Elisabeth Frank

Alterations in central neuropeptide expression, release, and receptor binding in rats bred for high anxiety: critical role of vasopressin.

To model aspects of trait anxiety/depression, Wistar rats were bred for extremes in either hyper (HAB)- or hypo(LAB)-anxiety as measured on the elevated plus-maze and in a variety of additional behavioral tests. Similar to psychiatric patients, HAB rats prefer passive stress-coping strategies, indicative of depression-like behavior, show hyper-reactivity of the hypothalamo-pituitary–adrenal axis, and a pathological response to the dexamethasone/corticotropin-releasing hormone (CRH) challenge test. Here we tested central mRNA expression, release patterns, and receptor binding of neuropeptides critically involved in the regulation of both anxiety-related behavior and the HPA axis. Thus, CRH, arginine-8-vasopressin (AVP), and oxytocin (OXT) were studied in brains of HAB and LAB males both under basal conditions and after exposure to a mild emotional stressor. In HAB rats, CRH mRNA was decreased in the bed nucleus of the stria terminalis only. While no significant difference in CRH1-receptor binding was found in any brain area, CRH2-receptor binding was elevated in the hypothalamic paraventricular nucleus (PVN), the ventromedial hypothalamus, and the central amygdala of HABs compared to LABs. AVP, but not OXT, mRNA expression as well as release of the neuropeptide, were higher in the PVN of HABs, whereas AVP V1a-receptor binding failed to show significant differences in any brain region studied. Remarkably, intra-PVN treatment of HABs with the AVP V1-receptor antagonist d (CH2)5 Tyr (Me) AVP resulted in a decrease in anxiety/depression-related behavior. The elevated expression and release of AVP within the PVN of HAB rats together with the behavioral effects of the AVP V1-receptor antagonist suggest a critical involvement of this neuropeptide in neuroendocrine and behavioral phenomena associated with trait anxiety/depression.

Candidate genes of anxiety-related behavior in HAB/LAB rats and mice: focus on vasopressin and glyoxalase-I.

Two animal models of trait anxiety, HAB/LAB rats and mice, are described, representing inborn extremes in anxiety-related behavior. The comprehensive phenotypical characterization included basal behavioral features, stress-coping strategies and neuroendocrine responses upon stressor exposure with HAB animals being hyper-anxious, preferring passive coping, emitting more stressor-induced ultrasonic vocalization calls and showing typical peculiarities of the hypothalamic-pituitary-adrenocortical axis and line-specific patterns of Fos expression in the brain indicative of differential neuronal activation. In most cases, unselected Wistar rats and CD1 mice, respectively, displayed intermediate behaviors. In both HAB/LAB rats and mice, the behavioral phenotype has been found to be significantly correlated with the expression of the neuropeptide arginine vasopressin (AVP) at the level of the hypothalamic paraventricular nucleus (PVN). Additional receptor antagonist approaches in HABs confirmed that intra-PVN release of AVP is likely to contribute to hyper-anxiety and depression-like behavior. As shown exemplarily in HAB rats and LAB mice, single nucleotide polymorphisms (SNPs) in regulatory structures of the AVP gene underlie AVP-mediated phenotypic phenomena; in HAB rats, a SNP in the promoter of the AVP gene leads to reduced binding of the transcriptional repressor CBF-A, thus causing AVP overexpression and overrelease. Conversely, in LAB mice, a SNP in the AVP gene seems to cause an amino acid exchange in the signal peptide, presumably leading to a deficit in bioavailable AVP likely to underlie the total hypo-anxiety of LAB mice in combination with signs of central diabetes insipidus. Another feature of LAB mice is overexpression of glyoxalase-I. The functional characterization of this enzyme will determine its involvement in anxiety-related behavior beyond that of a reliable biomarker. The further identification of quantitative trait loci, candidate genes (and their products) and SNPs will not only help to explain inter-individual variation in emotional behavior, but will also reveal novel targets for anxiolytic and antidepressive interventions.

