RNA expression profiling in depressed patients suggests retinoid-related orphan receptor alpha as a biomarker for antidepressant response.

Response to antidepressant treatment is highly variable with some patients responding within a few weeks, whereas others have to wait for months until the onset of clinical effects. Gene expression profiling may be a tool to identify markers of antidepressant treatment response and new potential drug targets. In a first step, we selected 12 male, age- and severity-matched pairs of remitters and nonresponders, and analyzed expression profiles in peripheral blood at admission and after 2 and 5 weeks of treatment using Illumina expression arrays. We identified 127 transcripts significantly associated with treatment response with a minimal P-value of 9.41 × 10(-)(4) (false discovery rate-corrected). Analysis of selected transcripts in an independent replication sample of 142 depressed inpatients confirmed that lower expression of retinoid-related orphan receptor alpha (RORa, P=6.23 × 10(-4)), germinal center expressed transcript 2 (GCET2, P=2.08 × 10(-2)) and chitinase 3-like protein 2 (CHI3L2, P=4.45 × 10(-2)) on admission were associated with beneficial treatment response. In addition, leukocyte-specific protein 1 (LSP1) significantly decreased after 5 weeks of treatment in responders (P=2.91 × 10(-2)). Additional genetic, in vivo stress responsitivity data and murine gene expression findings corroborate our finding of RORa as a transcriptional marker of antidepressant response. In summary, using a genome-wide transcriptomics approach and subsequent validation studies, we identified several transcripts including the circadian gene transcript RORa that may serve as biomarkers indicating antidepressant treatment response.

Do social statuses affect the startle reflex in male mice?

Usual housing conditions lead to dominance hierarchy forming between male mice. The situation produces physiological and behavioural differences between dominants and subordinates. The goal of the present study was to assess stress responses, and possible changes in prepulse inhibition (PPI) of the startle reflex in dominant and subordinate male mice. Three weeks of daily social interactions led to stable aggressive dominance in 11 pairs of male NMRI mice. Stress levels were assessed by measuring faecal corticosterone metabolites (FCM), a non-invasive technique for monitoring hormonal changes in response to specific situations, with repeated sampling of each animal. The analysis of FCM levels showed greater stress in subordinate males at the beginning of the experiment, as the hierarchy was being established, but by the end of the experiment, FCM levels were reduced and similar in both dominants and subordinates. No significant differences were found in the startle response or PPI.

Stress and affective disorders: animal models elucidating the molecular basis of neuroendocrine-behavior interactions.

Profound dysfunctions in several neuroendocrine systems have been described in patients suffering from affective disorders such as major depression. In order to elucidate the mechanisms underlying these functional alterations, animal models including mice genetically modified by either direct gene-targeting or by selective breeding approaches have been used exceedingly, revealing valuable insights into neuroendocrine pathways conserved between rodents and men. This review focuses on altered function and regulation of the hypothalamic-pituitary-adrenocortical axis, including its involvement in emotionality and stress responsiveness. In this context, the corticotropin-releasing hormone system and disturbances in glucocorticoid receptor signaling seem to be of central importance. However, changes in the expression and release patterns of vasopressin, dopamine and serotonin have also been shown to contribute to variation in emotionality, stress coping, cognitive functions and social behaviors. Affective disorders show a high degree of complexity, involving a multitude of molecular, neuroendocrine, and behavioral alterations as well as an intense gene-environment interaction, making it difficult to dissociate the primary causes from secondary consequences of the disease. Thus, interdisciplinary research, as applied in the emerging field of systems biology, involving adequate animal models and combined methodologies can significantly contribute to our understanding regarding the transmission of genetic predispositions into clinically relevant endophenotypes. It is only with deep insight into the mechanisms by which the stress hormone systems are regulated that novel treatment strategies and promising targets for therapeutic interventions can be developed in the future. Such in-depth understanding is ultimately essential to realizing our goal of predictive, preventive, and personalized medicine.

Antidepressants recruit new neurons to improve stress response regulation.

