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Background: Current therapies targeting several neurotransmitter systems are only able to partially mitigate the symptoms of stress- and trauma-related disorder. Stress and trauma-related disorders lead to a prominent inflammatory response in humans, and in pre-clinical models. However, mechanisms underlying the induction of neuroinflammatory response in PTSD and anxiety disorders are not clearly understood. The present study investigated the mechanism underlying the activation of proinflammatory NLRP3 inflammasome and IL1β in mouse models of stress., Methods: We used two mouse models of stress, i.e., mice subjected to physical restraint stress with brief underwater submersion, and predator odor stress. Mice were injected with MCC950, a small molecule specific inhibitor of NLRP3 activation. To pharmacologically inhibit BTK, a specific inhibitor ibrutinib was used. To validate the observation from ibrutinib studies, a separate group of mice was injected with another BTK-specific inhibitor LFM-A13. Seven days after the induction of stress, mice were examined for anxious behavior using open field test (OFT), light-dark test (LDT), and elevated plus maze test (EPM). Following the behavior tests, hippocampus and amygdale were extracted and analyzed for various components of NLRP3-caspase 1-IL1β pathway. Plasma and peripheral blood mononuclear cells were also used to assess the induction of NLRP3-Caspase 1-IL-1β pathway in stressed mice., Results: Using two different pre-clinical models of stress, we demonstrate heightened anxious behavior in female mice as compared to their male counterparts. Stressed animals exhibited upregulation of proinflammatory IL1β, IL-6, Caspase 1 activity and NLRP3 inflammasome activation in brain, which were significantly higher in female mice. Pharmacological inhibition of NLRP3 inflammasome activation led to anxiolysis as well as attenuated neuroinflammatory response. Further, we observed induction of activated Bruton's tyrosine kinase (BTK), an upstream positive-regulator of NLRP3 inflammasome activation, in hippocampus and amygdala of stressed mice. Next, we conducted proof-of-concept pharmacological BTK inhibitor studies with ibrutinib and LFM-A13. In both sets of experiments, we found BTK inhibition led to anxiolysis and attenuated neuroinflammation, as indicated by significant reduction of NLRP3 inflammasome and proinflammatory IL-1β in hippocampus and amygdala. Analysis of plasma and peripheral blood mononuclear cells indicated peripheral induction of NLRP3-caspase 1-IL1β pathway in stressed mice., Conclusion: Our study identified BTK as a key upstream regulator of neuroinflammation, which drives anxiogenic behavior in mouse model of stress. Further, we demonstrated the sexually divergent activation of BTK, providing a clue to heightened neuroinflammation and anxiogenic response to stress in females as compared to their male counterparts. Our data from the pharmacological inhibition studies suggest BTK as a novel target for the development of potential clinical treatment of PTSD and anxiety disorders. Induction of pBTK and NLRP3 in peripheral blood mononuclear cells of stressed mice suggest the potential effect of stress on systemic inflammation., (© 2021. The Author(s).)

Excessive binge alcohol intake is a common drinking pattern in humans, especially during holidays. Cessation of the binge drinking often leads to aberrant withdrawal behaviors, as well as serious heart rhythm abnormalities (clinically diagnosed as Holiday Heart Syndrome (HHS)). In our HHS mouse model with well-characterized binge alcohol withdrawal (BAW)-induced heart phenotypes, BAW leads to anxiety-like behaviors and cognitive impairment. We have previously reported that stress-activated c-Jun NH(2)-terminal kinase (JNK) plays a causal role in BAW-induced heart phenotypes. In the HHS brain, we found that activation of JNK2 (but not JNK1 and JNK3) in the prefrontal cortex (PFC), but not hippocampus and amygdala, led to anxiety-like behaviors and impaired cognition. DNA methylation mediated by a crucial DNA methylation enzyme, DNA methyltransferase1 (DNMT1), is known to be critical in alcohol-associated behavioral deficits. In HHS mice, JNK2 in the PFC (but not hippocampus and amygdala) causally enhanced total genomic DNA methylation via increased DNMT1 expression, which was regulated by enhanced binding of JNK downstream transcriptional factor c-JUN to the DNMT1 promoter. JNK2-specific inhibition either by an inhibitor JNK2I or JNK2 knockout completely offset c-JUN-regulated DNMT1 upregulation and restored the level of DNA methylation in HHS PFC to the baseline levels seen in sham controls. Strikingly, either JNK2-specific inhibition or genetic JNK2 depletion or DNMT1 inhibition (by an inhibitor 5-Azacytidine) completely abolished BAW-evoked behavioral deficits. In conclusion, our studies revealed a novel mechanism by which JNK2 drives BAW-evoked behavioral deficits through a DNMT1-regulated DNA hypermethylation. JNK2 could be a novel therapeutic target for alcohol withdrawal treatment and/or prevention., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Alcohol use-associated disorders are highly comorbid with anxiety disorders; however, their mechanism remains unknown. The amygdala plays a central role in anxiety. We recently found that 7,8-dihydroxyflavone (7,8-DHF) significantly reduces withdrawal symptoms in a rat model of chronic intermittent alcohol (ethanol) exposure. This study aimed to determine the role of 7,8-DHF in regulating anxiety induced by chronic alcohol exposure and its associated underlying mechanism. Male C57BL/6J mice were exposed to chronic intermittent alcohol for 3 weeks followed by alcohol withdrawal for 12 h with or without 7,8-DHF administered intraperitoneally. All mice were tested using an open field test and elevated plus maze to assess anxiety-like behaviors. Synaptic activity and intrinsic excitability in basal and lateral amygdala (BLA) neurons were assessed using electrophysiological recordings. 7,8-DHF alleviated alcohol-induced anxiety-like behavior and attenuated alcohol-induced enhancement of activities in BLA pyramidal neurons. Furthermore, 7,8-DHF prevented alcohol withdrawal-evoked augmentation of glutamatergic transmission in the amygdala and had no effect on GABAergic transmission in the amygdala, as demonstrated by unaltered frequency and amplitude of spontaneous inhibitory postsynaptic currents. Microinjection of K252a, a tropomyosin-related kinase B (TrkB) antagonist, into the BLA blocked the effects of 7,8-DHF on anxiety-like behavior and neuronal activity in the BLA. Our findings suggest that 7,8-DHF alleviates alcohol-induced anxiety-like behavior induced by chronic alcohol exposure through regulation of glutamate transmission involving TrKB in the BLA.

Acyl-CoA dehydrogenase 10 (Acad10)-deficient mice develop impaired glucose tolerance, peripheral insulin resistance, and abnormal weight gain. In addition, they exhibit biochemical features of deficiencies of fatty acid oxidation, such as accumulation of metabolites consistent with abnormal mitochondrial energy metabolism and fasting induced rhabdomyolysis. ACAD10 has significant expression in mouse brain, unlike other acyl-CoA dehydrogenases (ACADs) involved in fatty acid oxidation. The presence of ACAD10 in human tissues was determined using immunohistochemical staining. To characterize the effect of ACAD10 deficiency on the brain, micro-MRI and neurobehavioral evaluations were performed. Acad10-deficient mouse behavior was examined using open field testing and DigiGait analysis for changes in general activity as well as indices of gait, respectively. ACAD10 protein was shown to colocalize to mitochondria and peroxisomes in lung, muscle, kidney, and pancreas human tissue. Acad10-deficient mice demonstrated subtle behavioral abnormalities, which included reduced activity and increased time in the arena perimeter in the open field test. Mutant animals exhibited brake and propulsion metrics similar to those of control animals, which indicates normal balance, stability of gait, and the absence of significant motor impairment. The lack of evidence for motor impairment combined with avoidance of the center of an open field arena and reduced vertical and horizontal exploration are consistent with a phenotype characterized by elevated anxiety. These results implicate ACAD10 function in normal mouse behavior, which suggests a novel role for ACAD10 in brain metabolism., Competing Interests: The authors have declared that no competing interests exist.

Tissue-nonspecific alkaline phosphatase (TNAP) is a ubiquitously expressed enzyme that is best known for its role during mineralization processes in bones and skeleton. The enzyme metabolizes phosphate compounds like inorganic pyrophosphate and pyridoxal-5'-phosphate to provide, among others, inorganic phosphate for the mineralization and transportable vitamin B6 molecules. Patients with inherited loss of function mutations in the ALPL gene and consequently altered TNAP activity are suffering from the rare metabolic disease hypophosphatasia (HPP). This systemic disease is mainly characterized by impaired bone and dental mineralization but may also be accompanied by neurological symptoms, like anxiety disorders, seizures, and depression. HPP characteristically affects all ages and shows a wide range of clinical symptoms and disease severity, which results in the classification into different clinical subtypes. This review describes the molecular function of TNAP during the mineralization of bones and teeth, further discusses the current knowledge on the enzyme's role in the nervous system and in sensory perception. An additional focus is set on the molecular role of TNAP in health and on functional observations reported in common laboratory vertebrate disease models, like rodents and zebrafish.