Impaired Repression at a Vasopressin Promoter Polymorphism Underlies Overexpression of Vasopressin in a Rat Model of Trait Anxiety

Two inbred rat lines have been developed that show either high (HAB) or low (LAB) anxiety-related behavior. The behavioral phenotype correlates with arginine vasopressin (AVP) expression at the level of the hypothalamic paraventricular nucleus (PVN), but not supraoptic nucleus, with HAB animals overexpressing the neuropeptide in both magnocellular and parvocellular subdivisions of the PVN. We detected a number of single nucleotide polymorphisms (SNPs) in the AVP locus that differ between the HAB and LAB animals, two of which were embedded in cis-regulatory elements. The HAB-specific allele of the AVP gene promoter occurs in 1.5% of outbred Wistar rats and is more transcriptionally active in vivo, as revealed by allele-specific transcription studies in cross-mated HAB/LAB F1 animals. Interestingly, one specific SNP [A(-1276)G] conferred reduced binding of the transcriptional repressor CArG binding factor A (CBF-A) in the HAB allele, the consequent differential regulation of the AVP promoter resulting in an overexpression of AVP in vitro and in vivo. Furthermore, CBF-A is highly coexpressed in AVP-containing neurons of the PVN supporting an important role for regulation of AVP gene expression in vivo. Taken together, our results demonstrate a role for an AVP gene polymorphism and CBF-A in elevated AVP expression in the PVN of HAB rats likely to contribute to their behavioral and neuroendocrine phenotype.

Viral vector-mediated gene transfer of the vole V1a vasopressin receptor in the rat septum: improved social discrimination and active social behaviour

This study explores the effects of enhancing vasopressin V1a receptor expression in the septum using viral vector‐mediated gene transfer on social discrimination and social interactions. Bilateral infusion of an adeno‐associated viral vector containing the prairie vole V1a receptor gene (V1aR‐AAV) regulated by a neuron‐specific enolase promoter resulted in a stable increase in V1a receptor binding density in the rat septum without affecting oxytocin receptor density. Control animals were infused with a vector expressing the lacZ gene. In a social discrimination paradigm, only V1aR‐AAV‐treated animals succeeded in discriminating a previously encountered from a novel juvenile after an interexposure interval (IEI) of more than 2 h, demonstrating the functional incorporation of the vole V1a receptor in the rat septal circuits underlying short‐term memory processes. Microdialysis administration of synthetic vasopressin during the first juvenile exposure, used to mimic intraseptal release patterns of the neuropeptide, produced similar prolongations in recognition (up to an IEI of 24 h) in both V1aR‐AAV and control animals. Septal microdialysis administration of a selective V1a, but not oxytocin, receptor antagonist in both groups prevented discrimination even after an IEI of as short as 0.5 h, confirming the specificity of the vole V1a receptor involvement in social discrimination abilities. In addition, active social interactions were found to be increased among V1aR‐AAV rats compared to controls. Viral vector‐mediated gene transfer provides a valuable tool for studies on the role of localized gene expression on behavioural parameters.

The vasopressin system--from antidiuresis to psychopathology.

Vasopressin is a neuropeptide with multiple functions. In addition to its predominantly antidiuretic action after peripheral secretion from the posterior pituitary, it seems to fulfill--together with its receptor subtype--all requirements for a neuropeptide system critically involved in higher brain functions, including cognitive abilities and emotionality. Following somatodendritic and axonal release in distinct brain areas, vasopressin acts as a neuromodulator and neurotransmitter in multiple and varying modes of interneuronal communication. Accordingly, changes in vasopressin expression and release patterns may have wide-spread consequences. As shown in mice, rats, voles, and humans, central vasopressin release along a continuum may be beneficial to the individual, serving to adjust physiology and behavior in stressful scenarios, possibly at the potential expense of increasing susceptibility to disease. Indeed, if over-expressed and over-released, it may contribute to hyper-anxiety and depression-like behaviors. A vasopressin deficit, in turn, may cause signs of both diabetes insipidus and total hypo-anxiety. The identification of genetic polymorphisms underlying these phenomena does not only explain individual variation in social memory and emotionality, but also help to characterize potential targets for therapeutic interventions. The capability of both responding to stressful stimuli and mediating genetic polymorphisms makes the vasopressin system a key mediator for converging (i.e., environmentally and genetically driven) behavioral regulation.