Recent research suggests an involvement of hippocampal neurogenesis in behavioral effects of antidepressants. However, the precise mechanisms through which newborn granule neurons might influence the antidepressant response remain elusive. Here, we demonstrate that unpredictable chronic mild stress in mice not only reduces hippocampal neurogenesis, but also dampens the relationship between hippocampus and the main stress hormone system, the hypothalamo-pituitary-adrenal (HPA) axis. Moreover, this relationship is restored by treatment with the antidepressant fluoxetine, in a neurogenesis-dependent manner. Specifically, chronic stress severely impairs HPA axis activity, the ability of hippocampus to modulate downstream brain areas involved in the stress response, the sensitivity of the hippocampal granule cell network to novelty/glucocorticoid effects and the hippocampus-dependent negative feedback of the HPA axis. Remarkably, we revealed that, although ablation of hippocampal neurogenesis alone does not impair HPA axis activity, the ability of fluoxetine to restore hippocampal regulation of the HPA axis under chronic stress conditions, occurs only in the presence of an intact neurogenic niche. These findings provide a mechanistic framework for understanding how adult-generated new neurons influence the response to antidepressants. We suggest that newly generated neurons may facilitate stress integration and that, during chronic stress or depression, enhancing neurogenesis enables a dysfunctional hippocampus to restore the central control on stress response systems, then allowing recovery.

Resilient emotionality and molecular compensation in mice lacking the oligodendrocyte-specific gene Cnp1.

Altered oligodendrocyte structure and function is implicated in major psychiatric illnesses, including low cell number and reduced oligodendrocyte-specific gene expression in major depressive disorder (MDD). These features are also observed in the unpredictable chronic mild stress (UCMS) rodent model of the illness, suggesting that they are consequential to environmental precipitants; however, whether oligodendrocyte changes contribute causally to low emotionality is unknown. Focusing on 2'-3'-cyclic nucleotide 3'-phosphodiesterase (Cnp1), a crucial component of axoglial communication dysregulated in the amygdala of MDD subjects and UCMS-exposed mice, we show that altered oligodendrocyte integrity can have an unexpected functional role in affect regulation. Mice lacking Cnp1 (knockout, KO) displayed decreased anxiety- and depressive-like symptoms (i.e., low emotionality) compared with wild-type animals, a phenotypic difference that increased with age (3-9 months). This phenotype was accompanied by increased motor activity, but was evident before neurodegenerative-associated motor coordination deficits (≤ 9-12 months). Notably, Cnp1(KO) mice were less vulnerable to developing a depressive-like syndrome after either UCMS or chronic corticosterone exposure. Cnp1(KO) mice also displayed reduced fear expression during extinction, despite normal amygdala c-Fos induction after acute stress, together implicating dysfunction of an amygdala-related neural network, and consistent with proposed mechanisms for stress resiliency. However, the Cnp1(KO) behavioral phenotype was also accompanied by massive upregulation of oligodendrocyte- and immune-related genes in the basolateral amygdala, suggesting an attempt at functional compensation. Together, we demonstrate that the lack of oligodendrocyte-specific Cnp1 leads to resilient emotionality. However, combined with substantial molecular changes and late-onset neurodegeneration, these results suggest the low Cnp1 seen in MDD may cause unsustainable and maladaptive molecular compensations contributing to the disease pathophysiology.

Intrahippocampal corticosterone response in mice selectively bred for extremes in stress reactivity: a microdialysis study.