Cyclooxygenases are a group of heme-containing isozymes (namely Cox-1 and Cox-2) that catalyze the conversion of arachidonic acid to largely bioactive prostaglandins (PGs). Cox-1 is the ubiquitous housekeeping enzyme, and the mitogen-inducible Cox-2 is activated to cause inflammation. Interestingly, Cox-2 is constitutively expressed in the brain at the postsynaptic dendrites and excitatory terminals of the cortical and spinal cord neurons. Neuronal Cox-2 is activated in response to synaptic excitation to yield PGE 2 , the predominant Cox-2 metabolite in the brain, which in turn stimulates the release of glutamate and neuronal firing in a retrograde fashion. Cox-2 is also engaged in the metabolism of new endocannabinoids from 2-arachidonoyl-glycerol to modulate their actions at presynaptic terminals. In addition to these interactions, the induction of neuronal Cox-2 is coupled to the trans-synaptic activation of the dopaminergic mesolimbic system and some serotoninergic receptors, which might contribute to the development of emotional behavior. Although much of the focus regarding the induction of Cox-2 in the brain has been centered on neuroinflammation-related neurodegenerative and psychiatric disorders, some evidence also suggests that Cox-2 release during neuronal signaling may be pivotal for the fine tuning of cortical networks to regulate behavior. This review compiles the evidence supporting the homeostatic role of neuronal Cox-2 in synaptic transmission and plasticity, since neuroinflammation is originally triggered by the induction of glial Cox-2 expression. The goal is to provide perspective on the roles of Cox-2 beyond neuroinflammation, such as those played in memory and anxiety, and whose evidence is still scant.

Intracellular signalling pathways have been extensively studied as therapeutic targets for the treatment of mental diseases. Our attention has been caught by two kinases potentially involved in anxiety, ERK1/2 and CaMKII. The study aimed to examine changes in the activation of ERK1/2 and CaMKII concerning anxiolytic-like behaviours in mice. To evaluate anxiety-related response in mice, we used the open field test and the elevated plus maze test. Behavioural studies were complemented with the immunoblotting analysis to identify proteins of interest in the cortex, hippocampus, and striatum. We analysed the phosphorylation status of ERK1/2 and CaMKII in mice treated with a well-known anxiolytic drug - diazepam. Next, the blockade of ERK1/2 pathway by SL-327, a selective MEK1/2 inhibitor, was checked for anxiolytic action. Finally, the co-administration of subeffective doses of diazepam and SL-327 was investigated for a potential synergistic anxiolytic effect. Anxiolytic effects of acute diazepam are accompanied by decreased p-ERK1/2 and upregulation of p-CaMKII. Subchronic treatment with SL-327 leads to the manifestation of anxiolytic-like behaviours and changes in the phosphorylation status of both kinases in a diazepam-like manner. Co-administration of subeffective doses of SL-327 and diazepam induces anxiolysis, which is CaMKII-independent and correlates to selectively decreased phosphoactive ERK1/2 in the hippocampus. The MEK-ERK pathway is significantly involved in anxiolytic action of diazepam and its prolonged inhibition produces anxiolytic-like phenotype in mice. ERK inhibition could be used to manage anxiety symptoms in a benzodiazepine-sparing regimen for treatment of anxiety., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Growing evidence demonstrates that Oxaliplatin (OXA) is commonly associated with neurotoxicity that leads to emotional and cognitive impairments. The aim of the present study was to evaluate the OXA and Na + , K + -ATPase interaction and to correlate anxious behavior and cognitive impairment induced by this chemotherapeutic in Swiss mice. Also, considering the pharmacological modulation of Na + , K + -ATPase as a potential target for OXA-induced neurotoxicity, the therapeutic potential of 7-chloro-4-(phenylselanyl) quinoline (4-PSQ) was evaluated. Mice received OXA (10 mg kg -1 ) or vehicle by intraperitoneal route (days 0 and 2). Oral administration of 4-PSQ (1 mg kg -1 ) or vehicle was performed from days 2-14. Behavioral tasks started from day 12 onwards. On day 15, the animals were sacrificed, and the tissues collected. The effects of OXA and 4-PSQ on activity and expression level of Na + , K + -ATPase in the hippocampus and cerebral cortex, and the plasmatic corticosterone levels were determined. The findings demonstrated a significant positive correlation between anxious behavior and cognitive impairment induced by OXA. OXA caused an increase on the plasmatic corticosterone levels and reduced activity and expression level of Na + , K + -ATPase. 4-PSQ reduced both anxious behavior and cognitive impairment induced by OXA. 4-PSQ effect seems to be due to the modulation of Na + , K + -ATPase and reduction of corticosterone levels. Our results helped to expand knowledge about the mechanisms involved in the physiopathology of the OXA-induced neurotoxicity and strongly indicated that 4-PSQ may be a good prototype for the treatment of anxious behavior and cognitive impairment induced by OXA exposure., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cytochrome P450 C19 (CYP2C19) metabolizes exogenous and endogenous compounds. Although CYP2C19 is highly expressed in the liver, it is also expressed in the brain during early life. Previous human and animal studies have linked CYP2C19 genotype-predicted enzyme activity to hippocampal volumes, depressive symptoms, and anxiety-like behaviors. We examined these promising associations in a general community sample comprising 386 Caucasian adults with no history of psychiatric or neurological illnesses. Contrary to previous findings, CYP2C19 genotype-predicted enzyme activity was not associated with hippocampal volumes, nor depressive and anxiety symptoms. Interstudy differences in CYP2C19 frequencies and/or study methodology may explain this discrepancy., Competing Interests: Declaration of Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Changes in sphingolipid metabolism have been suggested to contribute to the pathophysiology of major depression. In this study, we investigated the activity of acid and neutral sphingomyelinases (ASM, NSM) and ceramidases (AC, NC), respectively, in twelve brain regions of female rats selectively bred for high (HAB) versus low (LAB) anxiety-like behavior. Concomitant with their highly anxious and depressive-like phenotype, HAB rats showed increased activity of ASM and NSM as well as of AC and NC in multiple brain regions associated with anxiety- and depressive-like behavior, including the lateral septum, hypothalamus, ventral hippocampus, ventral and dorsal mesencephalon. Strong correlations between anxiety-like behavior and ASM activity were found in female HAB rats in the amygdala, ventral hippocampus and dorsal mesencephalon, whereas NSM activity correlated with anxiety levels in the dorsal mesencephalon. These results provide novel information about the sphingolipid metabolism, especially about the sphingomyelinases and ceramidases, in major depression and comorbid anxiety.

Anxiety and depression impact dramatically on public health, underlying the importance of alternative cost-effective treatments. Previous studies have shown that biophysical treatment can significantly reduce anxiety symptoms and recently, salivary alpha-amylase (SAA) has been identified as an objective correlate of the sympathetic-parasympathetic imbalance related to increased stress burden, defined as allostatic load. The aim of this study was to evaluate the effect of biophysical therapy on SAA levels, in addition to the Depression Anxiety Stress Scale (DASS)-21 questionnaire. Twenty-four workers (sales representatives) presenting with mild anxiety/stress symptoms (Generalized Anxiety Disorder 7-item scale of > 5) were randomized to biophysical treatment ( N = 12) or placebo control ( N = 12). The biophysical group underwent electromagnetic information transfer through an aqueous system procedure, with daily self-administration for one month. SAA collection and the DASS-21 questionnaire were undertaken at baseline and after one month in all patients. Clinical characteristics and baseline DASS-21 subscale scores were similar between placebo and biophysical group at baseline. After one month, patients receiving biophysical therapy had significantly reduced SAA levels compared to the placebo group (27.8 ± 39.4 vs. 116.8 ± 114.9 U/mL, p = 0.019). All three DASS-21 subscales, depression (9.3 ± 5.1 vs. 5.7 ± 5.5, p = 0.1), anxiety (6.7 ± 25 vs. 3.7 ± 2.2, p = 0.0049) and stress (10.8 ± 4.2 vs. 7.3 ± 3.7, p = 0.041) were also decreased after biophysical treatment compared to placebo after one month. Our findings suggest that biophysical therapy can benefit workers with mild (subclinical) anxiety/stress. These results were also validated by the concomitant reduction of SAA levels and an improvement in DASS-21 subscales. The underlying molecular mechanisms of this therapy remain to be characterized.