TMEM132D, a new candidate for anxiety phenotypes: evidence from human and mouse studies

The lifetime prevalence of panic disorder (PD) is up to 4% worldwide and there is substantial evidence that genetic factors contribute to the development of PD. Single-nucleotide polymorphisms (SNPs) in TMEM132D, identified in a whole-genome association study (GWAS), were found to be associated with PD in three independent samples, with a two-SNP haplotype associated in each of three samples in the same direction, and with a P-value of 1.2e−7 in the combined sample (909 cases and 915 controls). Independent SNPs in this gene were also associated with the severity of anxiety symptoms in patients affected by PD or panic attacks as well as in patients suffering from unipolar depression. Risk genotypes for PD were associated with higher TMEM132D mRNA expression levels in the frontal cortex. In parallel, using a mouse model of extremes in trait anxiety, we could further show that anxiety-related behavior was positively correlated with Tmem132d mRNA expression in the anterior cingulate cortex, central to the processing of anxiety/fear-related stimuli, and that in this animal model a Tmem132d SNP is associated with anxiety-related behavior in an F2 panel. TMEM132D may thus be an important new candidate gene for PD as well as more generally for anxiety-related behavior.

Impaired extinction of learned fear in rats selectively bred for high anxiety – evidence of altered neuronal processing in prefrontal-amygdala pathways

The impaired extinction of acquired fear is a core symptom of anxiety disorders, such as post‐traumatic stress disorder, phobias or panic disorder, and is known to be particularly resistant to existing pharmacotherapy. We provide here evidence that a similar relationship between trait anxiety and resistance to extinction of fear memory can be mimicked in a psychopathologic animal model. Wistar rat lines selectively bred for high (HAB) or low (LAB) anxiety‐related behaviour were tested in a classical cued fear conditioning task utilizing freezing responses as a measure of fear. Fear acquisition was similar in both lines. In the extinction trial, however, HAB rats showed a marked deficit in the attenuation of freezing responses to repeated auditory conditioned stimulus presentations as compared with LAB rats, which exhibited rapid extinction. To gain information concerning the putatively altered neuronal processing associated with the differential behavioural response between HAB and LAB rats, c‐Fos expression was investigated in the main prefrontal‐amygdala pathways important for cued fear extinction. HAB compared to LAB rats showed an attenuated c‐Fos response to repeated conditioned stimulus presentations in infralimbic and cingulate cortices, as well as in the lateral amygdala, but facilitated the c‐Fos response in the medial part of the central amygdala. In conclusion, the present results support the notion that impaired extinction in high anxiety rats is accompanied by an aberrant activation profile in extinction‐relevant prefrontal‐amygdala circuits. Thus, HAB rats may represent a clinically relevant model to study the mechanisms and potential targets to accelerate delayed extinction processes in subjects with enhanced trait anxiety.

Genetic predisposition to anxiety-related behavior determines coping style, neuroendocrine responses, and neuronal activation during social defeat

Genetic background may influence an individuals susceptibility to, and subsequent coping strategy for, an acute stressor. When exposed to social defeat (SD), rats bred for high (HAB) or low (LAB) trait anxiety, which also differ in depression-like behavior, showed highly divergent passive and active coping behaviors, respectively. HABs spent more time freezing and emitted more ultrasound vocalization calls during SD than LABs, which spent more time rearing and grooming. Although the behavioral data confirmed the prediction that heightened trait anxiety would make rats more prone to experience stress, adrenocorticotropin and corticosterone were secreted to a higher extent in LABs than in HABs. In the latter, Fos expression upon SD was enhanced in the amygdala and hypothalamic areas compared with LABs, whereas it was diminished in prefrontal and brainstem areas.

