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Differential expression of myelin-related genes and changes in myelin thickness have been demonstrated in mice after chronic psychosocial stress, a risk factor for anxiety disorders. To determine whether and how stress affects structural remodeling of nodes of Ranvier, another form of myelin plasticity, we developed a 3D reconstruction analysis of node morphology in C57BL/6NCrl and DBA/2NCrl mice. We identified strain-dependent effects of chronic social defeat stress on node morphology in the medial prefrontal cortex (mPFC) gray matter, including shortening of paranodes in C57BL/6NCrl stress-resilient and shortening of node gaps in DBA/2NCrl stress-susceptible mice compared to controls. Neuronal activity has been associated with changes in myelin thickness. To investigate whether neuronal activation is a mechanism influencing also node of Ranvier morphology, we used DREADDs to repeatedly activate the ventral hippocampus-to-mPFC pathway. We found reduced anxiety-like behavior and shortened paranodes specifically in stimulated, but not in the nearby non-stimulated axons. Altogether, our data demonstrate (1) nodal remodeling of the mPFC gray matter axons after chronic stress and (2) axon-specific regulation of paranodes in response to repeated neuronal activity in an anxiety-associated pathway. Nodal remodeling may thus contribute to aberrant circuit function associated with anxiety disorders., (© 2023. The Author(s).)

Microglia are immune brain cells implicated in stress-related mental illnesses including posttraumatic stress disorder (PTSD). Their role in the pathophysiology of PTSD, and on neurobiological systems that regulate stress, is not completely understood. We tested the hypothesis that microglia activation, in fronto-limbic brain regions involved in PTSD, would be elevated in participants with occupation-related PTSD. We also explored the relationship between cortisol and microglia activation. Twenty participants with PTSD and 23 healthy controls (HC) completed positron emission tomography (PET) scanning of the 18-kDa translocator protein (TSPO), a putative biomarker of microglia activation using the probe [ 18 F]FEPPA, and blood samples for measurement of cortisol. [ 18 F]FEPPA V T was non-significantly elevated (6.5-30%) in fronto-limbic regions in PTSD participants. [ 18 F]FEPPA V T was significantly higher in PTSD participants reporting frequent cannabis use compared to PTSD non-users (44%, p = 0.047). Male participants with PTSD (21%, p = 0.094) and a history of early childhood trauma (33%, p = 0.116) had non-significantly higher [ 18 F]FEPPA V T . Average fronto-limbic [ 18 F]FEPPA V T was positively related to cortisol (r = 0.530, p = 0.028) in the PTSD group only. Although we did not find a significant abnormality in TSPO binding in PTSD, findings suggest microglial activation might have occurred in a subgroup who reported frequent cannabis use. The relationship between cortisol and TSPO binding suggests a potential link between hypothalamic-pituitary-adrenal-axis dysregulation and central immune response to trauma which warrants further study., (© 2023. The Author(s).)

Gestational stress can increase postpartum depression in women. To treat maternal depression, fluoxetine (FLX) is most commonly prescribed. While FLX may be effective for the mother, at high doses it may have adverse effects on the fetus. As environmental enrichment (EE) can reduce maternal stress effects, we hypothesized that a subthreshold dose of FLX increases the impact of EE to reduce anxiety and depression-like behavior in postpartum dams exposed to gestational stress. We evaluated this hypothesis in mice and to assess underlying mechanisms we additionally measured hypothalamic-pituitary-adrenal (HPA) axis function and brain levels of the hormone oxytocin, which are thought to be implicated in postpartum depression. Gestational stress increased anxiety- and depression-like behavior in postpartum dams. This was accompanied by an increase in HPA axis function and a decrease in whole-brain oxytocin levels in dams. A combination of FLX and EE remediated the behavioral, HPA axis and oxytocin changes induced by gestational stress. Central administration of an oxytocin receptor antagonist prevented the remediating effect of FLX + EE, indicating that brain oxytocin contributes to the effect of FLX + EE. These findings suggest that oxytocin is causally involved in FLX + EE mediated remediation of postpartum stress-related behaviors, and HPA axis function in postpartum dams.

Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by abnormal expansion of glutamine-encoding CAG repeats in the Ataxin-1 (ATXN1) gene. SCA1 is characterized by progressive motor deficits, cognitive decline, and mood changes including anxiety and depression, with longer number of repeats correlating with worse disease outcomes. While mouse models have been very useful in understanding etiology of ataxia and cognitive decline, our understanding of mood symptoms in SCA1 has lagged. It remains unclear whether anxiety or depression stem from an underlying brain pathology or as a consequence of living with an untreatable and lethal disease. To increase our understanding of the etiology of SCA1 mood alterations, we used the elevated-plus maze, sucrose preference and forced swim tests to assess mood in four different mouse lines. We found that SCA1 knock-in mice exhibit increased anxiety that correlated with the length of CAG repeats, supporting the idea that underlying brain pathology contributes to SCA1-like anxiety. Additionally, our results support the concept that increased anxiety is caused by non-cerebellar pathology, as Purkinje cell specific SCA1 transgenic mice exhibit decreased anxiety-like behavior. Regarding the molecular mechanism, partial loss of ATXN1 may play a role in anxiety, based on our results for Atxn1 haploinsufficient and null mice.

The peripartum period is accompanied by numerous physiological and behavioural adaptations organised by the maternal brain. These changes are essential for adequate expression of maternal behaviour, thereby ensuring proper development of the offspring. The corticotropin-releasing factor (CRF) plays a key role in a variety of behaviours accompanying stress, anxiety, and depression. There is also evidence that CRF contributes to maladaptations during the peripartum period. We investigated the effects of CRF in the bed nucleus of the stria terminalis (BNST) of lactating mice during maternal care and analysed locomotor activity and anxiety-like behaviour in the offspring. The BNST has been implicated in anxiety behaviour and regulation of the stress response. The effects of intra-BNST CRF administration were compared with those induced by the limited bedding (LB) procedure, a model that produces altered maternal behaviour. BALB/cJ dams were exposed to five infusions of CRF or saline into the BNST in the first weeks after birth while the LB dams were exposed to limited nesting material from postnatal days (P) 2-9. Maternal behaviour was recorded in intercalated days, from P1-9. Offspring anxiety-like behaviour was assessed during adulthood using the open-field, elevated plus-maze, and light/dark tests. Both intra-BNST CRF and LB exposure produced altered maternal care, represented by decreased arched-back nursing and increased frequency of exits from the nest. These changes in maternal care resulted in robust sex-based differences in the offspring's behavioural responses during adulthood. Females raised by CRF-infused dams exhibited increased anxiety-like behaviour, whereas males presented a significant decrease in anxiety. On the other hand, both males and females raised by dams exposed to LB showed higher locomotor activity. Our study demonstrates that maternal care is impaired by intra-BNST CRF administrations, and these maladaptations are similar to exposure to adverse early environments. These procedures, however, produce distinct phenotypes in mice during young adulthood and suggest sex-based differences in the susceptibility to poor maternal care.