The hypothalamic-pituitary-adrenocortical (HPA) axis is one of the major stress hormone systems, and glucocorticoids (GCs) play a pivotal role in homeostatic processes throughout the body and brain. A dysregulation of the HPA axis, leading to an aberrant secretion of GCs, is associated with affective disorders such as major depression. In the present study, three mouse lines selectively bred for high (HR), intermediate (IR) or low (LR) stress reactivity were used to elucidate the temporal dynamics of intrahippocampal corticosterone (CORT) in response to a standardised stressor. In particular, we addressed the question of whether the distinct differences in HPA axis reactivity between the three mouse lines, as determined by plasma CORT measurements, are present in the central nervous system as well, and if the respective endophenotype is brought about by alterations in blood-brain barrier (BBB) functionality. We applied in vivo microdialysis in the hippocampus, demonstrating that the concentrations of CORT released from the adrenals in response to restraint stress are not only distinctly different in the plasma, but can also be found in the central nervous system, although the differences between the three mouse lines were attenuated, particularly between IR and LR animals. Additionally, a time lag of approximately 60 min was observed in all three lines regarding intrahippocampal peak concentrations of CORT after the onset of the stressor. Furthermore, we showed that the penetration and clearance of CORT in the hippocampal tissue was not affected by differences in BBB functionality because the multidrug resistance 1 P-glycoprotein (Mdr1 Pgp) was equally expressed in HR, IR and LR mice. Furthermore, we could exclude surgical damage of the BBB because peripherally-injected dexamethasone, which is a high affinity substrate for the Mdr1 Pgp and therefore restricted from entering the brain, could only be detected in the plasma and was virtually absent in the brain.

Modeling psychotic and cognitive symptoms of affective disorders: Disrupted latent inhibition and reversal learning deficits in highly stress reactive mice.

Increased stress reactivity has repeatedly been reported in patients suffering from psychiatric diseases including schizophrenia and major depression. These disorders also have other symptoms in common, such as cognitive deficits and psychotic-like behavior. We have therefore investigated if increased stress reactivity is associated with these phenotypic endpoints in an animal model of affective disorders. The stress reactivity mouse model used in this study consists of three CD-1-derived mouse lines, that have been selectively bred for high (HR), intermediate (IR) or low (LR) stress reactivity. Male mice from these three breeding lines were subjected to a reversal learning task and latent inhibition (Li) was assessed using a conditioned taste aversion paradigm. Furthermore, as the dopaminergic system is involved in both Li and reversal learning, the dopamine 1 receptor (D1R), dopamine 2 receptor (D2R) and dopamine transporter (DAT) mRNA expression levels were assessed in relevant brain areas of these animals. The results demonstrate that HR mice show perseveration in the reversal learning task and have disrupted Li. Furthermore, compared to LR mice, HR mice have decreased D2R mRNA levels in the ventral tegmental area, as well as decreased D1R mRNA levels in the cingulate cortex, and an increased expression of D2R mRNA in the nucleus accumbens. Taken together, these results demonstrate that the HR mice display cognitive deficits associated with psychotic-like behavior, similar to those observed in patients suffering from schizophrenia and major depression and could be utilized in the search for better treatment strategies for these symptoms of psychiatric disorders.

Faecal glucocorticoid metabolites: how to express yourself - comparison of absolute amounts versus concentrations in samples from a study in laboratory rats.

During the last two decades, measurement of faecal cortisol or corticosterone metabolites (FCM) has become one of the most important tools to non-invasively monitor stress in animals. However, to reliably assess an animal's adrenocortical activity, a careful validation of this technique for each species and sex investigated is obligatory. Usually results in these validation studies and in subsequent applications are expressed as concentration (FCM(conc)). Nevertheless, some authors express their results as absolute amounts (FCM(abs)) and claim this to be more accurate. A physiological validation to prove this assumption, however, is still missing as well as information about the influence of the intervals set for faecal sampling, although the chosen intervals might play an important role. Since FCM(conc) and FCM(abs) may differ and therefore lead to different conclusions, our study aimed to gain fundamental and scientifically valid information about these parameters by re-analysing a set of data obtained in a study on laboratory rats. The data basis used was derived from four validation experiments performed in male and female rats: an adrenocorticotrophic hormone challenge test, a dexamethasone (Dex) suppression test, an investigation of the diurnal variation (DV) of glucocorticoids and the stress response in reaction to the injection procedure itself (for details see Lepschy et al. Non-invasive measurement of adrenocortical activity in male and female rats. Lab Anim 2007;41:372-87). Faecal samples were collected in short time intervals and the exact amount of faeces voided during each sampling interval was documented. Throughout all performed tests strong positive correlations between FCM(conc) and FCM(abs) were found (median of r(s) > 0.72). In males, for all calculated sampling intervals (4, 8 and 12 h) pharmacological stimulation, suppression and the DV of adrenocortical activity were reflected accurately using both FCM(conc) and FCM(abs). In females, suppression of FCM by Dex was also clearly reflected in both systems. However, pharmacological stimulation was only reflected accurately by means of FCM(conc), which clearly limits the usability of FCM(abs). Thus, using the data of physiological validation experiments, we clearly demonstrate for the first time advantages and disadvantages of presenting results as FCM(conc) or FCM(abs). Based on our findings in laboratory animals such as rats, giving results as FCM(conc) seems to be more appropriate and FCM(abs) - if at all - might only be used as an addition.

