High Anxiety-related Behavior Research Articles

Microglial sex differences in innate high anxiety and modulatory effects of minocycline

Microglia modulate synaptic refinement in the central nervous system (CNS). We have previously shown that a mouse model with innate high anxiety-related behavior (HAB) displays higher CD68+ microglia density in the key regions of anxiety circuits compared to mice with normal anxiety-related behavior (NAB) in males, and that minocycline treatment attenuated the enhanced anxiety of HAB male. Given that a higher prevalence of anxiety is widely reported in females compared to males, little is known concerning sex differences at the cellular level. Herein, we address this by analyzing microglia heterogeneity and function in the HAB and NAB brains of both sexes. Single-cell RNA sequencing revealed ten distinct microglia clusters varied by their frequency and gene expression profile. We report striking sex differences, especially in the major microglia clusters of HABs, indicating a higher expression of genes associated with phagocytosis and synaptic engulfment in the female compared to the male. On a functional level, we show that female HAB microglia engulfed a greater amount of hippocampal vGLUT1+ excitatory synapses compared to the male. We moreover show that female HAB microglia engulfed more synaptosomes compared to the male HAB in vitro. Due to previously reported effects of minocycline on microglia, we finally administered oral minocycline to HABs of both sexes and showed a significant reduction in the engulfment of synapses by female HAB microglia. In parallel to our microglia-specific findings, we further showed an anxiolytic effect of minocycline on female HABs, which is complementary to our previous findings in the male HABs. Our study, therefore, identifies the altered function of synaptic engulfment by microglia as a potential avenue to target and resolve microglia heterogeneity in mice with innate high anxiety.

Why do mice squeak? Toward a better understanding of defensive vocalization

SummaryAlthough mice mostly communicate in the ultrasonic range, they also emit audible calls. We demonstrate that mice selectively bred for high anxiety-related behavior (HAB) have a high disposition for emitting sonic calls when caught by the tail. The vocalization was unrelated to pain but sensitive to anxiolytics. As revealed by manganese-enhanced MRI, HAB mice displayed an increased tonic activity of the periaqueductal gray (PAG). Selective inhibition of the dorsolateral PAG not only reduced anxiety-like behavior but also completely abolished sonic vocalization. Calls were emitted at a fundamental frequency of 3.8 kHz, which falls into the hearing range of numerous predators. Indeed, playback of sonic vocalization attracted rats if associated with a stimulus mouse. If played back to HAB mice, sonic calls were repellent in the absence of a conspecific but attractive in their presence. Our data demonstrate that sonic vocalization attracts both predators and conspecifics depending on the context.

Astrocytes Prime an Early Synaptic Pruning and Remodeling in the Prefrontal Cortex Upon Antidepressant Treatment

Brains of major depressive disorder (MDD) patients show aberrant density and morphology of astrocytes and an altered glutamatergic activity in the prefrontal cortex (PFC), also present in an animal model of MDD, the high anxiety-related behavior (HAB) rats.

GDF15 promotes simultaneous astrocyte remodeling and tight junction strengthening at the blood-brain barrier.

Perivascular astrocyte processes (PAP) surround cerebral endothelial cells (ECs) and modulate the strengthening of tight junctions to influence blood-brain barrier (BBB) permeability. Morphologically altered astrocytes may affect barrier properties and trigger the onset of brain pathologies. However, astrocyte-dependent mediators of these events remain poorly studied. Here, we show a pharmacologically driven elevated expression and release of growth/differentiation factor 15 (GDF15) in rat primary astrocytes and cerebral PAP. GDF15 has been shown to possess trophic properties for motor neurons, prompting us to hypothesize similar effects on astrocytes. Indeed, its increased expression and release occurred simultaneously to morphological changes of astrocytes in vitro and PAP, suggesting modulatory effects of GDF15 on these cells, but also neighboring EC. Administration of recombinant GDF15 was sufficient to promote astrocyte remodeling and enhance barrier properties between ECs in vitro, whereas its pharmacogenetic abrogation prevented these effects. We validated our findings in male high anxiety-related behavior rats, an animal model of depressive-like behavior, with shrunk PAP associated with reduced expression of the junctional protein claudin-5, which were both restored by a pharmacologically induced increase in GDF15 expression. Thus, we identified GDF15 as an astrocyte-derived trigger of astrocyte process remodeling linked to enhanced tight junction strengthening at the BBB.