Adolescent alcohol exposure may increase anxiety-like behaviors by altering central monoaminergic functions and other important neuronal pathways. The present study was designed to investigate the anxiolytic effect of 0.5% γ-oryzanol (GORZ) and its neurochemical and molecular mechanisms under chronic 10% ethanol consumption. Five-week-old ICR male mice received either control (14% casein, AIN 93 M) or GORZ (14% casein, AIN 93 M + 0.5% GORZ) diets in this study. We showed that GORZ could potentially attenuate alcohol-induced anxiety-like behaviors by significantly improving the main behavioral parameters measured by the elevated plus maze test. Moreover, GORZ treatment significantly restored the alcohol-induced downregulation of 5-hydroxytryptophan and 5-hydroxyindole acetic acid in the hippocampus and improved homovanillic acid levels in the cerebral cortex. Furthermore, a recovery increase in the level of 3-methoxy-4-hydroxyphenylglycol both in the hippocampus and cerebral cortex supported the anxiolytic effect of GORZ. The significant elevation and reduction in the hippocampus of relative mRNA levels of brain-derived neurotrophic factor and interleukin 1β, respectively, also showed the neuroprotective role of GORZ in ethanol-induced anxiety. Altogether, these results suggest that 0.5% GORZ is a promising neuroprotective drug candidate with potential anxiolytic, neurogenic, and anti-neuroinflammatory properties for treating adolescent alcohol exposure.

The current report provides an updated review of sleep disturbance in posttraumatic stress disorder and anxiety-related disorders. First, this review provides a summary description of the unique and overlapping clinical characteristics and physiological features of sleep disturbance in specific DSM anxiety-related disorders. Second, this review presents evidence of a bidirectional relationship between sleep disturbance and anxiety-related disorders, and provides a model to explain this relationship by integrating research on psychological and neurocognitive processes with a current understanding of neurobiological pathways. A heuristic neurobiological framework for understanding the bidirectional relationship between abnormalities in sleep and anxiety-related brain pathways is presented. Directions for future research are suggested.

Pharmacological research in mice and human genetic analyses suggest that the kallikrein-kinin system (KKS) may regulate anxiety. We examined the role of the KKS in anxiety and stress in both species. In human genetic association analysis, variants in genes for the bradykinin precursor (KNG1) and the bradykinin receptors (BDKRB1 and BDKRB2) were associated with anxiety disorders (p < 0.05). In mice, however, neither acute nor chronic stress affected B1 receptor gene or protein expression, and B1 receptor antagonists had no effect on anxiety tests measuring approach-avoidance conflict. We thus focused on the B2 receptor and found that mice injected with the B2 antagonist WIN 64338 had lowered levels of a physiological anxiety measure, the stress-induced hyperthermia (SIH), vs controls. In the brown adipose tissue, a major thermoregulator, WIN 64338 increased expression of the mitochondrial regulator Pgc1a and the bradykinin precursor gene Kng2 was upregulated after cold stress. Our data suggests that the bradykinin system modulates a variety of stress responses through B2 receptor-mediated effects, but systemic antagonists of the B2 receptor were not anxiolytic in mice. Genetic variants in the bradykinin receptor genes may predispose to anxiety disorders in humans by affecting their function.

The personality trait neuroticism is associated with increased vulnerability to anxiety and mood disorders, conditions linked with abnormal serotonin neurotransmission and emotional processing. The interaction between neuroticism and serotonin during emotional processing is however not understood. Here we investigate how individual neuroticism scores influence the neural response to negative emotional faces and their sensitivity to serotonergic tone. Twenty healthy participants performed an emotional face task under functional MRI on three occasions: increased serotonin tone following infusion of a selective serotonin reuptake inhibitor (SSRI), decreased serotonin tone following acute tryptophan depletion (ATD) protocol, and no serotonin challenge (control). During the task, participants performed a gender-discrimination task of neutral, fearful or angry facial expressions. Individual variations in neuroticism scores were associated with neural response of subgenual anterior cingulate cortex to fearful facial expressions. The association was however opposite under the two serotoninergic challenges. The fear-related response in this region and individual neuroticism scores correlated negatively during citalopram challenge and positively during ATD. Thus, neuroticism scores were associated with the relative impact of serotonin challenges on fear processing in subgenual anterior cingulate cortex. This finding may link to a neural mechanism for the variable therapeutic effect of SSRI treatment observed in clinical populations.

Prenatal distress is associated with adverse outcomes in affected offspring. Alterations in placental glucocorticoid signalling and subsequent foetal overexposure to glucocorticoids have been implicated as an underlying mechanism. Infant sex is emerging as an important factor in disease susceptibility. This study aimed to examine the effects of maternal distress across pregnancy on birth outcomes and placental glucocorticoid genes in a sex-dependent manner. Participants completed psychological distress questionnaires throughout pregnancy. Placental HSD11B2, NR3C1 and FKBP51 were analysed by real time PCR and cortisol was measured in new-born hair. Second trimester stress was negatively correlated with birthweight in males and positively correlated with placental NR3C1 mRNA in females. Second trimester anxiety was negatively correlated with birthweight and placental FKBP51 mRNA in females. In mediation analysis, placental FKBP51 mRNA expression was found to mediate the link between prenatal anxiety and birthweight. New-born cortisol was negatively correlated with second trimester anxiety and positively correlated with female placental FKBP51 mRNA levels. Again, FKBP51 mRNA was found to mediate the link between anxiety and new-born cortisol. These results highlight a role for FKBP51 in the placental response to prenatal distress in females. The precise role that placental FKBP51 has in foetal and infant development has not been extensively studied and warrants further investigations.