The aim of the present study was to investigate the effect of rutin administration (100 mg/kg/day) to pentylenetetrazol (PTZ)-treated Balb-c mice (60 mg/kg/day), with respect to anxiety-like behavior using both open-field and elevated plus-maze (EPM) tests, and acetylcholinesterase (AChE) activity in salt-soluble (SS) fraction and detergent-soluble (DS) fraction of the cerebral cortex, hippocampus, striatum, midbrain, and diencephalon. Our results demonstrated that the administration of PTZ in 3 doses and the induction of seizures increased significantly anxiety behavior of mice and reduced significantly DS-AChE activity in all brain regions examined, while the reduction in the SS fraction was brain region-specific. Rutin administration to normal mice did not affect their behavior, while it induced a brain region-specific reduction in SS-AChE and a significant decrease in DS-AChE in all brain regions. We demonstrated for the first time that pretreatment of PTZ-mice with rutin (PTZ + Rutin group) prevented the manifestation of anxiety and induced interestingly a further significant reduction on the SS- and DS-AChE activities only in the cerebral cortex and striatum, in comparison with PTZ group. Our results show that rutin exhibits an important anxiolytic effect and an anticholinesterase activity in specific brain areas in the seizure model of PTZ., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Accumulating evidence from preclinical and clinical studies indicates prenatal exposure to stress or excess glucocorticoids can affect offspring brain. HDAC2 is an important target of glucocorticoid. Here we detected HDAC2 expression in male offspring hippocampus from gestational restraint stressed rat during development and the relationship between HDAC2 expression and behaviors and neurogenesis in male offspring. Pregnant rats received restrained stress during the last week of pregnancy. Expressions of HDAC2 in offspring hippocampus were detected on postnatal 0 day (P0) and 60 days (P60). Neurogenesis was evaluated by Doublecortin (DCX) staining on P60. Anxiety-like behavior and cognition were detected in open field, elevated plus maze, novel object recognition test, and Barnes maze. We found that HDAC2 expression in the hippocampus of male prenatally stressed offspring (MPSO) was similar to the male control offspring on P0, but significantly lower on P60. Corresponding to the decreased expression of HDAC2 in MPSO hippocampus at P60, neurogenesis in the dentate gyrus of MPSO was significantly lower than the control male offspring. And MPSO also showed greater anxiety and poorer learning and memories abilities than control male offspring. These showed that HDAC2 could partly explain the effects of gestational stress on male offspring behaviors., (Copyright © 2018. Published by Elsevier B.V.)

Background: Patients undergoing surgery often feel anxious. Accumulating evidence indicated that presurgical anxiety was related to the more severe postsurgical pain. An animal model was established that exposed Sprague-Dawley rats to a single-prolonged stress (SPS) procedure to induce presurgical anxiety-like behaviors. The experiment revealed that presurgical anxiety not only aggravated but also prolonged postsurgical pain. However, the underlying mechanisms were unknown., Methods: The rats in group C + Cort, group I + Cort, group A + Cort, and group AI + Cort were injected with corticosterone. The rats in group C + RU486, group I + RU486, group A + RU486, and group AI + RU486 were injected with mifepristone (RU486). The rats in group C + GSK650394 and group AI + GSK650394 were injected with GSK650394. The rats in group C + FC1 and group AI + FC1 were injected with fluorocitrate (FC) 30 minutes before SPS, 30 minutes before incision, and on postoperative days 1, 2, 3, 4, and 5. The rats in group C + FC2 and group AI + FC2 were injected with FC on postoperative days 7, 8, 9, 10, 11, 12, and 13. The paw withdrawal mechanical threshold was assessed 24 hours before SPS and from postoperative days 1 to 28. The level of corticosterone was determined by enzyme-linked immunosorbent assay. The expression of serum/glucocorticoid regulated kinase 1 (SGK1), interleukin-1β, and tumor necrosis factor-α was visualized by Western blot. The concentrations of adenosine triphosphate (ATP) were measured by ATP assay kit., Results: This study showed SPS elevated plasma glucocorticoids and ATP release from astrocytes, which meant the mechanical pain hypersensitivity in presurgical anxiety-induced postsurgical hyperalgesia was dependent on GCs-SGK1-ATP signaling pathway. SGK1 protein level in astrocytes was increased in response to the glucocorticoid stimuli and enhanced the extracellular release of ATP. Furthermore, spinal astrocytes played a key role in the maintenance. Targeting spinal astrocytes in maintenance phase prevented the pathological progression., Conclusions: These data suggested an important signaling pathway that affected the pain sensitivity after operation caused by presurgical anxiety.

Background: The cholinergic system has an important role in mood regulation. Cholinesterase inhibitor pesticides (e.g. organophosphates) appear to increase depression and anxiety symptoms in the few existing animal and human studies. Human studies have not described such associations using biomarkers of exposure and studies among children are needed., Methods: We studied 529 adolescents (ages 11-17y) in agricultural communities in the Ecuadorian Andes (ESPINA study). Acetylcholinesterase (AChE) activity was measured in a finger-stick sample. Anxiety and depression symptoms were assessed using the CDI-2 and MASC-2 (greater scores reflect greater internalizing symptoms). Models adjusted for age, gender, hemoglobin, income among others., Results: The median age was 14.38y and 51% were female. The mean (SD) of the following parameters were: AChE 3.7 U/mL (0.55), depression T-score 53.0 (9.4) and anxiety T-score: 57.6 (9.8). Lower AChE activity (reflecting greater cholinesterase inhibitor exposure) was associated with higher depression symptoms (difference per SD decrease of AChE [β [95% CI:]]: 1.09 [0.02, 2.16]), was stronger among girls (β = 1.61) than boys (β = 0.69), and among younger (<14.38y, β = 1.61) vs. older children (β = 0.57). The associations were strongest among girls <14.38y (β = 3.30 [0.54, 6.05], OR for elevated symptoms per SD decrease in AChE = 2.58 [1.26, 5.27]). No associations were observed with anxiety scores. Analyses of AChE change between 2008 and 2016 concurred with these findings., Discussion: We observed associations between a biomarker of pesticide exposure and children's depression symptoms. Lower AChE activity may create risk for depression in teenagers, particularly among girls during early adolescence., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

UBA6 is an alternative enzyme for ubiquitin activation in vertebrates that plays a pivotal role in early mouse development. Previously, we reported that the Uba6 brain-specific knockout (NKO) mouse is a novel autism spectrum disorder (ASD) mouse model that displays decreased social behavior and communication. To determine the therapeutic impact of environmental stimulation in ASDs, we investigated the behavioral and molecular changes of the NKO and control mice after exposure to environmental enrichment and paired housing in different developmental phases. Our results demonstrated that early paired housing could diminish the ASD phenotypes of NKO mice such as impaired nest building and social interaction and anxiety. Additionally, increased histone acetylation in the amygdala was observed in NKO mice after paired housing without a change in Ube3a levels. Our data suggest that paired housing at an early time point can play a crucial role in ameliorating ASD behavior and can be applied in other ASD animal models or clinical settings., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Phosphodiesterase-2 (PDE2) is a cyclic nucleotide phosphodiesterase and is highly expressed in the amygdala, which suggests its important role in anxiety-like behavior., Aims: The present study examined whether reduced PDE2A expression in the central nucleus of the amygdala (CeA) produces anxiolytic-like effects in mice., Methods: PDE2A knockdown in amygdaloid (AR5) cells or the CeA was established using a lentiviral vector-based siRNA system. The anxiety-like behaviors were detected by the elevated plus maze (EPM) and hole-board tests in mice. The related proteins involved in cAMP/cGMP-dependent signaling, such as specific marker VASP ser239 , CREB ser133 and BDNF were detected by immunoblot analysis., Results: PDE2A inhibition in AR-5 cells resulted in increases in cAMP/cGMP-related pVASP ser239 and pCREB ser133 . Behavioral tests showed that PDE2A knockdown in the CeA induced anxiolytic-like effects as evidenced by the increases in percentages of open-arm entries and time spent in the open arms in the EPM test, and the increases in head dips and time spent in head dipping in the hole-board test. However, these anxiolytic-like effects were antagonized by pre-treatment of soluble guanylyl cyclase inhibitor ODQ or adenylate cyclase inhibitor SQ. Furthermore, PDE2A knockdown significantly increased pVASP Ser239 , pCREB Ser133 and decreased BDNF expression in the amygdala. Pre-intra-CeA of ODQ or SQ reversed or partially prevented the effects of PDE2A knockdown on these proteins., Conclusions: The results suggest that PDE2A plays a crucial role in the regulation of anxiety by the cGMP/cAMP-dependent pVASP-pCREB-BDNF signaling pathway.

Soluble epoxide hydrolase (sEH), an enzyme with COOH-terminal hydrolase and NH 2 -terminal lipid phosphatase activities, is expressed in regions of the brain such as the cortex, white matter, hippocampus, substantia nigra, and striatum. sEH is involved in the regulation of cerebrovascular and neuronal function upon pathological insults. However, the physiological significance of sEH and its underlying mechanism in modulating brain function are not fully understood. In this study, we investigated the role of sEH in anxiety and potential underlying mechanisms in mice. Western blot for protein phosphorylation and expression was performed. Immunohistochemical analyses and Nissl and Golgi staining were performed for histological examination. Mouse behaviors were evaluated by open field activity, elevated plus maze, classical fear conditioning, social preference test, and Morris water maze. Our results demonstrated that the expression of sEH was upregulated during postnatal development in wild-type (WT) mice. Genetic deletion of sEH (sEH -/- ) in mice resulted in anxiety-like behavior and disrupted social preference. Increased olfactory bulb (OB) size and altered integrity of neurites were observed in sEH -/- mice. In addition, ablation of sEH in mice decreased protein expression of tyrosine hydroxylase and reduced dopamine production in the brain. Moreover, the level of phosphorylated calmodulin kinase II (CaMKII) and glycogen synthase kinase 3 α/β (GSK3α/β) was higher in sEH -/- mice than in WT mice. Collectively, these findings suggest that sEH is a key player in neurite outgrowth of neurons, OB development in the brain, and the development of anxiety-like behavior, by regulating the CaMKII-GSK3α/β signaling pathway.