Increased stress reactivity is associated with cognitive deficits and decreased hippocampal brain-derived neurotrophic factor in a mouse model of affective disorders.

Cognitive deficits are a common feature of major depression (MD), with largely unknown biological underpinnings. In addition to the affective and cognitive symptoms of MD, a dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is commonly observed in these patients. Increased plasma glucocorticoid levels are known to render the hippocampus susceptible to neuronal damage. This structure is important for learning and memory, creating a potential link between HPA axis dysregulation and cognitive deficits in depression. In order to further elucidate how altered stress responsiveness may contribute to the etiology of MD, three mouse lines with high (HR), intermediate (IR), or low (LR) stress reactivity were generated by selective breeding. The aim of the present study was to investigate whether increased stress reactivity is associated with deficits in hippocampus-dependent memory tests. To this end, we subjected mice from the HR, IR, and LR breeding lines to tests of recognition memory, spatial memory, and depression-like behavior. In addition, measurements of brain-derived neurotrophic factor (BDNF) in the hippocampus and plasma of these animals were conducted. Our results demonstrate that HR mice exhibit hippocampus-dependent memory deficits along with decreased hippocampal, but not plasma, BDNF levels. Thus, the stress reactivity mouse lines are a promising animal model of the cognitive deficits in MD with the unique feature of a genetic predisposition for an altered HPA axis reactivity, which provides the opportunity to explore the progression of the symptoms of MD, predisposing genetic factors as well as new treatment strategies.

Excretion of catecholamines in rats, mice and chicken.

Stress assessment favours methods, which do not interfere with an animal's endocrine status. To develop such non-invasive methods, detailed knowledge about the excretion of hormone metabolites in the faeces and urine is necessary. Our study was therefore designed to generate basic information about catecholamine excretion in rats, mice and chickens. After administration of (3)H-epinephrine or (3)H-norepinephrine to male and female rats, mice and chickens, all voided excreta were collected for 4 weeks, 3 weeks or for 10 days, respectively. Peak concentrations of radioactivity appeared in one of the first urinary samples of mice and rats and in the first droppings in chickens 0.2-7.2 h after injection. In rats, between 77.3 and 95.6% of the recovered catecholamine metabolites were found in the urine, while in mice, a mean of 76.3% were excreted in the urine. Peak concentrations in the faeces were found 7.4 h post injection in mice, and after about 16.4 h in rats (means). Our study provides valuable data about the route and the profile of catecholamine excretion in three frequently used species of laboratory animals. This represents the first step in the development of a reliable, non-invasive quantification of epinephrine and norepinephrine to monitor sympatho-adrenomedullary activity, although promising results for the development of a non-invasive method were found only for the chicken.

Non-invasive measurement of adrenocortical activity in male and female rats.