Psychosocial stress contributes to the development of psychiatric disorders. Repeated social defeat (RSD) is a murine stressor that causes a release of inflammatory monocytes into circulation. Moreover, RSD-induced anxiety-like behavior is dependent on the recruitment of these monocytes to the brain. Activation of the endocannabinoid (ECB) system may modulate both neuroendocrine and inflammatory responses mediated by stress. Therefore, we hypothesized that a cannabinoid receptor agonist would attenuate RSD-induced inflammation, anxiety, and stress sensitization. To test this hypothesis, mice received an injection of the synthetic cannabinoid 1/2 receptor agonist, WIN55,212-2 (WIN; 1 mg/kg, intraperitoneally) daily for six consecutive days, 30 min before each exposure to RSD. Anxiety-like behavior, immune activation, neuroinflammation, and microglial reactivity were determined 14 h after RSD. RSD-induced anxiety-like behavior in the open field and in the EPM was reversed by WIN55,212-2. Moreover, WIN55,212-2 reduced the accumulation of inflammatory monocytes in circulation and brain after RSD and attenuated RSD-induced interleukin-1β (IL-1β) messenger RNA (mRNA) expression in microglia/macrophages. Increased ex vivo reactivity of microglia/monocytes to lipopolysaccharides (LPS) after RSD was also attenuated by WIN55,212-2. Next, fear expression, extinction, and recall were evaluated 24 and 48 h, respectively, after contextual fear conditioning, which took place 7 days after RSD. Here, RSD caused prolonged fear expression and impaired fear extinction recall, which was associated with increased IL-1β mRNA in the brain. Moreover, these stress-induced effects were reversed by WIN55,212-2. In conclusion, activation of cannabinoid receptors limited the immune and neuroinflammatory responses to RSD and reversed the short-term and long-term behavioral deficits associated with RSD.

Anxiety disorders significantly impair quality of life. However, limited knowledge of the underlying mechanisms impedes the development of effective therapeutics. Previous studies have suggested that the expression of the Nptx2 gene is associated with anxiety, but the neurobiological processes underlying this association remain unclear. We generated multiple mouse models with knockout or overexpression of Nptx2 in specific brain regions and during different developmental stages to assess anxiety, adult neurogenesis, and glucocorticoid-related gene expression. Our results provide evidence that Nptx2 expression in the adult hippocampus regulates anxiety in mice. Eliminating Nptx2 expression in either the developing mouse brain or in adulthood leads to increased anxiety levels. The increase in anxiety was evident in hippocampus-specific Nptx2 knockout mice, but not in an amygdala specific knockouts. Gene expression analysis revealed increased expression of glucocorticoid receptor target genes in Nptx2 knockout mice after acute stress. Overexpression of Nptx2 in the hippocampus alleviates stress-induced anxious behaviors and reverses the changes in expression of glucocorticoid receptor related genes. In conclusion, we demonstrate that Nptx2 in the hippocampus performs a critical role in modulating anxiety, hippocampal cell proliferation, and glucocorticoid receptor related gene expression. Our results suggest Nptx2 may be a potential target for anxiolytic therapeutics.

Chronic psychosocial stress is a well-established risk factor for neuropsychiatric diseases. Abnormalities in brain activity have been demonstrated in patients with stress-related disorders. Global brain activation patterns during chronic stress exposure are less well understood but may have strong modifying effects on specific brain circuits and thereby influence development of stress-related pathologies. We determined neural activation induced by chronic social defeat stress, a mouse model of psychosocial stress. To assess chronic activation with an unbiased brain-wide focus we used manganese-enhanced magnetic resonance imaging (MEMRI) and immunohistochemical staining of ∆FOSB, a transcription factor induced by repeated neural activity. One week after 10-day social defeat we observed significantly more activation in several brain regions known to regulate depressive and anxiety-like behaviour, including the prefrontal cortex, bed nucleus of stria terminalis, ventral hippocampus and periaqueductal grey in stressed compared to control mice. We further established that the correlation of ∆FOSB positive cells between specific brain regions was altered following chronic social defeat. Chronic activation of these neural circuits may relate to persistent brain activity changes occurring during chronic psychosocial stress exposure, with potential relevance for the development of anxiety and depression in humans.

An abnormal neuronal activity in the amygdala is involved in the pathogenesis of anxiety disorders. However, little is known about the mechanisms. High-anxiety mice and low-anxiety mice, representing the innate extremes of anxiety-related behaviors, were first grouped according to their anxiety levels in the elevated plus maze test. We found that the mRNA for endothelin-1 (ET1) and ET1 B-type receptors (ETBRs) in the amygdala was down-regulated in high-anxiety mice compared with low-anxiety mice. Knocking down basolateral amygdala (BLA) ET1 expression enhanced anxiety-like behaviors, whereas over-expressing ETBRs, but not A-type receptors (ETARs), had an anxiolytic effect. The combined down-regulation of ETBR and ET1 had no additional anxiogenic effect compared to knocking down the ETBR gene alone, suggesting that BLA ET1 acts through ETBRs to regulate anxiety-like behaviors. To explore the mechanism underlying this phenomenon further, we verified that most of the ET1 and the ET1 receptors in the BLA were expressed in pyramidal neurons. The ET1-ETBR signaling pathway decreased the firing frequencies and threshold currents for the action potentials of BLA pyramidal neurons but did not alter BLA synaptic neurotransmission. Together, these results indicate that amygdalar ET1-ETBR signaling could attenuate anxiety-like behaviors by directly decreasing the excitability of glutamatergic neurons.

Generalized anxiety disorder (GAD) is a common chronic condition that is understudied compared to other psychiatric disorders. An altered adrenergic function has been reported in GAD, however direct evidence for genetic susceptibility is missing. This study evaluated the associations of gene variants in adrenergic receptors (ADRs) with GAD, with the involvement of stressful events. Data were obtained from 844 French community-dwelling elderly aged 65 or over. Anxiety disorders were assessed using the Mini-International Neuropsychiatry Interview, according to DSM-IV criteria. Eight single-nucleotide polymorphisms (SNPs) involved with adrenergic function were genotyped; adrenergic receptors alpha(1A) (ADRA1A), alpha(2A) (ADRA2A), and beta2 (ADRB2) and transcription factor TCF7L2. Questionnaires evaluated recent stressful life events as well as early environment during childhood and adolescence. Using multivariate logistic regression analyses four SNPs were significantly associated with GAD. A 4-fold modified risk was found with ADRA1A rs17426222 and rs573514, and ADRB2 rs1042713 which remained significant after Bonferroni correction. Certain variants may moderate the effect of adverse life events on the risk of GAD. Replication in larger samples is needed due to the small case number. This is the first study showing that ADR variants are susceptibility factors for GAD, further highlighting the critical role of the adrenergic nervous system in this disorder.