Inborn vs. acquired anxiety in cross-breeding and cross-fostering HAB/LAB mice bred for extremes in anxiety-related behavior.

This study focused on genetically determined versus acquired factors in shaping anxiety-related behavior by combining cross-breeding and cross-fostering approaches. Via cross-breeding of HAB (high anxiety-related behavior) female and LAB (low anxiety-related behavior) male mice, we obtained F1 hybrids with intermediate anxiety levels carrying genetic characteristics of both parental lines. Pups were raised either by their biological HAB (noncross-fostered control) or foster LAB (cross-fostered) mothers. Compared to controls, 6-week-old offspring raised by LAB mothers showed lower levels of anxiety in the elevated plus-maze and open field, but not the light-dark box, tests. No differences were found in the forced swim test reflecting active versus passive coping. The behavioral changes were associated with increased stress-induced concentrations of plasma corticosterone in cross-fostered animals. The expression of the corticotropin-releasing hormone receptor type I and glucocorticoid receptor genes did not differ in limbic and hypothalamic brain areas between cross-fostered and control mice. The data suggest that LAB-typical maternal care may partially shift behavioral and neuroendocrine characteristics of F1 crosses carrying both HAB and LAB alleles from intermediate toward reduced anxiety-related behavior. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

82. Astrocytic EphrinA Impacts the Distribution of Synaptic AMPA Receptors in Health and Depressive-Like Disorder

Brains of major depressive disorder (MDD) patients show aberrant density and morphology of astrocytes and an altered glutamatergic activity in the prefrontal cortex (PFC), also present in an animal model of MDD, the high anxiety-related behavior (HAB) rats.

Plasma phospholipid profiling of a mouse model of anxiety disorder by hydrophilic interaction liquid chromatography coupled to high-resolution mass spectrometry.

Glycerophospholipids (PLs), as amphipathic small molecules and the main constituents of biological membranes, play an important role in several cellular processes, even though their accurate identification from complex biological samples remains a challenge. In this paper, we report a fast and comprehensive HILIC-ESI-MS method for the analysis of glycerophospholipid classes using high-resolution mass spectrometry in negative mode. The final method enabled the quantitative analysis of 130 endogenous PL species in mouse plasma. The application of the method developed was to find differences of plasma PL composition in a mouse model of anxiety disorder. In the case of four PL classes and 35 PL species, significant differences were observed comparing low anxiety-related behavior with high anxiety-related behavior groups. The most characteristic trend was up-regulation in both the PL classes and PL species, and decreases were only detected in two phosphatidylcholines among 35 species in mice having elevated anxiety.

Comprehensive phospholipid and sphingomyelin profiling of different brain regions in mouse model of anxiety disorder using online two-dimensional (HILIC/RP)-LC/MS method

A novel online system including two-dimensional liquid chromatography coupled to high-resolution mass spectrometry (2D-LC/MS) was developed and applied for comprehensive phospholipid (PL) and sphingomyelin (SM) profiling of dorsal hippocampus (DHPC), ventral (VHPC) and prefrontal cortex (PFC) brain regions in a mouse model of anxiety disorder. In the first dimension, lipid classes were distinguished by hydrophilic interaction liquid chromatography (HILIC), while the second dimensional separation of individual PL and SM species was achieved by reversed-phase (RP) chromatography. For the enrichment of lipid species in diluted HILIC effluent, two RP trapping columns were used separately. The developed fully-automated 2D method allowed the quantitative analysis of over 150 endogenous PL and SM species in mouse brain regions within 40min. The developed method was applied in a pilot study, which aimed to find alteration of PL and SM composition in a mouse model of anxiety disorder. In the case of 37 PL and SM species, significant differences were observed between high anxiety-related behavior (AX) and low anxiety-related behavior (nAX) mice. In mice having elevated anxiety, the most typical trend was the downregulation of PL species, in particular, in VHPC.

Graded photochemical spinal cord injury results in chronic hyperalgesia and depression-like behaviour but no anxiety exacerbation in female BALB/c mice.

