Sergey V. Sotnikov

Bidirectional rescue of extreme genetic predispositions to anxiety: impact of CRH receptor 1 as epigenetic plasticity gene in the amygdala.

The continuum of physiological anxiety up to psychopathology is not merely dependent on genes, but is orchestrated by the interplay of genetic predisposition, gene x environment and epigenetic interactions. Accordingly, inborn anxiety is considered a polygenic, multifactorial trait, likely to be shaped by environmentally driven plasticity at the genomic level. We here took advantage of the extreme genetic predisposition of the selectively bred high (HAB) and low anxiety (LAB) mouse model exhibiting high vs low anxiety-related behavior and tested whether and how beneficial (enriched environment) vs detrimental (chronic mild stress) environmental manipulations are capable of rescuing phenotypes from both ends of the anxiety continuum. We provide evidence that (i) even inborn and seemingly rigid behavioral and neuroendocrine phenotypes can bidirectionally be rescued by appropriate environmental stimuli, (ii) corticotropin-releasing hormone receptor 1 (Crhr1), critically involved in trait anxiety, shows bidirectional alterations in its expression in the basolateral amygdala (BLA) upon environmental stimulation, (iii) these alterations are linked to an increased methylation status of its promoter and, finally, (iv) binding of the transcription factor Yin Yang 1 (YY1) to the Crhr1 promoter contributes to its gene expression in a methylation-sensitive manner. Thus, Crhr1 in the BLA is critically involved as plasticity gene in the bidirectional epigenetic rescue of extremes in trait anxiety.

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.

Real-Time Imaging of Amygdalar Network Dynamics In Vitro Reveals a Neurophysiological Link to Behavior in a Mouse Model of Extremes in Trait Anxiety

In humans and numerous other mammalian species, individuals considerably vary in their level of trait anxiety. This well known phenomenon is closely related to the etiology of several psychiatric disorders, but its neurophysiological basis remains poorly understood. Here, we applied voltage-sensitive dye imaging to brain slices from animals of the high (HAB), normal (NAB), and low (LAB) trait anxiety mouse model and investigated whether evoked neuronal activity propagations from the lateral (LA) to the central (CeA) amygdala differ in their relative strength among HAB, NAB, and LAB mice. For this purpose, we divided a real-time measure of neuronal population activity in the CeA by a respective measure obtained for the LA. This calculation yielded the metric “CeA/LA activity.” Our data clearly demonstrate a positive correlation between trait anxiety levels evaluated by the elevated plus-maze test and CeA/LA activity. Moreover, we found reduced CeA/LA activity in HAB mice, which responded with decreased anxiety levels to an environmental enrichment and, inversely, detected increased anxiety levels and CeA/LA activity in LAB mice that experienced chronic mild stress. We did not observe differences in the spread of neuronal activity in the motor and visual cortex among HAB, NAB, and LAB animals. Collectively, these findings provide evidence that, in mammals, interindividual variability in trait anxiety is causally linked to individual variations in the physiological constitution of the LA-to-CeA circuitry that give rise to a differential regulation of neuronal signal flow through this fundamental input–output network of the amygdala.

Genetic predisposition to anxiety-related behavior predicts predator odor response

While rodents have a keen sense of smell and largely depend on olfactory cues for operating in their environment, most of the widely used tests to assess anxiety-related behavior largely ignore the olfactory system, being primarily based on fear of brightly lit, novel and open spaces. Here, we aimed at testing whether the genetic predisposition to anxiety predicts the predator odor response in mice. In the first experiment, using the 3-chamber avoidance test in CD-1 mice, trimethylthiazoline (TMT), a synthetic fox fecal odor, was shown to induce stronger behavioral and neuroendocrine effects than cat odor and butyric acid, respectively, and was therefore chosen as aversive odor for the following series of experiments. In this series, bidirectionally, selectively inbred CD-1 mice with either high (HAB), intermediate (NAB) or low (LAB) anxiety-related behavior responded differently to TMT, with HABs spending significantly less time than both NABs and LABs in the chamber harbouring the predator odor. Importantly, this result is not confounded by any deficit of the olfactory system, as LAB and NAB mice, while not or only moderately responding to TMT, responded to both the pleasant odor of female urine and the repugnant odor of butyric acid. Probably due to the strength of TMT, a similar increase in corticosterone levels upon predator odor exposure was observed in all three groups. Together, the results suggest that, dependent on the genetic predisposition to extremes in anxiety-related behavior, mice differentially interpret the odor of a potential predator, making this type of avoidance behavior highly predictable.

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.

Polymorphism in Tmem132d regulates expression and anxiety-related behavior through binding of RNA polymerase II complex

TMEM132D is a candidate gene, where risk genotypes have been associated with anxiety severity along with higher mRNA expression in the frontal cortex of panic disorder patients. Concurrently, in a high (HAB) and low (LAB) trait anxiety mouse model, Tmem132d was found to show increased expression in the anterior cingulate cortex (aCC) of HAB as compared to LAB mice. To understand the molecular underpinnings underlying the differential expression, we sequenced the gene and found two single-nucleotide polymorphisms (SNPs) in the promoter differing between both lines which could explain the observed mRNA expression profiles using gene reporter assays. In addition, there was no difference in basal DNA methylation in the CpG Island that encompasses the HAB vs. LAB Tmem132d promoter region. Furthermore, we found significantly higher binding of RNA polymerase II (POLR2A) to the proximal HAB-specific SNP (rs233264624) than the corresponding LAB locus in an oligonucleotide pull-down assay, suggesting increased transcription. Virus mediated overexpression of Tmem132d in the aCC of C57BL/6 J mice could confirm its role in mediating an anxiogenic phenotype. To model gene–environmental interactions, HAB mice exposed to enriched environment (HAB-EE) responded with decreased anxiety levels but, had enhanced Tmem132d mRNA expression as compared to standard-housed HAB (HAB-SH) mice. While LAB mice subjected to unpredictable chronic mild stress (LAB-UCMS) exhibited higher anxiety levels and had lower mRNA expression compared to standard-housed LAB (LAB-SH) mice. Chromatin immunoprecipitation revealed significantly higher binding of POLR2A to rs233264624 in HAB-EE, while LAB-UCMS had lower POLR2A binding at this locus, thus explaining the enhanced or attenuated expression of Tmem132d compared to their respective SH controls. To further investigate gene–environment interactions, DNA methylation was assessed using Illumina 450 K BeadChip in 74 panic disorder patients. Significant methylation differences were observed in two CpGs (cg26322591 and cg03283235) located in TMEM132D depending on the number of positive life events supporting the results of an influence of positive environmental cues on regulation of Tmem132d expression in mice.