Large numbers of women undergo antidepressant treatment during pregnancy; however, long-term consequences for their offspring remain largely unknown. Rodents exposed to serotonin transporter (SERT)-inhibiting antidepressants during development show changes in adult emotion-like behavior. These changes have been equated with behavioral alterations arising from genetic reductions in SERT. Both models are highly relevant to humans yet they vary in their time frames of SERT disruption. We find that anxiety-related behavior and, importantly, underlying serotonin neurotransmission diverge between the two models. In mice, constitutive loss of SERT causes life-long increases in anxiety-related behavior and hyperserotonemia. Conversely, early exposure to the antidepressant escitalopram (ESC; Lexapro) results in decreased anxiety-related behavior beginning in adolescence, which is associated with adult serotonin system hypofunction in the ventral hippocampus. Adult behavioral changes resulting from early fluoxetine (Prozac) exposure were different from those of ESC and, although somewhat similar to SERT deficiency, were not associated with changes in hippocampal serotonin transmission in late adulthood. These findings reveal dissimilarities in adult behavior and neurotransmission arising from developmental exposure to different widely prescribed antidepressants that are not recapitulated by genetic SERT insufficiency. Moreover, they support a pivotal role for serotonergic modulation of anxiety-related behavior.

Evidence in animal models suggests IL-1 family cytokines interact with central endogenous opioid neurotransmitter systems, inducing or perpetuating pathological states such as persistent pain syndromes, depression, substance use disorders, and their comorbidity. Understanding these interactions in humans is particularly relevant to understanding pathological states wherein this neurotransmitter system is implicated (ie, persistent pain, mood disorders, substance use disorders, etc). Here, we examined relationships between IL-1β, IL-1ra, and functional measures of the endogenous opioid system in 34 healthy volunteers, in the absence and presence of a standardized sustained muscular pain challenge, a psychophysical challenge with emotionally and physically stressful components. Mu-opioid receptor availability in vivo was examined with [(11)C]carfentanil positron emission tomography (PET) scanning. Sex and neuroticism impacted IL-1 family cytokines; higher baseline IL-1β and IL-1ra was identified in females with lower neuroticism. Higher baseline IL-1β was also associated with reduced μ-opioid receptor availability (amygdala) and greater pain sensitivity. The pain challenge increased IL-1β in females with high neuroticism. Strong associations between IL-1ra (an anti-nociceptive cytokine) and μ-opioid receptor activation (VP/NAcc) were identified during the pain challenge and the resulting analgesic effect of μ-opioid receptor activation was moderated by changes in IL-1β whereby volunteers with greater pain induced increase in IL-1β experienced less endogenous opioid analgesia. This study demonstrates the presence of relationships between inflammatory factors and a specific central neurotransmitter system and circuitry, of relevance to understanding interindividual variations in regulation of responses to pain and other physical and emotional stressors.

The serotonin 1A receptor (5-HT1A) has a major role in modulating the effects of serotonin on mood and behavior. Previous studies have shown that knockout of 5-HT1A selectively in the raphe leads to higher levels of anxiety during adulthood. However, it remains unclear whether this phenotype is due to variation in receptor levels specifically during development or throughout life. To test the hypothesis that developmental sensitivity may underlie the effects of 5-HT1A on anxiety, we used an inducible transgenic system to selectively suppress 5-HT1A levels in serotonergic raphe neurons from post-natal days (P) 14 to P30, with a maximal reduction of 40% at P21 and return to regular levels by P30. This developmental decrease in receptor levels has long-lasting consequences, increasing anxiety and decreasing social investigation in adulthood. In addition, post-natal knockdown of autoreceptors leads to long-term increases in the excitability of serotonergic neurons, which may represent a mechanism underlying the effects of post-natal receptor variation on behavior later in life. Finally, we also examined the interplay between receptor variation and juvenile exposure to stress (applied from P14 to P21). Similar to receptor knockdown, juvenile exposure to stress led to increased anxiety phenotypes but did not exacerbate 5-HT1A knockdown-mediated anxiety levels. This work indicates that the effects of 5-HT1A autoreceptors on anxiety and social behaviors are developmentally mediated and suggests that natural variations in the expression of 5-HT1A may act during development to influence individual anxiety levels and contribute to susceptibility to anxiety disorders.

Chronic stress is the primary environmental risk factor for the development and exacerbation of affective disorders, thus understanding the neuroadaptations that occur in response to stress is a critical step in the development of novel therapeutics for depressive and anxiety disorders. Brain endocannabinoid (eCB) signaling is known to modulate emotional behavior and stress responses, and levels of the eCB 2-arachidonoylglycerol (2-AG) are elevated in response to chronic homotypic stress exposure. However, the role of 2-AG in the synaptic and behavioral adaptations to chronic stress is poorly understood. Here, we show that stress-induced development of anxiety-like behavior is paralleled by a transient appearance of low-frequency stimulation-induced, 2-AG-mediated long-term depression at GABAergic synapses in the basolateral amygdala, a key region involved in motivation, affective regulation, and emotional learning. This enhancement of 2-AG signaling is mediated, in part, via downregulation of the primary 2-AG-degrading enzyme monoacylglycerol lipase (MAGL). Acute in vivo inhibition of MAGL had little effect on anxiety-related behaviors. However, chronic stress-induced anxiety-like behavior and emergence of long-term depression of GABAergic transmission was prevented by chronic MAGL inhibition, likely via an occlusive mechanism. These data indicate that chronic stress reversibly gates eCB synaptic plasticity at inhibitory synapses in the amygdala, and in vivo augmentation of 2-AG levels prevents both behavioral and synaptic adaptations to chronic stress.