Neuropathic pain (NP) is present in 40-to-50% of spinal cord injured patients. It tends to chronicity and correlates with lower quality-of-life. Moreover, the role of NP in the eventual exacerbation of anxiety- and depression-like behaviours during its development and chronification in genetically susceptible individuals remains unclear. Thus, although solely few animal models are available, new specific models are needed to complete the array of chances to assay new therapeutic strategies with the aim of treating chronic NT and its associated mood disorders. The present study was conceived to evaluate hyperalgesic responses and anxiety- and depression-like behaviours after graded photochemical spinal cord injury (SCI) up to chronic phase. BALB/c strain was used: it expresses a phenotype characterized by high innate anxiety levels, allowing to elucidate whether NP may exacerbate mood disorders at SCI chronic phase. After different photoinduction-times on exposed spinal cord, the mice developed a graded chronic hyperalgesia with minor to non-existent motor dysfunction. Behavioural data suggest that whilst hyperalgesia associated to SCI does not exacerbate BALB/c anxiety-like behaviours, it may result in depression-like behaviour at SCI chronic phase. Our study demonstrates that chronic central hyperalgesia may exacerbate despair-like behaviour at the SCI chronic phase in a mouse model of high anxiety-related behaviour. This implies that photochemical-SCI may be a suitable model to study the comorbidity between chronic NP and mood disorders.

P.2.006 - Sex-dependent attenuation of deficits in behaviour and microglia in high anxiety-related behaviour rats by minocycline

P.2.006 - Sex-dependent attenuation of deficits in behaviour and microglia in high anxiety-related behaviour rats by minocycline

Fluoxetine normalizes disrupted light-induced entrainment, fragmented ultradian rhythms and altered hippocampal clock gene expression in an animal model of high trait anxiety- and depression-related behavior

ABSTRACTIntroduction Disturbances of circadian rhythms are a key symptom of mood and anxiety disorders. Selective serotonin reuptake inhibitors (SSRIs) - commonly used antidepressant drugs – also modulate aspects of circadian rhythmicity. However, their potential to restore circadian disturbances in depression remains to be investigated.Materials and methods The effects of the SSRI fluoxetine on genetically based, depression-related circadian disruptions at the behavioral and molecular level were examined using mice selectively bred for high anxiety-related and co-segregating depression-like behavior (HAB) and normal anxiety/depression behavior mice (NAB).Results The length of the circadian period was increased in fluoxetine-treated HAB as compared to NAB mice while the number of activity bouts and light-induced entrainment were comparable. No difference in hippocampal Cry2 expression, previously reported to be dysbalanced in untreated HAB mice, was observed, while Per2 and Per3 mRNA levels were higher in HAB mice under fluoxetine treatment.Discussion The present findings provide evidence that fluoxetine treatment normalizes disrupted circadian locomotor activity and clock gene expression in a genetic mouse model of high trait anxiety and depression. An interaction between the molecular mechanisms mediating the antidepressant response to fluoxetine and the endogenous regulation of circadian rhythms in genetically based mood and anxiety disorders is proposed.

Selective Mitochondrial Targeting Exerts Anxiolytic Effects In Vivo.

Current treatment strategies for anxiety disorders are predominantly symptom-based. However, a third of anxiety patients remain unresponsive to anxiolytics highlighting the need for more effective, mechanism-based therapeutic approaches. We have previously compared high vs low anxiety mice and identified changes in mitochondrial pathways, including oxidative phosphorylation and oxidative stress. In this work, we show that selective pharmacological targeting of these mitochondrial pathways exerts anxiolytic effects in vivo. We treated high anxiety-related behavior (HAB) mice with MitoQ, an antioxidant that selectively targets mitochondria. MitoQ administration resulted in decreased anxiety-related behavior in HAB mice. This anxiolytic effect was specific for high anxiety as MitoQ treatment did not affect the anxiety phenotype of C57BL/6N and DBA/2J mouse strains. We furthermore investigated the molecular underpinnings of the MitoQ-driven anxiolytic effect and found that MitoQ treatment alters the brain metabolome and that the response to MitoQ treatment is characterized by distinct molecular signatures. These results indicate that a mechanism-driven approach based on selective mitochondrial targeting has the potential to attenuate the high anxiety phenotype in vivo, thus paving the way for translational implementation as long-term MitoQ administration is well-tolerated with no reported side effects in mice and humans.

Intranasally applied neuropeptide S shifts a high-anxiety electrophysiological endophenotype in the ventral hippocampus towards a "normal"-anxiety one.