Aberrant activation of toll-like receptor (TLR)s results in persistent and prolonged neuroinflammation and has been implicated in the pathogenesis and exacerbation of psychiatric and neurodegenerative disorders. TLR3 coordinates the innate immune response to viral infection and recent data have demonstrated that inhibiting fatty acid amide hydrolase (FAAH), the enzyme that primarily metabolizes anandamide, modulates TLR3-mediated neuroinflammation. However, the physiological and behavioural consequences of such modulation are unknown. The present study examined the effect of URB597, a selective FAAH inhibitor, on neuroinflammation, physiological and behavioural alterations following administration of the TLR3 agonist and viral mimetic poly I:C to female rats. URB597 attenuated TLR3-mediated fever, mechanical and cold allodynia, and anxiety-like behaviour in the elevated plus maze and open field arena. There was no effect of URB597 on TLR3-mediated decreases in body weight and no effect in the sucrose preference or forced swim tests. URB597 attenuated the TLR3-mediated increase in the expression of CD11b and CD68, markers of microglia/macrophage activation. In summary, these data demonstrate that enhancing FAAH substrate levels suppresses TLR3-mediated microglia/macrophage activation and associated changes in fever, nociceptive responding and anxiety-related behaviour. These data provide further support for FAAH as a novel therapeutic target for neuroinflammatory disorders., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Modulation of behavioural responses by neuronal signalling pathways remains incompletely understood. Signalling via mitogen-activated protein (MAP) kinase cascades regulates multiple neuronal functions. Here, we show that neuronal p38α, a MAP kinase of the p38 kinase family, has a critical and specific role in modulating anxiety-related behaviour in mice. Neuron-specific p38α-knockout mice show increased levels of anxiety in behaviour tests, yet no other behavioural, cognitive or motor deficits. Using CRISPR-mediated deletion of p38α in cells, we show that p38α inhibits c-Jun N-terminal kinase (JNK) activity, a function that is specific to p38α over other p38 kinases. Consistently, brains of neuron-specific p38α-knockout mice show increased JNK activity. Inhibiting JNK using a specific blood-brain barrier-permeable inhibitor reduces JNK activity in brains of p38α-knockout mice to physiological levels and reverts anxiety behaviour. Thus, our results suggest that neuronal p38α negatively regulates JNK activity that is required for specific modulation of anxiety-related behaviour.

Background: Anhedonia (loss of pleasure) is characterized by low responsiveness to rewards and, by virtue of being one of the two core symptoms of depression, by altered responses to stress. We investigated the effect of an acute stress experience (i.e., a tandem skydive) that was expected to elicit both intense fear and intense euphoria in a sample of anhedonic young adults., Objective: (1) To examine individual differences in alpha-amylase reactivity to and recovery from a tandem skydive in anhedonic young adults; (2) to investigate whether trait depressive and anxiety problems, trait positive affect (PA), i.e., level of pleasure and reward responsiveness, and state anxiety, PA and self-esteem prior to the skydive were associated with alpha-amylase reactivity and recovery patterns; (3) to investigate whether alpha-amylase reactivity and recovery patterns were associated with pre- to post-jump changes in state anxiety, PA, and self-esteem., Method: Participants were 61 individuals with persistent anhedonia (Mage = 21.38, 78.7% female), who filled out a baseline questionnaire at the start of the study, and momentary questionnaires (3 times per day) before and after the tandem skydive. Alpha-amylase was measured at four time points by means of salivettes (2 before and 2 after the skydive)., Results: Alpha-amylase reactivity and recovery patterns were highly similar across individuals, although mean levels varied greatly. No associations were found between any of the trait and state measures and reactivity and recovery. Only state self-esteem was affected by the reactivity and recovery patterns, in that individuals who showed high reactivity and low recovery experienced decreases in self-esteem after the skydive., Conclusions: Alpha-amylase patterns following a tandem skydive in anhedonic individuals are highly similar to patterns previously found in healthy individuals. Although replication is warranted, our findings tentatively suggest that a strong stress response that cannot be downregulated well predicts a decrease in self-esteem., Competing Interests: The authors have declared that no competing interests exist.

Mammalian asparagine endopeptidase (AEP) is a lysosomal cysteine protease that cleaves protein substrates on the C-terminal side of asparagine. The expression and activity of AEP are closely related to many pathological conditions that include cancer, atherosclerosis and inflammation. It has been validated that the level of AEP is elevated in aged human and neurodegenerative diseases like Alzheimer's disease (AD). Mood disorder is one of the most emotional symptoms that can be seen in AD patients, which leads us to assume that AEP can modulate affective behaviors. AEP knockout (AEP KO) and wildtype (WT) mice were used in this study, and a series of behavioral tests were performed to establish a potential link between AEP and psychiatric disorders. It was demonstrated that AEP KO mice displayed lower anxiety-like behavior and more advance exploratory behavior in open-field and hole-board tests. AEP KO mice reduced depressive-like behaviors in the forced swim and tail suspension tests. Morris water maze (MWM) test showed that the abilities of spatial learning and memory were elevated in AEP-deletion mice compared with those of WT mice. Furthermore, the enhanced synaptic plasticity (LTP and DPT) as well as the increased expressions of SYP and PSD-95 proteins in hippocampus were showed in AEP KO mice. Otherwise, the level of BDNF protein was reduced and the level of NF-κB p65 protein was increased in hippocampus and frontal cortex of AEP KO mice. These data highlight the importance of studying AEP in the anxiety and depression behaviors and the spatial learning and memory., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Several pieces of evidence indicate that elastase-2 (ELA2; chymotrypsin-like ELA2) is an alternative pathway to the generation of angiotensin II (ANGII). Elastase-2 knockout mice (ELA2KO) exhibit alterations in the arterial blood pressure and heart rate. However, there is no data on the behavioral consequences of ELA2 deletion. In this study, we addressed this question, submitting ELA2KO and wild-type (WT) mice to several models sensitive to anxiety- and depression-like, memory, and repetitive behaviors. Our data indicates a higher incidence of barbering behavior in ELA2KO compared to WT, as well as an anxiogenic phenotype, evaluated in the elevated plus maze (EPM). While a decrease in locomotor activity was observed in ELA2KO in EPM, this feature was not the main source of variation in the other parameters analyzed. The marble-burying test (MBT) indicated increase in repetitive behavior, observed by a higher number of buried marbles. The actimeter test indicated a decrease in total activity and confirmed the increase in repetitive behavior. The spatial memory was tested by repeated exposure to the actimeter in a 24-h interval. Both ELA2KO and WT exhibited decreased activity compared to the first exposure, without any distinction between the genotypes. However, when submitted to the cued fear conditioning, ELA2KO displayed lower levels of freezing behavior in the extinction session when compared to WT, but no difference was observed during the conditioning phase. Increased levels of BDNF were found in the prefrontal cortex but not in the hippocampus of ELA2KO mice compared to WT. Finally, in silico analysis indicates that ELA2 is putatively able to cleave BDNF, and incubation of the purified enzyme with BDNF led to the degradation of the latter. Our data suggested an anxiogenic- and antidepressant-like phenotype of ELA2KO, possibly associated with increased levels of BDNF in the prefrontal cortex.

Post-stroke anxiety (PSA) is a common neuropsychiatric affective disorder occurring after stroke. The purpose of this study was to investigate the association between anxiety and the serum levels of oxidative stress markers at admission. First-ever or recurrent ischemic stroke patients were consecutively recruited into the study and followed up 1 month. Patients were divided into PSA and non-PSA group according DSM-IV criteria for anxiety due to stroke. Overall, 49 patients (24.1%) were diagnosed anxiety. Serum GPX (glutathione peroxidase), CAT (catalase), SOD (superoxide dismutase), and MDA (malondialdehyde) were significantly higher in patients with anxiety than patients without anxiety. The HAM-A scores had a significant positive association with MDA levels. In multivariate logistic regression analysis, serum antioxidant enzymes and MDA were independent predictors of PSA. An increased risk of PSA was associated with serum MDA levels ≥ 3.0 nmol/ml (adjusted OR 8.68, 95% CI 3.02-24.95; P < 0.001) after adjusting for relevant confounders such as social support and treatments at admission. Elevated serum levels of lipid oxidation products and antioxidant enzymes at admission were associated with anxiety 1 month after stroke, suggesting that these alterations might participate in the pathophysiology of anxiety symptoms in stroke patients.