Recent studies point to a role of neuropeptide-S (NPS) in the etiology of anxiety disorders. In animal models, NPS and its receptor (NPSR) were shown to be highly expressed in the amygdala, a central structure in the fear circuit, also known to be hyper-responsive in anxiety disorders. Recently, a functional polymorphism in the NPSR gene (rs324981 A/T) has been associated with panic disorder and anxiety sensitivity. However, the role of NPSR gene variation in the modulation of fear-related amygdala responsiveness remains to be clarified. In 79 healthy subjects genotyped for NPSR rs324981, amygdala responses were assessed by means of fMRI. The participants were presented with fear-relevant faces in a robust emotion-processing paradigm frequently used to study amygdala responsiveness. We observed a strong association of NPSR T-alleles with right amygdala responsiveness to fear-relevant faces. The association peak was located in the BLA. Furthermore, responsiveness to aversive stimuli within this BLA cluster predicted a participant's self-reported harm avoidance but not depression level. We conclude that NPSR genotype is associated with increased amygdala responsiveness to fear-relevant stimuli. Thereby, NPSR rs324981 apparently causes an indirect effect on anxiety-related traits and potentially contributes to the pathogenesis of anxiety disorders by shaping fear-related limbic activity.

A minority of individuals experiencing traumatic events develop anxiety disorders. The reason for the lack of correspondence between the prevalence of exposure to psychological trauma and the development of anxiety is unknown. Extracellular proteolysis contributes to fear-associated responses by facilitating neuronal plasticity at the neuron-matrix interface. Here we show in mice that the serine protease neuropsin is critical for stress-related plasticity in the amygdala by regulating the dynamics of the EphB2-NMDA-receptor interaction, the expression of Fkbp5 and anxiety-like behaviour. Stress results in neuropsin-dependent cleavage of EphB2 in the amygdala causing dissociation of EphB2 from the NR1 subunit of the NMDA receptor and promoting membrane turnover of EphB2 receptors. Dynamic EphB2-NR1 interaction enhances NMDA receptor current, induces Fkbp5 gene expression and enhances behavioural signatures of anxiety. On stress, neuropsin-deficient mice do not show EphB2 cleavage and its dissociation from NR1 resulting in a static EphB2-NR1 interaction, attenuated induction of the Fkbp5 gene and low anxiety. The behavioural response to stress can be restored by intra-amygdala injection of neuropsin into neuropsin-deficient mice and disrupted by the injection of either anti-EphB2 antibodies or silencing the Fkbp5 gene in the amygdala of wild-type mice. Our findings establish a novel neuronal pathway linking stress-induced proteolysis of EphB2 in the amygdala to anxiety.

Anxiety disorders are increasingly prevalent in society; hence, there is a need to improve on existing treatments for such disorders. Fibroblast growth factor-2 (FGF2), a mitogen that is involved in brain development and regeneration, has been shown to both facilitate long-term extinction of fear and reduce stress-precipitated relapse in rats. Extinction is the laboratory analog of exposure-based therapies in humans. In this study, we continued to investigate the clinical potential of FGF2 as a pharmacological enhancer of extinction by examining its effect on renewal, a common type of relapse. In all experiments, rats were trained to fear a white noise-conditioned stimulus, and then this learned fear was extinguished the following day. Rats received systemic injections of FGF2 or vehicle immediately after extinction training. At test, on the day after extinction training, levels of freezing elicited by the white noise in either the extinction context or the original training context were measured. FGF2-treated rats showed less renewal of fear when tested in the original training context than did vehicle-treated rats. This pattern occurred even when vehicle rats were given double the amount of extinction training, and when FGF2-treated rats were given equivalent exposure to the extinction context. These results show that FGF2 facilitates long-term extinction and attenuates relapse, and thus highlight its potential as a novel pharmacological adjunct to exposure therapy.

Cannabinoid receptor 1 (CB1) gene (CNR1) knockout mice are prone to develop anhedonic and helpless behavior after chronic mild stress. In humans, the CB1 antagonist rimonabant increases the risk of depressed mood disorders and anxiety. These studies suggest the hypothesis that genetic variation in CB1 receptor function influences the risk of depression in humans in response to stressful life events. In a population sample (n=1269), we obtained questionnaire measures of personality (Big Five Inventory), depression and anxiety (Brief Symptom Inventory), and life events. The CNR1 gene was covered by 10 SNPs located throughout the gene to determine haplotypic association. Variations in the CNR1 gene were significantly associated with a high neuroticism and low agreeableness phenotype (explained variance 1.5 and 2.5%, respectively). Epistasis analysis of the SNPs showed that the previously reported functional 5' end of the CNR1 gene significantly interacts with the 3' end in these phenotypes. Furthermore, current depression scores significantly associated with CNR1 haplotypes but this effect diminished after covariation for recent life events, suggesting a gene x environment interaction. Indeed, rs7766029 showed highly significant interaction between recent negative life events and depression scores. The results represent the first evidence in humans that the CNR1 gene is a risk factor for depression--and probably also for co-morbid psychiatric conditions such as substance use disorders--through a high neuroticism and low agreeableness phenotype. This study also suggests that the CNR1 gene influences vulnerability to recent psychosocial adversity to produce current symptoms of depression.

Extinction of learned fear is facilitated by the partial NMDA agonist D-cycloserine (DCS). However, some studies suggest that the involvement of NMDA in learning differs depending on whether learning is for the first or second time. The current study aimed to extend these findings by examining the role of NMDA in extinction for the first and the second time. Specifically, the present series of experiments used Pavlovian fear conditioning and extinction paradigms to compare the effect of DCS on extinction of fear to a light CS the first and second time around. As found previously, DCS facilitated extinction of learned fear (Experiment 1). A novel finding, however, was that DCS did not facilitate the re-extinction of fear to this same CS following retraining (Experiments 2A and 2B). Finally, it was demonstrated that the transition from NMDA-dependent to NMDA-independent extinction was stimulus specific (Experiment 3). That is, rats were first trained to fear a CS (light); this fear was then extinguished. Following this, rats were then retrained to fear the same CS (light) or a new CS (white noise). When given a second extinction session, DCS was found to facilitate extinction of the new CS but not the original CS. The results of this series of experiments suggest that the role of NMDA in extinction depends on whether extinction is new learning (first extinction) or retrieval of a previous extinction memory (re-extinction).