The neurobiological basis of pathological anxiety and the improvement of its pharmacological treatment are a matter of intensive investigation. Here, using electrophysiological techniques in brain slices from animals of the high anxiety-related behavior (HAB) and normal anxiety-related behavior (NAB) mouse model, we show that basal neurotransmission at ventral hippocampal CA3-CA1 synapses is weaker in HAB compared to NAB mice. We further demonstrate that paired-pulse facilitation (PPF) and long-term potentiation (LTP) at these synapses are more pronounced in slices from HAB animals. Based on previous findings, we also examined whether intranasal delivery of neuropeptide S (NPS), which increasingly emerges as a potential novel treatment option for anxiety symptoms occurring in a variety of diseases like anxiety disorders, posttraumatic stress disorder, and major depression, impacts on the high-anxiety electrophysiological endophenotype in HAB mice. Strikingly, we detected enhanced basal neurotransmission and reduced PPF and LTP in slices from NPS-treated HAB animals. Collectively, our study uncovers a multifaceted high-anxiety neurophysiological endophenotype in the murine ventral hippocampus and provides the first evidence that an intranasally applied neuropeptide can shift such an endophenotype in an anxiety-regulating brain structure towards a “normal”-anxiety one.

Selective breeding for high anxiety introduces a synonymous SNP that increases neuropeptide S receptor activity.

Neuropeptide S (NPS) has generated substantial interest due to its anxiolytic and fear-attenuating effects in rodents, while a corresponding receptor polymorphism associated with increased NPS receptor (NPSR1) surface expression and efficacy has been implicated in an increased risk of panic disorder in humans. To gain insight into this paradox, we examined the NPS system in rats and mice bred for high anxiety-related behavior (HAB) versus low anxiety-related behavior, and, thereafter, determined the effect of central NPS administration on anxiety- and fear-related behavior. The HAB phenotype was accompanied by lower basal NPS receptor (Npsr1) expression, which we could confirm via in vitro dual luciferase promoter assays. Assessment of shorter Npsr1 promoter constructs containing a sequence mutation that introduces a glucocorticoid receptor transcription factor binding site, confirmed via oligonucleotide pull-down assays, revealed increased HAB promoter activity-an effect that was prevented by dexamethasone. Analogous to the human NPSR1 risk isoform, functional analysis of a synonymous single nucleotide polymorphism in the coding region of HAB rodents revealed that it caused a higher cAMP response to NPS stimulation. Assessment of the behavioral consequence of these differences revealed that intracerebroventricular NPS reversed the hyperanxiety of HAB rodents as well as the impaired cued-fear extinction in HAB rats and the enhanced fear expression in HAB mice, respectively. These results suggest that alterations in the NPS system, conserved across rodents and humans, contribute to innate anxiety and fear, and that HAB rodents are particularly suited to resolve the apparent discrepancy between the preclinical and clinical findings to date.

Endogenous vasopressin, innate anxiety, and the emission of pro-social 50-kHz ultrasonic vocalizations during social play behavior in juvenile rats

Although the involvement of the neuropeptide arginine vasopressin (AVP) in rodent social interaction is already extensively characterized, little is known about its role in social communication. Rats communicate in the ultrasonic range by means of ultrasonic vocalizations (USV). Depending on developmental stage and affective state, rats emit various distinct types of USV, with appetitive 50-kHz USV being induced by positive social interactions, like juvenile social play, probably serving an affiliative communicative function, namely to (re)establish or induce social proximity. In rats and mice selectively bred for low (LAB) and high (HAB) anxiety-related behavior, the emission of isolation-induced distress USV during maternal deprivation as pups correlates with innate high levels of hypothalamic AVP availability. Moreover, male LAB and HAB rats express deficits in social approach towards conspecifics, together with high and/or abnormal forms of aggression when confronted with harmless opponents, possibly due to a lack of social communication skills. The aim of this study was therefore (1) to investigate and characterize social play behavior and concomitant pro-social 50-kHz USV emission in male and female, juvenile LAB and HAB rats and to compare them to non-selected Wistar (NAB) rats; and (2) to link these findings pharmacologically to the central AVP system via applying an AVP 1a receptor (V1aR) antagonist (0.75 μg; Manning compound) or synthetic AVP (1 ng) into the lateral ventricle of male juvenile NAB rats. Our results show that reduced social play behavior in highly anxious male and female, juvenile HAB rats is accompanied by low amounts of pro-social 50-kHz USV, as compared to respective LAB and NAB rats, possibly reflecting a lack of positive affective states in expectation of or following social interactions in these individuals. Secondly, although synthetic AVP did not alter social play behavior and pro-social 50-kHz USV, we demonstrated for the first time that a blockade of the central AVP system not only reduces juvenile social play behavior, but at the same time pro-social 50-kHz USV emission rates, indicating an involvement of the social neuropeptide in regulating affiliative communication in rodents.