This study aimed to investigate the effect of exercise and nitric oxide synthase (NOS) inhibition on memory, anxiety, and protein levels of brain-derived neurotrophic factor (BDNF) and P70S6 kinase (P70S6K). Twenty-month-old rats were divided into 6 groups: a control group, 2 groups treated with l-nitro-arginine methyl ester (L-NAME) (25 or 100 mg/kg) for 63 days, 2 groups treated with L-NAME (25 or 100 mg/kg) for 63 days plus 2 months of exercise, and 1 group treated with exercise. Behavioral tests were conducted to determine the anxiolytic and memory-improving role of exercise and NOS inhibition. BDNF, P70S6K, and cleaved caspase-3 protein levels in the hippocampus and prefrontal cortex were evaluated by Western blotting. Exercise and L-NAME (25 mg/kg) or their combination had an anxiolytic effect and improved spatial memory in old rats compared with the control or exercised group, respectively. Exercise and treatment with a low dose of L-NAME (25 mg/kg) each increased BDNF and P70S6K in the hippocampus and prefrontal cortex compared with levels in control rats. In comparison with exercise alone, co-treatment with exercise and a low dose of L-NAME (25 mg/kg) also increased BDNF and P70S6K in the hippocampus. The neuronal level of cleaved caspase-3 was reduced in the L-NAME (25 mg/kg) + exercise group compared with the exercised group. The L-NAME (100 mg/kg) + exercise treatment had no positive behavioral or molecular effects compared with exercise alone. The protective role of NOS inhibition and aerobic exercise against aging is probably modulated via BDNF and P70S6K in the brain.

Elevated levels of oxidative stress and neuronal inflammation in the hypothalamus or ventral midbrain, respectively, represent common denominators for obesity and Parkinson's Disease (PD). However, little is known about defense mechanisms that protect neurons in these regions from oxidative damage. Here, we aimed to assess whether murine Gpx4, a crucial antioxidant enzyme that protects neurons from membrane damage and ferroptosis, is critical for the protection from neuronal inflammation in two distinct pathophysiologic diseases, namely metabolic dysfunction in diet-induced obesity or PD. Gpx4 was deleted from either AgRP or POMC neurons in the hypothalamus, essential for metabolic homeostasis, or from dopaminergic neurons in the ventral midbrain, governing behaviors such as anxiety or voluntary movement. To induce a pro-inflammatory environment, AgRP and POMC neuron-specific Gpx4 knockout mice were subjected to high-fat high-sucrose (HFHS) diet. To exacerbate oxidative stress in dopaminergic neurons of the ventral midbrain, we systemically co-deleted the PD-related gene DJ-1. Gpx4 was dispensable for the maintenance of cellular health and function of POMC neurons, even in mice exposed to obesogenic conditions. In contrast, HFHS-fed mice with Gpx4 deletion from AgRP neurons displayed increased body adiposity. Gpx4 expression and activity were diminished in the hypothalamus of HFHS-fed mice compared to standard diet-fed controls. Gpx4 deletion from dopaminergic neurons induced anxiety behavior, and diminished spontaneous locomotor activity when DJ-1 was co-deleted. Overall, these data suggest a physiological role for Gpx4 in balancing metabolic control signals and inflammation in AgRP but not POMC neurons. Moreover, Gpx4 appears to constitute an important rheostat against neuronal dysfunction and PD-like symptoms in dopaminergic circuitry within the ventral midbrain., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background: There is increasing evidence suggesting that the Locus Coeruleus plays a role in pain-related anxiety. Indeed, we previously found that prolonged arthritis produces anxiety-like behavior in rats, along with enhanced expression of phosphorylated extracellular signal-regulated kinase 1/2 (a marker of plasticity) in the Locus Coeruleus. However, it is unknown how this effect correlates with the electrophysiological activity of Locus Coeruleus neurons or pain-related anxiety., Methods: Using the complete Freund's adjuvant model of monoarthritis in male Sprague-Dawley rats, we studied the behavioral attributes of pain and anxiety as well as Locus Coeruleus electrophysiology in vivo 1 (MA1W) and 4 weeks (MA4W) after disease induction., Results: The manifestation of anxiety in MA4W was accompanied by dampened tonic Locus Coeruleus activity, which was coupled to an exacerbated evoked Locus Coeruleus response to noxious stimulation of the inflamed and healthy paw. When a mitogen-activating extracellular kinase inhibitor was administered to the contralateral Locus Coeruleus of MA4W, the phosphorylated extracellular signal-regulated kinase 1/2 levels in the Locus Coeruleus were restored and the exaggerated evoked response was blocked, reversing the anxiogenic-like behavior while pain hypersensitivity remained unaltered., Conclusion: As phosphorylated extracellular signal-regulated kinase 1/2 blockade in the Locus Coeruleus relieved anxiety and counteracted altered LC function, we propose that phosphorylated extracellular signal-regulated kinase 1/2 activation in the Locus Coeruleus plays a crucial role in pain-related anxiety., (© The Author 2017. Published by Oxford University Press on behalf of CINP.)

Background: Major vessel hemorrhage in endoscopic, endonasal skull-base surgery is a rare but potentially fatal event. Surgical simulation models have been developed to train surgeons in the techniques required to manage this complication. This mixed-methods study aims to quantify the stress responses the model induces, determine how realistic the experience is, and how it changes the confidence levels of surgeons in their ability to deal with major vascular injury in an endoscopic setting., Methods: Forty consultant surgeons and surgeons in training underwent training on an endoscopic sheep model of jugular vein and carotid artery injury. Pre-course and post-course questionnaires providing demographics, experience level, confidence, and realism scores were taken, based on a 5-point Likert scale. Objective markers of stress response including blood pressure, heart rate, and salivary alpha-amylase levels were measured., Results: Mean "realism" score assessed posttraining showed the model to be perceived as highly realistic by the participants (score 4.02). Difference in participant self-rated pre-course and post-course confidence levels was significant (p < 0.0001): mean pre-course confidence level 1.66 (95% confidence interval [CI], 1.43 to 1.90); mean post-course confidence level 3.42 (95% CI, 3.19 to 3.65). Differences in subjects' heart rates (HRs) and mean arterial blood pressures (MAPs) were significant between injury models (p = 0.0008, p = 0.0387, respectively). No statistically significant difference in salivary alpha-amylase levels pretraining and posttraining was observed., Conclusion: Results from this study indicate that this highly realistic simulation model provides surgeons with an increased level of confidence in their ability to deal with the rare but potentially catastrophic event of major vessel injury in endoscopic skull-base surgery., (© 2017 ARS-AAOA, LLC.)

Defects in p21-activated kinase (PAK) lead to dendritic spine abnormalities and are sufficient to cause cognition impairment. The decrease in PAK in the brain of Alzheimer's disease (AD) patients is suspected to underlie synaptic and dendritic disturbances associated with its clinical expression, particularly with symptoms related to frontal cortex dysfunction. To investigate the role of PAK combined with Aβ and tau pathologies (3xTg-AD mice) in the frontal cortex, we generated a transgenic model of AD with a deficit in PAK activity (3xTg-AD-dnPAK mice). PAK inactivation had no effect on Aβ40 and Aβ42 levels, but increased the phosphorylation ratio of tau in detergent-insoluble protein fractions in the frontal cortex of 18-month-old heterozygous 3xTg-AD mice. Morphometric analyses of layer II/III pyramidal neurons in the frontal cortex showed that 3xTg-AD-dnPAK neurons exhibited significant dendritic attrition, lower spine density and longer spines compared to NonTg and 3xTg-AD mice. Finally, behavioral assessments revealed that 3xTg-AD-dnPAK mice exhibited pronounced anxious traits and disturbances in social behaviors, reminiscent of fronto-dependent symptoms observed in AD. Our results substantiate a critical role for PAK in the genesis of neuronal abnormalities in the frontal cortex underlying the emergence of psychiatric-like symptoms in AD.