Amygdala opioids such as enkephalin appear to play some role in the control of anxiety and the anxiolytic effects of benzodiazepines, although the opioid receptor subtypes mediating such effects are unclear. This study compared the influences of mu-opioid receptor (MOR) activation in the central nucleus of the amygdala (CEA) on unconditioned fear or anxiety-like responses in two models, the elevated plus maze, and the defensive burying test. The role of MORs in the anxiolytic actions of the benzodiazepine agonist diazepam was also examined using both models. Either the MOR agonist [D-Ala(2), NMe-Phe(4), Gly-ol(5)]-enkephalin (DAMGO), or the MOR antagonists Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP) or beta-funaltrexamine (FNA) were bilaterally infused into the CEA of rats before testing. The results show that microinjection of DAMGO in the CEA decreased open-arm time in the plus maze, whereas CTAP increased open-arm behaviors. In contrast, DAMGO injections in the CEA reduced burying behaviors and increased rearing following exposure to a predator odor, suggesting a shift in the behavioral response in this context. Amygdala injections of the MOR agonist DAMGO or the MOR antagonist CTAP failed to change the anxiolytic effects of diazepam in either test. Our results demonstrate that MOR activation in the central amygdala exerts distinctive effects in two different models of unconditioned fear or anxiety-like responses, and suggest that opioids may exert context-specific regulation of amygdalar output circuits and behavioral responses during exposure to potential threats (open arms of the maze) vs discrete threats (predator odor).

There is increasing recognition that many psychiatric disorders including anxiety disorders are neurodevelopmental in their origins. Here, we review and integrate data from human studies and from animal models that point to a critical period during which neural circuits that mediate anxiety develop. We then postulate that this highly plastic critical period is a time of heightened responsiveness that is particularly susceptible to adverse events. We discuss these concepts in the context the current heightened interest in gene by environment interactions in psychiatric illness emphasizing the importance of the temporal relationship between gene action and environmental milieu.

Preclinically, the combination of an SSRI and 5-HT autoreceptor antagonist has been shown to reduce the time to onset of anxiolytic activity compared to an SSRI alone. In accordance with this, clinical data suggest the coadministration of an SSRI and (+/-) pindolol can decrease the time to onset of anxiolytic/antidepressant activity. Thus, the dual-acting novel SSRI and 5-HT(1A/B) receptor antagonist, SB-649915-B, has been assessed in acute and chronic preclinical models of anxiolysis. SB-649915-B (0.1-1.0 mg/kg, i.p.) significantly reduced ultrasonic vocalization in male rat pups separated from their mothers (ED(50) of 0.17 mg/kg). In the marmoset human threat test SB-649915-B (3.0 and 10 mg/kg, s.c.) significantly reduced the number of postures with no effect on locomotion. In the rat high light social interaction (SI), SB-649915-B (1.0-7.5 mg/kg, t.i.d.) and paroxetine (3.0 mg/kg, once daily) were orally administered for 4, 7, and 21 days. Ex vivo inhibition of [(3)H]5-HT uptake was also measured following SI. SB-649915-B and paroxetine had no effect on SI after 4 days. In contrast to paroxetine, SB-649915-B (1.0 and 3.0 mg/kg, p.o., t.i.d.) significantly (p<0.05) increased SI time with no effect on locomotion, indicative of an anxiolytic-like profile on day 7. Anxiolysis was maintained after chronic (21 days) administration by which time paroxetine also increased SI significantly. 5-HT uptake was inhibited by SB-649915-B at all time points to a similar magnitude as that seen with paroxetine. In conclusion, SB-649915-B is acutely anxiolytic and reduces the latency to onset of anxiolytic behavior compared to paroxetine in the SI model.

Gamma-aminobutyric acid (GABA) transporter subtype 1 (GAT1), which transports extracellular GABA into presynaptic neurons, plays an important regulatory role in the function of GABAergic systems. However, the contributions of the GAT1 in regulating mental status are not fully understood. In this paper, we observed the behavioral alterations of GAT1 knockout (GAT1(-/-)) mice using several depression- and anxiety-related models (eg, the forced-swimming test and the tail-suspension test for testing depression-related behaviors; the open-field test, the dark-light exploration test, the emergence test, and the elevated plus maze (EPM) test for anxiety-related behaviors). Here we found that GAT1(-/-) mice showed a lower level of depression- and anxiety-like behaviors in comparison to wild-type mice. Furthermore, GAT1(-/-) mice exhibited measurable insensitivity to selected antidepressants and anxiolytics such as fluoxetine, amitriptyline, buspirone, diazepam, and tiagabine in the tail-suspension test and/or the EPM test. Moreover, the basal level of corticosterone was found to be significantly lower in GAT1(-/-) mice. These results showed that the absence of GAT1 affects mental status through enhancing the GABAergic system, as well as modifying the serotonergic system and the hypothalamic-pituitary-adrenal (HPA) activity in mice.

Benzodiazepine receptor inverse agonists reduce food intake in males, but their actions in females, in whom stress-related eating disorders are more common, as well as their behavioral mode of action remain unclear. The consummatory effects of benzodiazepine receptor ligands have alternately been hypothesized to reflect changes in the hedonic evaluation of food or secondary effects of anxiety-related or cognitive properties. To test the anorectic mode of action of benzodiazepine inverse agonists, the effects of FG 7142 on feeding microstructure were studied in nondeprived female Wistar rats (n=32). Microstructure analysis used a novel meal definition that recognizes prandial drinking. On pharmacologically synchronized diestrus I, rats were pretreated (-30 min dark onset) with the benzodiazepine partial inverse agonist FG 7142 (i.p. 0, 3.75, 7.5, 15 mg/kg) in a between-subjects design. FG 7142 delayed the onset of (16-541%), decreased the amount eaten (36-52%) and drunk (63-87%), and reduced the time spent drinking (59-87%) within the first nocturnal meal. Dose-dependent incremental anorexia continued 6 h into the dark cycle, whereas FG 7142 did not suppress the quantity, duration or rate of drinking past the first meal. Treated rats ate smaller meals (17-42%) of normal duration. This reflected that FG 7142 slowed feeding within meals (9-38%) by decreasing the regularity and maintenance of feeding from pellet-to-pellet. FG 7142 did not influence postprandial satiety; meal frequency and inter-meal intervals were unaffected. FG 7142 anorexia was blocked by the benzodiazepine receptor antagonist flumazenil in a 2:1 molar ratio (n=17 rats). The very early, nonspecific (+10 min), but not subsequent (2.5, 4.5 h) feeding-specific phase, of FG 7142 anorexia was mirrored by anxiogenic-like behavior in FG 7142-treated (7.5 mg/kg) female rats (n=48) in the elevated plus-maze. Thus, benzodiazepine receptor inverse agonists preferentially lessen the maintenance of feeding in female rats, effects opposite to those of palatable food.