Increased conditioned place preference for cocaine in high anxiety related behavior (HAB) mice is associated with an increased activation in the accumbens corridor

Anxiety disorders and substance use disorders are strongly associated in humans. Accordingly, a widely held but controversial concept in the addiction field, the so-called “self-medication hypothesis,” posits that anxious individuals are more vulnerable for drug dependence because they use drugs of abuse to alleviate their anxiety. We tested this hypothesis under controlled experimental conditions by quantifying the conditioned place preference (CPP) to 15 mg/kg i.p. cocaine given contingently (COCAINE) in CD1 mice selectively bred for high anxiety-related behavior (HAB) vs. normal anxiety-related behavior (NAB). Cocaine was conditioned to the initially non-preferred compartment in an alternate day design (cocaine vs. saline, four pairings each). HAB and NAB mice were also tested for the effects of non-contingent (NONCONT) cocaine administration. HAB mice showed a slightly higher bias for one of the conditioning compartments during the pretest than NAB mice that became statistically significant (p = 0.045) only after pooling COCAINE and NONCONT groups. Cocaine CPP was higher (p = 0.0035) in HAB compared to NAB mice. The increased cocaine CPP was associated with an increased expression of the immediate early genes (IEGs) c-Fos and Early Growth Related Protein 1 (EGR1) in the accumbens corridor, i.e., a region stretching from the anterior commissure to the interhemispheric border and comprising the medial nucleus accumbens core and shell, the major island of Calleja and intermediate part of the lateral septum, as well as the vertical limb of the diagonal band and medial septum. The cocaine CPP-induced EGR1 expression was only observed in D1- and D2-medium spiny neurons, whereas other types of neurons or glial cells were not involved. With respect to the activation by contingent vs. non-contingent cocaine EGR1 seemed to be a more sensitive marker than c-Fos. Our findings suggest that cocaine may be more rewarding in high anxiety individuals, plausibly due to an anxiolytic effect.

Epigenetic regulation of corticotropin-releasing hormone receptor 1: implication for anxiety-related disorders

Recent literature corroborates that both, genes and environment, are crucial determinants contributing to psychiatric disorders. The selectively bred mouse models of enhanced fear and anxiety-related behavior provide a unique opportunity to study the interaction of a rigid genetic predisposition with environmental factors and are used to identify targets contributing to pathological anxiety. Here, we studied gene × environment (G×E) interactions using a mouse model of high (HAB) vs. low (LAB) anxiety-related behavior. By applying enriched environment (EE) and chronic mild stress (CMS), we succeeded in shifting the phenotypes of HAB and LAB mice towards “normal” anxiety. In this bidirectional shift, Crhr1 was identified to be a key player. Increased methylation of CpG1 within the Crhr1 promoter region was shown to be critically involved in regulating the binding affinity of the transcription factor Ying-Yang 1 (YY1). The interplay between YY1 expression and DNA methylation might be the mechanism underlying the differences in Crhr1 expression after EE and CMS. Other epigenetic mechanisms contributing to Crhr1 expression are discussed here.

Blunted HPA axis reactivity reveals glucocorticoid system dysbalance in a mouse model of high anxiety-related behavior.

Depression and anxiety disorders are often characterized by altered hypothalamic-pituitary-adrenal (HPA) axis re-/activity. However, the presence of a molecular link between dysbalanced neuroendocrine regulation and psychopathologies is not yet fully established. Earlier, we reported that high (HAB), normal (NAB) and low (LAB) anxiety-related behavior mice express divergent anxiety-related and passive/active coping phenotypes. Here, we studied mechanisms that might contribute to the different HPA axis reactivity observed in HAB, NAB and LAB mice and their involvement in the regulation of anxiety-related behavior and passive/active coping style. We found that HAB mice respond with significantly reduced corticosterone (CORT) secretion to an acute stressful stimulus and a blunted response in the Dex/CRH test compared to NAB and LAB mice. At the molecular level, higher expression of the glucocorticoid receptor (GR/Nr3c1) and decreased corticotropin-releasing hormone receptor 1 (CRHR1) expression were observed in the pituitary of HAB mice. We further analyzed whether these stress mediators differed between the HAB, NAB and LAB lines in limbic system-associated brain regions and whether their interplay contributes to the phenotype. Interestingly, not only in the pituitary but also in almost all brain regions investigated, GR expression was significantly higher in HAB mice. In contrast, the amount of CORT in the brain structures analyzed was significantly lower in these animals. The expression of CRHR1 varied in the prefrontal cortex only. Since glucocorticoids regulate both GR and CRHR1, we treated HAB and NAB mice chronically with CORT. After 6 weeks of administration, reduced anxiety- and depression-like behaviors were observed in HAB mice, whereas increased anxiety was found in NABs. In both groups, GR, but not CRHR1, were significantly reduced. Taken together, our study proposes HAB mice as an animal model of simultaneous features of increased anxiety-related and depression-like behaviors with blunted HPA axis reactivity suggesting a dysregulated GR/CORT system as one key mechanism behind their phenotype.