A major aspect of mammalian aging is the decline in functional competence of many self-renewing cell types, including adult-born neuronal precursors. Since age-related senescence of self-renewal occurs simultaneously with chronic up-regulation of the p38MAPKalpha (p38α) signaling pathway, we used the dominant negative mouse model for attenuated p38α activity (DN-p38α AF/+ ) in which Thr180 and Tyr182 are mutated (T→A/Y→F) to prevent phosphorylation activation (DN-p38α AF/+ ) and kinase activity. As a result, aged DN-p38α AF/+ mice are resistant to age-dependent decline in proliferation and regeneration of several peripheral tissue progenitors when compared to wild-type littermates. Aging is the major risk factor for non-inherited forms of Alzheimer's disease (AD); environmental and genetic risk factors that accelerate the senescence phenotype are thought to contribute to an individual's relative risk. In the present study, we evaluated aged DN-p38α AF/+ and wildtype littermates in a series of behavioral paradigms to test if p38α mutant mice exhibit altered baseline abnormalities in neurological reflexes, locomotion, anxiety-like behavior, and age-dependent cognitive decline. While aged DN-p38α AF/+ and wildtype littermates appear equal in all tested baseline neurological and behavioral parameters, DN-p38α AF/+ exhibit superior context discrimination fear conditioning. Context discrimination is a cognitive task that is supported by proliferation and differentiation of adult-born neurons in the dentate gyrus of the hippocampus. Consistent with enhanced context discrimination in aged DN-p38α AF/+ , we discovered enhanced production of adult-born neurons in the dentate gyrus of DN-p38α AF/+ mice compared to wildtype littermates. Our findings support the notion that p38α inhibition has therapeutic utility in aging diseases that affect cognition, such as AD., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Although Metformin, a first-line antidiabetic drug, can ameliorate ischemia/reperfusion (I/R) induced brain damage, but how metformin benefits injured hippocampus and the mechanisms are still largely unknown. Therefore, the aim of this study was to investigate the neuroprotective mechanisms of metformin against ischemic brain damage induced by cerebral I/R and to explore whether the Akt-mediated down-regulation of the phosphorylation of JNK3 signaling pathway contributed to the protection provided by metformin. Transient global brain ischemia was induced by 4-vessel occlusion in adult male Sprague-Dawley rats. The open field tasks and Morris water maze were used to assess the effect of metformin on anxiety-like behavioral and cognitive impairment after I/R. Cresyl Violet staining was used to examine the survival of hippocampal CA1 pyramidal neurons. Immunoblotting was performed to measure the phosphorylation of Akt1, JNK3, c-Jun and the expression of cleaved caspase-3. Through ischemia/reperfusion (I/R) rat model, we found that metformin could attenuate the deficits of hippocampal related behaviors and inhibit cell apoptosis. The western blot data showed that metformin could promote the activation of Akt1 and reduce the phosphorylation of JNK3 and c-Jun as well as elevation of cleaved caspase-3 in I/R brains. PI3K inhibitor reversed all the protective effects, further indicating that metformin protect hippocampus from ischemic damage through PI3K/Akt1/JNK3/c-Jun signaling pathway., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cerebral ischemia/reperfusion injury can result in neuronal death, which further results in brain damage and can even lead to death. Although recent studies showed that rosmarinic acid (RA) exerts neuroprotective effects and attenuates ischemia-induced brain injury and neuronal cell death, little is known about the precise mechanisms that occur during cerebral ischemia/reperfusion (I/R). Therefore, the aim of this study was to examine the underlying mechanism of the neuroprotective effects of RA against ischemic brain injury induced by cerebral I/R. Transient global brain ischemia was induced by 4-vessel occlusion in adult male Sprague-Dawley rats. We randomly divided rats into five groups: sham, I/R, I/R+RA, I/R+Vehicle and I/R+RA+LY. Open-field, closed-field and Morris water maze tests were carried our separately to examine the anxiety and cognitive behavior of each group. Cresyl violet staining was used to examine the survival of hippocampal CA1 pyramidal neurons. The levels of p-Akt, p-JNK3 and cleaved caspase-3 in the hippocampus were also examined by Western blotting. Our results showed that administration of RA protected locomotive ability, relieved anxiety behavior and protected cognitive ability in cerebral I/R-injured rats. Additionally, RA significantly protected neurons in the hippocampal CA1 region against cerebral I/R-induced damage. Furthermore, RA increased the phosphorylation of Akt1, downregulated the phosphorylation of JNK3 and reduced the expression of cleaved caspase-3. Finally, the Akt inhibitor LY294002 reversed all the protective effects of RA, indicating that RA protects neurons in the hippocampal CA1 region from ischemic damage through the Akt/JNK3/caspase-3 signaling pathway., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The current study evaluated the effects of Xiao Yao San (XYS) on anxiety-like behaviors and sought to determine whether the c-Jun N-terminal kinase (JNK) signaling pathway is involved. A total of 40 rats were divided into 5 groups (n=8): the control group (deionized water, per os (p.o.)), the model group (deionized water, p.o.), the SP600125 group (surgery), the per se group (surgery), and the XYS group (3.9 g/kg/d, p.o.). A 1% dimethyl sulfoxide (DMSO) citrate buffer solution (2 µL/ventricle/d) and SP600125 (10 µg/ventricle, 2 µL/ventricle/d) were separately and bilaterally injected into the rats of the two surgery groups via the ventricular system of the brain. All but the control group underwent 14 d of chronic immobilization stress (CIS; 3 h/d). On day 15, the body weights of all of the rats were measured; additionally, the rats were subjected to the elevated plus maze (EPM) and novelty suppressed feeding (NSF) tests. Finally, JNK signaling pathway indices, including phosphorylated JNK (P-JNK), JNK, phosphorylated c-Jun (P-c-Jun) and cytochrome C (Cyt-C), were examined. After modeling, the body weight and behavioral analyses of the model rats indicated that this modeling method induced anxiety-like behaviors. P-JNK, JNK, and P-c-Jun were altered in the hippocampus of the model rats. After 14 d of treatment with XYS and SP600125, rat body weight and behaviors as well as P-JNK, JNK, and P-c-Jun had changed. However, no significant difference in Cyt-C was found. XYS improves the anxiety-like behaviors induced by CIS, which might be related to the JNK signaling pathway in the hippocampus.

The present study investigated the protective effect of quercetin (Querc) on memory, anxiety-like behavior and impairment of ectonucleotidases and acetylcholinesterase (AChE) activities in brain of streptozotocin-induced diabetic rats (STZ-diabetes). The type 1 diabetes mellitus was induced by an intraperitoneal injection of 70mg/kg of streptozotocin (STZ), diluted in 0.1M sodium-citrate buffer (pH 4.5). Querc was dissolved in 25% ethanol and administered by gavage at the doses of 5, 25 and 50mg/kg once a day during 40days. The animals were distributed in eight groups of ten animals as follows: vehicle, Querc 5mg/kg, Querc 25mg/kg, Querc 50mg/kg, diabetes, diabetes plus Querc 5mg/kg, diabetes plus Querc 25mg/kg and diabetes plus Querc 50mg/kg. Querc was able to prevent the impairment of memory and the anxiogenic-like behavior induced by STZ-diabetes. In addition, Querc prevents the decrease in the NTPDase and increase in the adenosine deaminase (ADA) activities in SN from cerebral cortex of STZ-diabetes. STZ-diabetes increased the AChE activity in SN from cerebral cortex and hippocampus. Querc 50mg/kg was more effective to prevent the increase in AChE activity in the brain of STZ-diabetes. Querc also prevented an increase in the malondialdehyde levels in all the brain structures. In conclusion, the present findings showed that Querc could prevent the impairment of the enzymes that regulate the purinergic and cholinergic extracellular signaling and improve the memory and anxiety-like behavior induced by STZ-diabetes., (Copyright © 2016. Published by Elsevier Masson SAS.)

Thyroid hormones (TH) are essential for normal development and function of mammalian central nervous system (CNS); TH dysregulation has been implicated in several cognitive and behavioral deficits related to dysfunctions of neurotransmitter systems. In the present study, we investigated the effects of adult onset hypothyroidism on the activity of acetylcholinesterase (AChE) and on related behavioral parameters. For this purpose we used adult male Balb/cJ mice that were divided randomly into euthyroid and hypothyroid animal groups. Animals were rendered hypothyroid through administration of 1% w/v KClO4 in their drinking water for 8weeks. At the end of the treatment, learning/memory procedures were examined through step-through passive avoidance task while fear/anxiety was assessed using elevated plus-maze (EPM) and open-field (OF) tests. AChE activity was determined colorimetrically in two different fractions, salt-soluble fraction (SS) (containing mainly the G1 isoform) and detergent-soluble fraction (DS) (containing mainly the G4 isoform) in cerebral cortex, cerebellum, midbrain, hippocampus and striatum. Our results indicate that adult onset hypothyroidism caused significant memory impairment and increased fear/anxiety. Moreover, the activity of both isoforms of AChE was reduced in all brain regions examined in a brain region- and isoform-specific manner., (Copyright © 2016. Published by Elsevier Inc.)

Major depressive disorder is often associated with deficits in social and cognitive functioning. Mice transgenic for acid sphingomyelinase (t-ASM) were previously shown to have a depressive-like phenotype, which could be normalized by antidepressant treatment. Here, we investigated whether t-ASM mice show deficits in social behavior and memory performance, and whether these possible deficits might be normalized by amitriptyline treatment. Our results revealed that ASM overexpression altered the behavior of mice in a sex-dependent manner. As such, t-ASM female, but not male, mice showed an impaired social preference and a depressive- and anxiogenic-like phenotype, which could be normalized by amitriptyline treatment. Both male and female t-ASM mice showed unaltered preference for social novelty, novel object recognition, and social and object discrimination abilities. Amitriptyline treatment impaired novel object recognition and object discrimination abilities in female, but not in male, wild-type mice, while female t-ASM mice showed unaltered novel object recognition and object discrimination abilities. This study suggests that female t-ASM mice represent a model of depression with comorbid anxiety and social deficits, without memory impairments. It further suggests that ASM overexpression has a protective role against the detrimental effects of amitriptyline on female, but not on male, non-social (object) memory., Competing Interests: The authors have declared that no competing interests exist.