Proteomics and Metabolomics Analysis of a Trait Anxiety Mouse Model Reveals Divergent Mitochondrial Pathways

BACKGROUND Although anxiety disorders are the most prevalent psychiatric disorders, no molecular biomarkers exist for their premorbid diagnosis, accurate patient subcategorization, or treatment efficacy prediction. To unravel the neurobiological underpinnings and identify candidate biomarkers and affected pathways for anxiety disorders, we interrogated the mouse model of high anxiety-related behavior (HAB), normal anxiety-related behavior (NAB), and low anxiety-related behavior (LAB) employing a quantitative proteomics and metabolomics discovery approach. METHODS We compared the cingulate cortex synaptosome proteomes of HAB and LAB mice by in vivo (15)N metabolic labeling and mass spectrometry and quantified the cingulate cortex metabolomes of HAB/NAB/LAB mice. The combined data sets were used to identify divergent protein and metabolite networks by in silico pathway analysis. Selected differentially expressed proteins and affected pathways were validated with immunochemical and enzymatic assays. RESULTS Altered levels of up to 300 proteins and metabolites were found between HAB and LAB mice. Our data reveal alterations in energy metabolism, mitochondrial import and transport, oxidative stress, and neurotransmission, implicating a previously nonhighlighted role of mitochondria in modulating anxiety-related behavior. CONCLUSIONS Our results offer insights toward a molecular network of anxiety pathophysiology with a focus on mitochondrial contribution and provide the basis for pinpointing affected pathways in anxiety-related behavior.

Differential effects of periodic maternal separation on adult stress coping in a rat model of extremes in trait anxiety

We studied interactions of genetic and environmental factors shaping adult emotionality and stress coping, and tested the hypothesis that repeated periodic maternal deprivation (PMD) exerts differential effects on adult behavioral and neuroendocrine stress responsiveness in dependence on the genetic predisposition to either hyper- or hypo-anxiety. Exposure of male Wistar rats bidirectionally bred for either high (HAB) or low (LAB) anxiety-related behavior to PMD between postnatal days 2 and 15 resulted in a behavioral approximation of the selected lines. This was reflected by test-dependent signs of reduced anxiety-related behavior in adult HAB rats and of enhanced levels of anxiety in LAB rats compared with their corresponding unstressed controls. In addition to behavioral parameters, differential effects of PMD were also seen with respect to the responsiveness of the hypothalamo-pituitary-adrenocortical axis to acute stressor exposure (novel environment) in adulthood. The corticotrophin (ACTH) and corticosterone hyper-responses seen in control rats of the HAB line compared with those of the LAB line became attenuated in PMD-HAB rats, whereas PMD did not significantly alter neuroendocrine responses in LAB rats. Thus, as a result of PMD, both ACTH and corticosterone responses became indistinguishable between HAB and LAB rats. Although HAB dams spent more time on the nest with the litter compared with LAB dams during the first 5 days postpartum, licking and grooming behavior did not differ between the lines prior to separation, and was found to be increased to the same extent in both HAB and LAB dams during the first hour immediately after reunion with the pups. In contrast to early life stress, exposure of adult HAB and LAB rats to a 10-day unpredictable stress schedule failed to alter their emotional measures. The mitigating effect of PMD on both behavioral and neuroendocrine parameters in rats representing extremes in trait anxiety might reflect an evolutionary benefit as the genetic variability among individuals of a species is sustained while allowing adequate responses to potentially dangerous stimuli in adulthood dependent on early life conditions.