Rats are widely used in biomedical research as animal models for human diseases. However, due to their small body size, blood sampling is complicated and invasive and thereby can seriously interfere with endocrine functions and possibly compromise the animals' welfare. Therefore, a non-invasive technique to monitor stress hormones in these animals is highly desired. Our study aimed to gain general information about corticosterone metabolism and excretion and to validate a 5alpha-pregnane-3beta,11beta,21-triol-20-one enzyme immunoassay (EIA) to reliably measure faecal corticosterone metabolites (CMs) in laboratory rats. In total, 18 rats were administered 2.3 MBq of (3)H-corticosterone intravenously and per os, respectively (intravenous: 6 males and 6 females; per os: 3 males and 3 females). Subsequently, all voided excreta were frequently collected for five days. About 75+/-9% of the recovered CMs were found in the faeces. Peak concentrations of radiolabelled steroids appeared in the urine after 1.7+/-0.6 h in males and after 6.0+/-3.5 h in females. In faeces, maxima were observed after 14.7+/-2.4 h in both sexes. In principle, the time course and delay for both routes of administration (intravenous or per os) were the same, except for a delay of peak concentrations in urine (4.5+/-2.1 h) in per os administered males. Using high-performance liquid chromatography (HPLC), faecal (3)H-CMs were characterized and differences were found between the sexes. In both sexes, corticosterone was extensively metabolized, but while males showed only minor variations in their CM patterns, those of females differed largely between individuals. To validate the mentioned EIA, we investigated the diurnal variation (DV) of glucocorticoids as well as effects of the injection procedure itself and conducted an adrenocorticotropic hormone challenge test and a dexamethasone suppression test, using six male and six female rats each. Our results demonstrated that pharmacological stimulation, suppression and DV of adrenocortical activity were accurately reflected by means of CM measurement in faeces. By successful physiological validation, we proved for the first time the suitability of an immunoassay to non-invasively monitor adrenocortical activity in rats of both sexes. This method opens up new perspectives for biomedical and pharmacological investigations as well as for animal welfare related issues.

Stress hormones in mammals and birds: comparative aspects regarding metabolism, excretion, and noninvasive measurement in fecal samples.

A multitude of endocrine mechanisms are involved in coping with challenges. Front-line hormones to overcome stressful situations are glucocorticoids (GCs) and catecholamines (CAs). These hormones are usually determined in plasma samples as parameters of adrenal activity and thus of disturbance. GCs (and CAs) are extensively metabolized and excreted afterwards. Therefore, the concentration of GCs (or their metabolites) can be measured in various body fluids or excreta. Above all, fecal samples offer the advantages of easy collection and a feedback-free sampling procedure. However, large differences exist among species regarding the route and time course of excretion, as well as the types of metabolites formed. Based on information gained from radiometabolism studies (reviewed in this paper), we recently developed and successfully validated different enzyme immunoassays that enable the noninvasive measurement of groups of cortisol or corticosterone metabolites in animal feces. The determination of these metabolites in fecal samples can be used as a powerful tool to monitor GC production in various species of domestic, wildlife, and laboratory animals.

Different types of oestrous cycle in two closely related South American rodents (Cavia aperea and Galea musteloides) with different social and mating systems.

A comparative approach was used to investigate two closely related South American rodent species: the wild cavy (Cavia aperea) and the yellow-toothed cavy (Galea musteloides). These species of wild guinea-pig inhabit different habitats and show divergent social and mating systems. Cavia have a polygynous mating system, whereas in Galea promiscuous mating occurs. These observations correspond to functional variations in sperm characteristics, which might be interpreted as adaptations to different reproductive patterns in the females of both species. Twenty-two female C. aperea and G. musteloides (11 of each species) were investigated to elucidate differences in the characteristics of the oestrous cycles. The experimental design consisted of three stages, in which the degree of contact between the sexes was successively changed. During these stages, the reproductive cycle of the females was monitored by examining the condition of the vaginal closure membrane, analysing vaginal smears and determining serum concentrations of oestrogens and progesterone. The results revealed different types of oestrous cycle in the two species. Female C. aperea showed periodical cycles with spontaneously occurring oestrous periods, ovulation and corpus luteum activity. In contrast, in female G. musteloides, oestrus was exclusively induced by the presence of a male. However, after the induction of oestrus, ovulation and corpus luteum activity occurred spontaneously without stimuli from copulation. To date this type of oestrous cycle has not been described for any other mammalian species. Thus, these data indicate that differences in the reproductive patterns of the females may have shaped the evolution of different sperm characteristics in the males.