Oxytocin (OT) is a neuropeptide synthesized in the paraventricular (PVN) and supraoptic nuclei (SON) in the hypothalamus. Although OT is more commonly known for its role in the milk-ejection reflex, in recent years research has indicated that OT participates in the expression of social behavior, memory processing, modulation of fear, and stress responses. The demonstration that OT influences affiliative behaviors, such as parental care and reproduction, and decreases anxiety has lead to speculations that it may have a role in mood disorders. Evidence from pharmacologic studies, pointing out the modulation of the OT system by serotonin, has argued in favor of OT as a mediator of serotonin reuptake inhibitors (SSRIs) antidepressant properties. In the present study, we investigated the distribution and overlap of OT-labeled cells and serotonin transporter (5-HTT) immunoreactive (IR) fibers in the Macaque hypothalamus, utilizing immunocytochemical and double-immunofluorescent techniques. Consistent with previous reports, the distribution of OT-labeled cells in the hypothalamus is confined to the PVN and SON. In these nuclei, we demonstrate that the distribution of 5-HTT-labeled fibers follows the distribution of OT-labeled cells. Overlap of OT-labeled neurons and 5-HTT-IR fibers occurs in the parvicellular, magnocellular, dorsal, and posterior subdivisions of the PVN. In the SON, 5-HTT-labeled fibers and OT-labeled cells overlap in the ventromedial subdivision and in the 'capsular' part of the dorsolateral SON. These findings provide neuroanatomic support for the idea that SSRIs' therapeutic effects on social affiliation and anxiety may be mediated in part through components of the OT system.

The endocannabinoids anandamide and 2-arachidonoyglycerol (2-AG) may contribute to the regulation of mood and emotion. In this study, we investigated the impact of the endocannabinoid transport inhibitor AM404 on three rat models of anxiety: elevated plus maze, defensive withdrawal and separation-induced ultrasonic vocalizations. AM404 (1-5 mg kg(-1), intraperitoneal (i.p.)) exerted dose-dependent anxiolytic-like effects in the three models. These behavioral effects were associated with increased levels of anandamide, but not 2-AG, in the prefrontal cortex and were prevented by the CB(1) cannabinoid antagonist rimonabant (SR141716A), suggesting that they were dependent on anandamide-mediated activation of CB(1) cannabinoid receptors. We also evaluated whether AM404 might influence motivation (in the conditioned place preference (CPP) test), sensory reactivity (acoustic startle reflex) and sensorimotor gating (prepulse inhibition (PPI) of the startle reflex). In the CPP test, AM404 (1.25-10 mg kg(-1), i.p.) elicited rewarding effects in rats housed under enriched conditions, but not in rats kept in standard cages. Moreover, AM404 did not alter reactivity to sensory stimuli or cause overt perceptual distortion, as suggested by its lack of effect on startle or PPI of startle. These results support a role of anandamide in the regulation of emotion and point to the anandamide transport system as a potential target for anxiolytic drugs.

We investigated the influence of a representative classical benzodiazepine on the regulation of the hypothalamic-pituitary-adrenocortical (HPA) axis activity both under basal conditions and stress. Adult male Wistar rats were intravenously administered with temazepam (0.5, 1, and 3 mg/kg body weight) and plasma concentrations of corticotropin (ACTH) and vasopressin (AVP) were measured in blood samples collected via chronically implanted jugular venous catheters. Simultaneously, the release of AVP within the hypothalamic paraventricular nucleus (PVN) was monitored via microdialysis. Plasma AVP levels remained unaffected by the different treatment conditions. Temazepam blunted the stressor exposure-induced secretion of ACTH in a dose-dependent manner. Concurrently, and also in a dose-dependent manner temazepam enhanced the intra-PVN release of AVP, known to originate from magnocellular neurons of the hypothalamic neurohypophyseal system. Furthermore, temazepam did not affect the in vitro secretion of ACTH from the adenohypophyseal cells. Taken together, the results of this study suggest that temazepam modulates the central nervous regulation of the HPA axis by altering intra-PVN AVP release. An increasingly released AVP of magnocellular origin seems to provide a negative tonus on ACTH secretion most probably via inhibiting the release of ACTH secretagogues from the median eminence into hypophyseal portal blood.

We examined the effect of glucocorticoid agonists on the extinction of conditioned fear in rats by using fear-potentiated startle. Systemic injection of glucocorticoid receptor agonists dexamethasone (DEX) (0.1, 0.5, and 1.0 mg/kg) and intra-amygdala infusion of RU28362 (0.5, 1.0, and 3.0 ng/side) prior to extinction training facilitated extinction of conditioned fear in a dose-dependent manner. Extinction of conditioned fear and circulating corticosterone levels were attenuated by administration of corticosteroid synthesis inhibitor metyrapone (25 mg/kg s.c.) 90 min before extinction training. The facilitation effect of DEX was dependent on repeated presentation of the conditioned stimulus rather than exposure to the experimental context, indicating this effect did not result from impaired expression of conditioned fear or accelerated forgetting. Intra-amygdaloid administration of the glucocorticoid receptor antagonist mifepristone (0.1, 0.2, and 0.5 ng/side, bilaterally) blocked extinction of conditioned fear and the facilitation effect of DEX in a dose-dependent manner. Mifepristone (2 ng/side) did not affect extinction but blocked the facilitating effect of DEX. Systemic administration of DEX after extinction training also facilitated extinction, suggesting that DEX may influence the memory consodilation phase of extinction. The Dose of dexamethsone or metyrapone used here did not influence fear-potentiated startle when administered before testing. Thus, it is unlikely that these drugs influenced extinction by increasing or disrupting CS processing. All results suggested that amygdaloid glucocorticoid receptors were involved in the extinction of conditioned fear.