Millions of levothyroxine-treated hypothyroid patients complain of impaired cognition despite normal TSH serum levels. This could reflect abnormalities in the type 2 deiodinase (D2)-mediated T4-to-T3 conversion, given their much greater dependence on the D2 pathway for T3 production. T3 normally reaches the brain directly from the circulation or is produced locally by D2 in astrocytes. Here we report that mice with astrocyte-specific Dio2 inactivation (Astro-D2KO) have normal serum T3 but exhibit anxiety-depression-like behavior as found in open field and elevated plus maze studies and when tested for depression using the tail-suspension and the forced-swimming tests. Remarkably, 4 weeks of daily treadmill exercise sessions eliminated this phenotype. Microarray gene expression profiling of the Astro-D2KO hippocampi identified an enrichment of three gene sets related to inflammation and impoverishment of three gene sets related to mitochondrial function and response to oxidative stress. Despite normal neurogenesis, the Astro-D2KO hippocampi exhibited decreased expression of four of six known to be positively regulated genes by T3, ie, Mbp (∼43%), Mag (∼34%), Hr (∼49%), and Aldh1a1 (∼61%) and increased expression of 3 of 12 genes negatively regulated by T3, ie, Dgkg (∼17%), Syce2 (∼26%), and Col6a1 (∼3-fold) by quantitative real-time PCR. Notably, in Astro-D2KO animals, there was also a reduction in mRNA levels of genes known to be affected in classical animal models of depression, ie, Bdnf (∼18%), Ntf3 (∼43%), Nmdar (∼26%), and GR (∼20%), which were also normalized by daily exercise sessions. These findings suggest that defects in Dio2 expression in the brain could result in mood and behavioral disorders.

Objective: To compare the effects of electroacupuncture (EA) preconditioning and post-conditioning on hippocampal neuronal nitric oxide synthase (nNOS) expression in rats with anxiety-like behavior, so as to explore the reasonable EA intervention time., Methods: Forty-two SD rats were randomly divided into 6 groups:normal 1, normal 2, anxiety model 1, an-xiety model 2, EA-pre-conditioning and EA-post-conditioning. The anxiety model was established by giving the rats with repeated foot shock stimulation (0.8 mA, 2-25 s/time, 10 times in 5 min) combined with isolation-raising. Before or after modeling, EA stimulation was applied to "Baihui" (GV 20) and "Yintang" (GV 29) for 20 min, once a day for 7 days. Elevated plus maze (EPM) tests were performed to determine the percentages of time spent in the open arms and the percentages of entries into the open arms in 5 min for evaluating the animals' anxiety-like behavioral activities. The reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect hippocampal nNOS mRNA expression and the immunohistochemical staining adopted to detect the expression of nNOS protein in the hippocampal CA 1 and CA 3 areas, respectively., Results: Compared with their own normal control group 1 and 2, both the percentage of time spent in the open arms and the percentage of entries into the open arms were significantly decreased in model group 1 and 2 ( P <0.05, P <0.01). After pre- and post-conditioning of EA, the decreased proportions of time spent and entries into the open arms were considerably increased ( P <0.05), suggesting an improvement of anxiety-like behavior activities after EA intervention. The expression levels of hippocampal nNOS mRNA and nNOS protein in the hippocampal CA 3 region were significantly higher in the model group 1 and 2 than in their own normal control group 1 and 2 ( P <0.01), but those of nNOS protein in the CA 1 area were markedly lower in the model group 1 and 2 than in their own normal control group 1 and 2 ( P <0.01). Following pre- and post-conditioning of EA, the increased expression levels of nNOS mRNA and protein of CA 3 area and the decreased level of nNOS protein of CA 1 area were all notably reversed ( P <0.05, P <0.01)., Conclusions: Both pre- and post-conditioning of EA can improve anxiety-like behavior in anxiety rats, which may be associated with its effects in down-regulating hippocampal nNOS expression.

Previous studies have shown that an enriched environment (EE) has an important effect on brain function via the neuronal nitric oxide synthase/nitric oxide (nNOS/NO) pathway in young and aged animals. However, whether EE induces its effect by altering nNOS expression levels and whether it lowers anxiety-like behaviors in aged mice remains unclear. Here, we show that nNOS expression levels increased with age in the hippocampus and cerebellum in aged mice, but not in the cortex. Moreover, EE reduced anxiety-like behaviors in aged mice and reduced nNOS expression levels in the cerebellum, but not in the cortex. The present study suggests that EE improves anxiety-like behaviors in aged mice by altering nNOS expression levels in the hippocampus or cerebellum., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Altered activity of glycogen synthase kinase-3 (GSK-3) is associated with psychiatric diseases and neurodegenerative diseases. GSK-3 is a key regulator in multiple aspects of neuronal differentiation in the brain. However, little is known about the role of GSK-3 in astrocyte development. To examine the role of GSK-3 in astrocytes, we generated a conditional knockout mouse using a glial fibrillary acidic protein (GFAP)-cre driver, in which the GSK-3 alpha and beta genes are deleted in astrocytes. We found that GFAP-cre-mediated GSK-3 deletion led to a larger brain. The number and size of astrocytes were increased in GSK-3 mutant brains. The levels of GFAP and phospho-STAT3, indicators of astrogenesis, were elevated in GSK-3 mutants. Furthermore, we found upregulation of astrocyte regulatory molecules such as phospho-AKT, phospho-S6, and cyclin D in GSK-3 mutant brains. Finally, GSK-3 mutant mice exhibited aberrant anxiety and social behavior. Our results suggest that GSK-3 plays a significant role in astrocyte development and behavioral control in mice.

Over-activation of the brain renin-angiotensin system (RAS) has been implicated in the etiology of anxiety disorders. Angiotensin converting enzyme 2 (ACE2) inhibits RAS activity by converting angiotensin-II, the effector peptide of RAS, to angiotensin-(1-7), which activates the Mas receptor (MasR). Whether increasing brain ACE2 activity reduces anxiety by stimulating central MasR is unknown. To test the hypothesis that increasing brain ACE2 activity reduces anxiety-like behavior via central MasR stimulation, we generated male mice overexpressing ACE2 (ACE2 KI mice) and wild type littermate controls (WT). ACE2 KI mice explored the open arms of the elevated plus maze (EPM) significantly more than WT, suggesting increasing ACE2 activity is anxiolytic. Central delivery of diminazene aceturate, an ACE2 activator, to C57BL/6 mice also reduced anxiety-like behavior in the EPM, but centrally administering ACE2 KI mice A-779, a MasR antagonist, abolished their anxiolytic phenotype, suggesting that ACE2 reduces anxiety-like behavior by activating central MasR. To identify the brain circuits mediating these effects, we measured Fos, a marker of neuronal activation, subsequent to EPM exposure and found that ACE2 KI mice had decreased Fos in the bed nucleus of stria terminalis but had increased Fos in the basolateral amygdala (BLA). Within the BLA, we determined that ∼62% of GABAergic neurons contained MasR mRNA and expression of MasR mRNA was upregulated by ACE2 overexpression, suggesting that ACE2 may influence GABA neurotransmission within the BLA via MasR activation. Indeed, ACE2 overexpression was associated with increased frequency of spontaneous inhibitory postsynaptic currents (indicative of presynaptic release of GABA) onto BLA pyramidal neurons and central infusion of A-779 eliminated this effect. Collectively, these results suggest that ACE2 may reduce anxiety-like behavior by activating central MasR that facilitate GABA release onto pyramidal neurons within the BLA., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Anxiety disorder is a state of mental discomfort while acute anxiety induces an enhancement of vigilance/arousal or increased anxious responses. Most of the previous studies investigated basic mechanisms for acute anxiety, while less information is available for prolonged or repetitive anxiety., Results: In the present study, we wanted to examine possible molecular mechanisms for behavioral anxiety after repeated exposures. Performing a paradigm of five sessions of the elevated plus-maze (EPM), we show that the repeated exposure to the EPM induces a long-lasting anxiety causing a gradual increase of anxiolytic activity, which is maintained for at least 21 days. Genetic deletion of AC8 (adenylyl cyclase 8) but not AC1 abolished long-lasting anxiety., Conclusions: Our results suggest that calcium-stimulated AC8 is required to sustain the long-lasting anxiety caused by repeated EPM testing, and we can identify in AC8 a novel target for treating anxiety-related mood disorders.

