dopamine (DA) systems can cause several mental disorders, including depression. We previously reported that dopamine D2 receptor knockout (D2R−/−) mice display increased anxiety and depression-like behaviors upon chronic stress. Here, we observed that chronic stress caused myelin loss in wild-type (WT) mice, while the myelin level in D2R−/− mice, which was already lower than that in WT mice, was not affected upon stress. Fewer mature oligodendrocytes (OLs) were observed in the corpus callosum of stressed WT mice, while in D2R−/− mice, both the control and stressed group displayed a decrease in the number of mature OLs. We observed a decrease in the number of active β-catenin (ABC)-expressing and TCF4-expressing cells among OL lineage cells in the corpus callosum of stressed WT mice, while such regulation was not found in D2R−/− mice. Administration of lithium normalized the behavioral impairments and myelin damage induced by chronic stress in WT mice, and restored the number of ABC-positive and TCF4-positive OLs, while such effect was not found in D2R−/− mice. Together, our findings indicate that chronic stress induces myelin loss through the Wnt/β-catenin signaling pathway in association with DA signaling through D2R.

Chronic mild stress impairs latent inhibition and induces region-specific neural activation in CHL1-deficient mice, a mouse model of schizophrenia

Schizophrenia is a neurodevelopmental disorder characterized by abnormal processing of information and attentional deficits. Schizophrenia has a high genetic component but is precipitated by environmental factors, as proposed by the ‘two-hit’ theory of schizophrenia. Here we compared latent inhibition as a measure of learning and attention, in CHL1-deficient mice, an animal model of schizophrenia, and their wild-type littermates, under no-stress and chronic mild stress conditions. All unstressed mice as well as the stressed wild-type mice showed latent inhibition. In contrast, CHL1-deficient mice did not show latent inhibition after exposure to chronic stress. Differences in neuronal activation (c-Fos-positive cell counts) were noted in brain regions associated with latent inhibition: Neuronal activation in the prelimbic/infralimbic cortices and the nucleus accumbens shell was affected solely by stress. Neuronal activation in basolateral amygdala and ventral hippocampus was affected independently by stress and genotype. Most importantly, neural activation in nucleus accumbens core was affected by the interaction between stress and genotype. These results provide strong support for a ‘two-hit’ (genes x environment) effect on latent inhibition in CHL1-deficient mice, and identify CHL1-deficient mice as a model of schizophrenia-like learning and attention impairments.

Gene deficiency and pharmacological inhibition of caspase-1 confers resilience to chronic social defeat stress via regulating the stability of surface AMPARs

Both inflammatory processes and glutamatergic systems have been implicated in the pathophysiology of mood-related disorders. However, the role of caspase-1, a classic inflammatory caspase, in behavioral responses to chronic stress remains largely unknown. To address this issue, we examined the effects and underlying mechanisms of caspase-1 on preclinical murine models of depression. We found that loss of caspase-1 expression in Caspase-1−/− knockout mice alleviated chronic stress-induced depression-like behaviors, whereas overexpression of caspase-1 in the hippocampus of wild-type (WT) mice was sufficient to induce depression- and anxiety-like behaviors. Furthermore, chronic stress reduced glutamatergic neurotransmission and decreased surface expression of glutamate receptors in hippocampal pyramidal neurons of WT mice, but not Caspase-1−/− mice. Importantly, pharmacological inhibition of caspase-1-interleukin-1β (IL-1β) signaling pathway prevented the depression-like behaviors and the decrease in surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) in stressed WT mice. Finally, the effects of chronic stress on both depression- and anxiety-like behaviors can be mimicked by exogenous intracerebroventricular (i.c.v.) administration of IL-1β in both WT and Caspase-1−/− mice. Taken together, our findings demonstrate that an increase in the caspase-1/IL-1β axis facilitates AMPAR internalization in the hippocampus, which dysregulates glutamatergic synaptic transmission, eventually resulting in depression-like behaviors. These results may represent an endophenotype for chronic stress-induced depression.