Enriched environment impacts trimethylthiazoline-induced anxiety-related behavior and immediate early gene expression: critical role of Crhr1.

It has been shown previously (Sotnikov et al., ) that mice selectively inbred for high anxiety-related behavior (HAB) vs. low anxiety-related behavior in the elevated plus maze differentially respond to trimethylthiazoline (TMT), a synthetic fox fecal odor. However, less is known about whether environmental factors can rescue these extreme phenotypes. Here, we found that an enriched environment (EE) provided during early adolescence induced anxiolytic effects in HAB (HAB-EE) mice, rescuing their strong avoidance behavior induced by TMT. In a series of experiments, the contribution of maternal, juvenile and adolescent behavior to the anxiolytic effects elicited by EE was investigated. At the molecular level, using c-fos expression mapping, we found that the activity of the medial and basolateral amygdala was significantly reduced in HAB-EE mice after TMT exposure. We further analysed the expression of Crhr1, as its amount in the amygdala has been reported to be important for the regulation of anxiety-related behavior after EE. Indeed, in situ hybridisation indicated significantly decreased Crhr1 expression in the basolateral and central amygdala of HAB-EE mice. To further test the involvement of Crhr1 in TMT-induced avoidance, we exposed conditional glutamatergic-specific Crhr1-knockout mice to the odor. The behavioral response of Crhr1-knockout mice mimicked that of HAB-EE mice, and c-fos expression in the amygdala after TMT exposure was significantly lower compared with controls, thereby further supporting a critical involvement of Crhr1 in environmentally-induced anxiolysis. Altogether, our results indicate that EE can rescue strong avoidance of TMT by HAB mice with Crhr1 expression in the amygdala being critically involved.

Environmental manipulations generate bidirectional shifts in both behavior and gene regulation in a crossbred mouse model of extremes in trait anxiety

Although gene-environment interactions are known to significantly influence psychopathology-related disease states, only few animal models cover both the genetic background and environmental manipulations. Therefore, we have taken advantage of the bidirectionally inbred high (HAB) and low (LAB) anxiety-related behavior mouse lines to generate HAB × LAB F1 hybrids that intrinsically carry both lines’ genetic characteristics, and subsequently raised them in three different environments—standard, enriched (EE) and chronic mild stress (CMS). Assessing genetic correlates of trait anxiety, we focused on two genes already known to play a role in HAB vs. LAB mice, corticotropin releasing hormone receptor type 1 (Crhr1) and high mobility group nucleosomal binding domain 3 (Hmgn3). While EE F1 mice showed decreased anxiety-related and increased explorative behaviors compared to controls, CMS sparked effects in the opposite direction. However, environmental treatments affected the expression of the two genes in distinct ways. Thus, while expression ratios of Hmgn3 between the HAB- and LAB-specific alleles remained equal, total expression resembled the one observed in HAB vs. LAB mice, i.e., decreased after EE and increased after CMS treatment. On the other hand, while total expression of Crhr1 remained unchanged between the groups, the relative expression of HAB- and LAB-specific alleles showed a clear effect following the environmental modifications. Thus, the environmentally driven bidirectional shift of trait anxiety in this F1 model strongly correlated with Hmgn3 expression, irrespective of allele-specific expression patterns that retained the proportions of basic differential HAB vs. LAB expression, making this gene a match for environment-induced modifications. An involvement of Crhr1 in the bidirectional behavioral shift could, however, rather be due to different effects of the HAB- and LAB-specific alleles described here. Both candidate genes therefore deserve attention in the complex regulation of anxiety-related phenotypes including environment-mediated effects.