The aim of this study is to assess neuron-specific enolase (NSE) levels and clinical features in subjects with major depressive disorder (MDD). This is a cross-sectional study with drug-naïve young adults with MDD (aged 18-29 years). Serum levels of NSE were assessed using the electrochemiluminescence method. MDD diagnosis, suicidal ideation, and time of disease were assessed using the Structured Clinical Interview for DSM-IV (SCID). The Hamilton Depression Rating Scale (HDRS) and Hamilton Anxiety Rating Scale (HARS) were used to assess depressive and anxiety symptoms. No relationship was observed between NSE levels and severity of depressive and anxiety symptoms, time of disease, and suicidal ideation. These results suggest that NSE serum levels were not associated with clinical features of MDD among drug-naïve young adults., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Nitric oxide (NO) mediated transmission in the dorsal raphe nucleus (DRN) has been shown to be involved in the modulation of anxiety-like behaviors. We investigated whether inhibition of nitric oxide synthase (NOS) in the DRN would prevent anxiety-like behavior induced by ethanol withdrawal. Male Wistar rats were treated with ethanol 2-6% (v/v) for a period of 21 days. Ethanol withdrawal was induced by abrupt discontinuation of the treatment. Experiments were performed 48 h after ethanol discontinuation. Rats with a guide cannula aimed at the DRN received intra-DRN injections of the non-selective NOS inhibitor NG-nitro-l-arginine methyl ester (l-NAME), selective neuronal NOS (nNOS) inhibitor N(ω)-propyl-l-arginine (NPLA), or selective inhibitor of inducible NOS (iNOS) N-([3-(aminomethyl)phenyl] methyl) ethanimidamidedihydrochloride (1400W). Five minutes later, the animals were tested in the elevated plus maze (EPM). Plasma ethanol levels were determined by gas chromatography. There was a reduction in plasma ethanol levels 48 h after ethanol withdrawal. Rats from the ethanol withdrawal group showed decreased exploration of the open arms of the EPM with no change in the exploration of enclosed arms. Intra-DRN treatment with l-NAME (100 nmoles/0.2 μL) and 1400W (1 nmol/0.2 μL), but not NPLA (10 nmoles/0.2 μL) in the DRN attenuated the decrease in the exploration of the open arms of the EPM induced by ethanol withdrawal. The major new finding of the present study is that iNOS in the DRN plays a role in the anxiety-like behavior induced by ethanol withdrawal., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cognitive dysfunction is a core feature of dementia and a prominent feature in psychiatric disease. As non-redundant regulators of intracellular cAMP gradients, phosphodiesterases (PDE) mediate fundamental aspects of brain function relevant to learning, memory, and higher cognitive functions. Phosphodiesterase-4B (PDE4B) is an important phosphodiesterase in the hippocampal formation, is a major Disrupted in Schizophrenia 1 (DISC1) binding partner and is itself a risk gene for psychiatric illness. To define the effects of specific inhibition of the PDE4B subtype, we generated mice with a catalytic domain mutant form of PDE4B (Y358C) that has decreased ability to hydrolyze cAMP. Structural modeling predictions of decreased function and impaired binding with DISC1 were confirmed in cell assays. Phenotypic characterization of the PDE4B(Y358C) mice revealed facilitated phosphorylation of CREB, decreased binding to DISC1, and upregulation of DISC1 and β-Arrestin in hippocampus and amygdala. In behavioral assays, PDE4B(Y358C) mice displayed decreased anxiety and increased exploration, as well as cognitive enhancement across several tests of learning and memory, consistent with synaptic changes including enhanced long-term potentiation and impaired depotentiation ex vivo. PDE4B(Y358C) mice also demonstrated enhanced neurogenesis. Contextual fear memory, though intact at 24 h, was decreased at 7 days in PDE4B(Y358C) mice, an effect replicated pharmacologically with a non-selective PDE4 inhibitor, implicating cAMP signaling by PDE4B in a very late phase of consolidation. No effect of the PDE4B(Y358C) mutation was observed in the prepulse inhibition and forced swim tests. Our data establish specific inhibition of PDE4B as a promising therapeutic approach for disorders of cognition and anxiety, and a putative target for pathological fear memory.

Background: Social defeat (SD) stress induces social avoidance and anxiety-like phenotypes. Amygdala is recognized as an emotion-related brain region such as fear, aversion and anxiety. It is conceivable to hypothesize that activation of amygdala is involved in SD-dependent behavioral defects., Results: SD model was established using C57BL/6J mice that were physically defeated by different CD-1 mice for 10 days. Stressed mice exhibited decreased social interaction level in social interaction test and significant anxiety-like behaviors in elevated plus maze and open field tests. Meanwhile, a higher phosphorylation of PKA and CREB with a mutually linear correlation, and increased Fos labeled cells in the basolateral amygdala (BLA) were observed. Activation of PKA in the BLA by 8-Br-cAMP, a PKA activitor, significantly upregulated pCREB and Fos expression. To address the role of PKA activation on SD stress-induced social avoidance and anxiety-like behaviors, 8-Br-cAMP or H-89, a PKA inhibitor, was continuously administered into the bilateral BLA by a micro-osmotic pump system during the 10-day SD period. Neither H-89 nor 8-Br-cAMP affected the social behavior. Differently, 8-Br-cAMP significantly relieved anxiety-like behaviors in both general and moderate SD protocols. H-89 per se did not have anxiogenic effect in naïve mice, but aggravated moderate SD stress-induced anxiety-like behaviors. The antidepressant clomipramine reduced SD-induced anxiety and up-regulated pPKA level in the BLA., Conclusions: These results suggest that SD-driven PKA activation in the basolateral amygdala is actually a compensatory rather than pathogenic response in the homeostasis, and modulating amygdaloid PKA may exhibit potency in the therapy of social derived disorders.

This review focuses on the genetic and other evidence supporting the notion that the cyclic AMP (cAMP) signaling pathway and its mediator, the protein kinase A (PKA) enzyme, which respond to environmental stressors and regulate stress responses, are central to the pathogenesis of disorders related to anxiety. We describe the PKA pathway and review in vitro animal studies (mouse) and other evidence that support the importance of PKA in regulating behaviors that lead to anxiety. Since cAMP signaling and PKA have been pharmacologically exploited since the 1940s (even before the identification of cAMP as a second messenger with PKA as its mediator) for a number of disorders from asthma to cardiovascular diseases, there is ample opportunity to develop therapies using this new knowledge about cAMP, PKA, and anxiety disorders., (© 2016 S. Karger AG, Basel.)

The etiology of depression remains still unclear. Recently, it has been proposed, that mitochondrial dysfunction may be associated with development of mood disorders, such as depression, bipolar disorder and anxiety disorders. Mitochondrial aldehyde dehydrogenase (ALDH2), an enzyme responsible for the detoxification of reactive aldehydes, is considered to exert protective function in mitochondria. We investigated the influence of Alda-1, a small-molecule activator of ALDH2, on depressive- and anxiety-like behaviors in an animal model of depression - the prenatally stressed rats - using behavioral, molecular and proteomic methods. Prolonged Alda-1 administration significantly increased the climbing time, tended to reduce the immobility time and increased the swimming time of the prenatally stressed rats in the forced swim test. Moreover, treatment of prenatally stressed rats with Alda-1 significantly increased number of entries into the open arms of the maze and the time spent therein, as assessed by elevated plus-maze test. Such actions were associated with reduction of plasma 4-HNE-protein content, decrease of TNF-α mRNA and increase of PGC-1α (regulator of mitochondrial biogenesis) mRNA level in the frontal cortex and hippocampus of the prenatally stressed rats as well as with normalization of peripheral immune parameters and significant changes in expression of 6 and 4 proteins related to mitochondrial functions in the frontal cortex and hippocampus, respectively. Collectively, ALDH2 activation by Alda-1 led to a significant attenuation of depressive- and anxiety-like behaviors in the prenatally stressed rats. The pattern of changes suggested mitoprotective effect of Alda-1, however the exact functional consequences of the revealed alterations require further investigation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The processes in the nervous and immune systems are closely interrelated. In particular, increased apoptosis was reported in lymphocytes of patients with depression. The aim of this study was to assess activities of proteases associated with cell death in lymphocytes of patient with personality disorders accompanied by depression and anxiety. In patients with personality disorders associated with organic brain dysfunction caspase activities were reduced in patients with depression and increased in patients with anxiety. The results may be useful for elucidation of pathogenetic mechanisms of personality disorders and in search of new biomarkers of these diseases.

Ogg1 and Mutyh DNA glycosylases cooperate to prevent mutations caused by 8-oxoG, a major premutagenic DNA lesion associated with cognitive decline. We have examined behavior and cognitive function in mice deficient of these glycosylases. Ogg1(-/-)Mutyh(-/-) mice were more active and less anxious, with impaired learning ability. In contrast, Mutyh(-/-) mice showed moderately improved memory. We observed no apparent change in genomic 8-oxoG levels, suggesting that Ogg1 and Mutyh play minor roles in global repair in adult brain. Notably, transcriptome analysis of hippocampus revealed that differentially expressed genes in the mutants belong to pathways known to be involved in anxiety and cognition. Esr1 targets were upregulated, suggesting a role of Ogg1 and Mutyh in repression of Esr1 signaling. Thus, beyond their involvement in DNA repair, Ogg1 and Mutyh regulate hippocampal gene expression related to cognition and behavior, suggesting a role for the glycosylases in regulating adaptive behavior., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)