Carriers of the short allele of a functional 5' promoter polymorphism of the serotonin transporter gene have increased anxiety-related temperamental traits, increased amygdala reactivity and elevated risk of depression. Here, we used multimodal neuroimaging in a large sample of healthy human subjects to elucidate neural mechanisms underlying this complex genetic association. Morphometrical analyses showed reduced gray matter volume in short-allele carriers in limbic regions critical for processing of negative emotion, particularly perigenual cingulate and amygdala. Functional analysis of those regions during perceptual processing of fearful stimuli demonstrated tight coupling as a feedback circuit implicated in the extinction of negative affect. Short-allele carriers showed relative uncoupling of this circuit. Furthermore, the magnitude of coupling inversely predicted almost 30% of variation in temperamental anxiety. These genotype-related alterations in anatomy and function of an amygdala-cingulate feedback circuit critical for emotion regulation implicate a developmental, systems-level mechanism underlying normal emotional reactivity and genetic susceptibility for depression.

Disturbances of neuronal excitability changes during the ovarian cycle may elevate seizure frequency in women with catamenial epilepsy and enhance anxiety in premenstrual dysphoric disorder (PMDD). The mechanisms underlying these changes are unknown, but they could result from the effects of fluctuations in progesterone-derived neurosteroids on the brain. Neurosteroids and some anxiolytics share an important site of action: tonic inhibition mediated by delta subunit-containing GABA(A) receptors (deltaGABA(A)Rs). Here we demonstrate periodic alterations in specific GABA(A)R subunits during the estrous cycle in mice, causing cyclic changes of tonic inhibition in hippocampal neurons. In late diestrus (high-progesterone phase), enhanced expression of deltaGABA(A)Rs increases tonic inhibition, and a reduced neuronal excitability is reflected by diminished seizure susceptibility and anxiety. Eliminating cycling of deltaGABA(A)Rs by antisense RNA treatment or gene knockout prevents the lowering of excitability during diestrus. Our findings are consistent with possible deficiencies in regulatory mechanisms controlling normal cycling of deltaGABA(A)Rs in individuals with catamenial epilepsy or PMDD.

Corticotropin-releasing hormone (CRH) is centrally involved in coordinating responses to a variety of stress-associated stimuli. Recent clinical data implicate CRH in the pathophysiology of human affective disorders. To differentiate the CNS pathways involving CRH and CRH receptor 1 (Crhr1) that modulate behavior from those that regulate neuroendocrine function, we generated a conditional knockout mouse line (Crhr1(loxP/loxP)Camk2a-cre) in which Crhr1 function is inactivated postnatally in anterior forebrain and limbic brain structures, but not in the pituitary. This leaves the hypothalamic-pituitary-adrenocortical (HPA) system intact. Crhr1(loxP/loxP)Camk2a-cre mutants showed reduced anxiety, and the basal activity of their HPA system was normal. In contrast to Crhr1 null mutants, conditional mutants were hypersensitive to stress corticotropin and corticosterone levels remained significantly elevated after stress. Our data clearly show that limbic Crhr1 modulates anxiety-related behavior and that this effect is independent of HPA system function. Furthermore, we provide evidence for a new role of limbic Crhr1 in neuroendocrine adaptation to stress.

The neuroendocrine and behavioral effects of chronic paroxetine treatment were investigated in two rat lines selectively bred for high anxiety-related behavior (HAB) or low anxiety-related behavior (LAB) emotionality. In addition to a characteristic behavioral phenotype with markedly passive stress-coping strategies, HAB rats show a hypothalamic vasopressinergic hyperdrive that is causally related to hypothalamic-pituitary-adrenocortical dysregulation as demonstrated in the combined dexamethasone (DEX)/corticotropin-releasing hormone (CRH) test. A total of 8 weeks of chronic paroxetine treatment induced a more active coping strategy in the forced swim test in HAB rats only. In contrast, paroxetine-treated LAB rats did not change their swimming behavior. To investigate the neuroendocrine alterations linked to these behavioral changes, a combined DEX/CRH test was performed. In HAB rats, the paroxetine-induced behavioral changes towards more active coping strategies were accompanied by a normalization of the CRH-stimulated increase in corticotropin (ACTH) and corticosterone secretion. Concomitantly, the hypothalamic vasopressinergic hyperdrive was found to be reduced in HAB but not LAB rats, as indicated by a decrease in vasopressin mRNA expression, whereas vasopressin 1a receptor binding was unaffected. These findings provide the first evidence that the vasopressinergic system is likely to be critically involved in the behavioral and neuroendocrine effects of antidepressant drugs. This novel mechanism of action of paroxetine on vasopressin gene regulation renders vasopressinergic neuronal circuits a promising target for the development of more causal antidepressant treatment strategies.

The antipanic utility of imipramine and monoamine oxidase inhibitors has led to hypotheses of noradrenergic and/or serotonin (5-HT)-related abnormalities underlying panic disorder (PD) and its agoraphobic complications. Further data support significant antipanic effects for agents with acute 5-HT reuptake blockade effects. It is unlikely that ameliorative effects observed following chronic administration of 5-HT drugs remain 5-HT specific. Despite a range of recently discovered 5-HT receptor subtypes, evidence for a specific receptor abnormality in PD is lacking. Preclinical studies suggest an important role for 5-HT effects in several animal models of anxiety, including separation-induced infant protest responses and respiratory modulation. Induction of anxiety with putative 5-HT agents such as meth-chloro-phenylpiperazine and fenfluramine lend further support to 5-HT involvement in anxiety states. Critical behavioral, physiologic, and neuroendocrine differences between putative 5-HT anxiogens and "classic" panicogens, such as lactate and carbon dioxide are discussed. We propose that 5-HT agents mediate antipanic effects through amelioration of a deranged internal evaluative mechanism along cybernetic lines, rather than simple augmentation or reduction of 5-HT function.

Regional glucose metabolic rates were measured in patients with panic disorder during the performance of auditory discrimination. Those regions examined by Reiman and colleagues in their blood flow study of panic disorder [Nature 310:683 (1984)] were examined with a higher resolution positron emission tomography (PET) scanner and with the tracer [F-18]-2-fluoro-2-deoxyglucose (FDG). In contrast to the blood flow findings of Reiman et al., we did not find global gray metabolic differences between patients with panic disorder and normal controls. Consistent with the findings of Reiman et al. [Nature; Am J Psychiatry 143:469 (1986)], we found hippocampal region asymmetry. We also found metabolic decreases in the left inferior parietal lobule and in the anterior cingulate (trend), as well as an increase in the metabolic rate of the medial orbital frontal cortex (trend) of panic disorder patients. It is unclear whether the continuous performance task (CPT) enhanced or diminished findings that would have been noted in a study performed without task.