A distinct transcriptional signature of antidepressant response in hippocampal dentate gyrus granule cells.

Major depressive disorder is the most prevalent mental illness worldwide, still its pharmacological treatment is limited by various challenges, such as the large heterogeneity in treatment response and the lack of insight into the neurobiological pathways underlying this phenomenon. To decode the molecular mechanisms shaping antidepressant response and to distinguish those from general paroxetine effects, we used a previously established approach targeting extremes (i.e., good vs poor responder mice). We focused on the dentate gyrus (DG), a subregion of major interest in the context of antidepressant mechanisms. Transcriptome profiling on micro-dissected DG granule cells was performed to (i) reveal cell-type-specific changes in paroxetine-induced gene expression (paroxetine vs vehicle) and (ii) to identify molecular signatures of treatment response within a cohort of paroxetine-treated animals. We identified 112 differentially expressed genes associated with paroxetine treatment. The extreme group comparison (good vs poor responder) yielded 211 differentially expressed genes. General paroxetine effects could be distinguished from treatment response-associated molecular signatures, with a differential gene expression overlap of only 4.6% (15 genes). Biological pathway enrichment and cluster analyses identified candidate mechanisms associated with good treatment response, e.g., neuropeptide signaling, synaptic transmission, calcium signaling, and regulation of glucocorticoid secretion. Finally, we examined glucocorticoid receptor (GR)-dependent regulation of selected response-associated genes to analyze a hypothesized interplay between GR signaling and good antidepressant treatment response. Among the most promising candidates, we suggest potential targets such as the developmental gene Otx2 or Htr2c for further investigations into antidepressant treatment response in the future.

Common genes associated with antidepressant response in mouse and man identify key role of glucocorticoid receptor sensitivity.

Response to antidepressant treatment in major depressive disorder (MDD) cannot be predicted currently, leading to uncertainty in medication selection, increasing costs, and prolonged suffering for many patients. Despite tremendous efforts in identifying response-associated genes in large genome-wide association studies, the results have been fairly modest, underlining the need to establish conceptually novel strategies. For the identification of transcriptome signatures that can distinguish between treatment responders and nonresponders, we herein submit a novel animal experimental approach focusing on extreme phenotypes. We utilized the large variance in response to antidepressant treatment occurring in DBA/2J mice, enabling sample stratification into subpopulations of good and poor treatment responders to delineate response-associated signature transcript profiles in peripheral blood samples. As a proof of concept, we translated our murine data to the transcriptome data of a clinically relevant human cohort. A cluster of 259 differentially regulated genes was identified when peripheral transcriptome profiles of good and poor treatment responders were compared in the murine model. Differences in expression profiles from baseline to week 12 of the human orthologues selected on the basis of the murine transcript signature allowed prediction of response status with an accuracy of 76% in the patient population. Finally, we show that glucocorticoid receptor (GR)-regulated genes are significantly enriched in this cluster of antidepressant-response genes. Our findings point to the involvement of GR sensitivity as a potential key mechanism shaping response to antidepressant treatment and support the hypothesis that antidepressants could stimulate resilience-promoting molecular mechanisms. Our data highlight the suitability of an appropriate animal experimental approach for the discovery of treatment response-associated pathways across species.

EV-3, an endogenous human erythropoietin isoform with distinct functional relevance.

Generation of multiple mRNAs by alternative splicing is well known in the group of cytokines and has recently been reported for the human erythropoietin (EPO) gene. Here, we focus on the alternatively spliced EPO transcript characterized by deletion of exon 3 (hEPOΔ3). We show co-regulation of EPO and hEPOΔ3 in human diseased tissue. The expression of hEPOΔ3 in various human samples was low under normal conditions, and distinctly increased in pathological states. Concomitant up-regulation of hEPOΔ3 and EPO in response to hypoxic conditions was also observed in HepG2 cell cultures. Using LC-ESI-MS/MS, we provide first evidence for the existence of hEPOΔ3 derived protein EV-3 in human serum from healthy donors. Contrary to EPO, recombinant EV-3 did not promote early erythroid progenitors in cultures of human CD34+ haematopoietic stem cells. Repeated intraperitoneal administration of EV-3 in mice did not affect the haematocrit. Similar to EPO, EV-3 acted anti-apoptotic in rat hippocampal neurons exposed to oxygen-glucose deprivation. Employing the touch-screen paradigm of long-term visual discrimination learning, we obtained first in vivo evidence of beneficial effects of EV-3 on cognition. This is the first report on the presence of a naturally occurring EPO protein isoform in human serum sharing non-erythropoietic functions with EPO.

Neuroprotection and endocytosis: erythropoietin receptors in insect nervous systems.

Erythropoietin (Epo) plays a dual role as an erythropoiesis-stimulating hormone and a locally produced cytoprotectant in various vertebrate tissues. Splice variants and engineered derivatives of Epo that mediate neuroprotection but do not stimulate erythropoiesis suggest that alternative receptors, different from the 'classical' homodimeric receptor involved in haematopoiesis, mediate neuroprotective Epo functions. Previous studies on grasshoppers demonstrated neuroprotective and neuroregenerative effects of Epo that involved similar transduction pathways as in mammals. To advance the characterization of yet unidentified neuroprotective Epo receptors, we studied the neuroprotective potency of the human non-erythropoietic Epo splice variant EV-3 in primary cultured locust brain neurons. We demonstrate that EV-3, like Epo, protects locust neurons from hypoxia-induced apoptotic death through activation of the Janus kinase/signal transducer and activator of transcription transduction pathway. Using the fluorescent dye FM1-43 to quantify endocytotic activity we show that both Epo and EV-3 increase the number of fluorescently labelled endocytotic vesicles. This reveals that binding of Epo to its neuroprotective receptor induces endocytosis, as it has been described for the mammalian homodimeric Epo-receptor expressed by erythroid progenitors. Reduction in Epo-stimulated endocytotic activity following pre-exposure to EV-3 indicated that both Epo and its splice variant bind to the same receptor on locust neurons. The shared neuroprotective potency of Epo and EV-3 in insect and mammalian neurons, in the absence of erythropoietic effects of EV-3 in mammals, suggests a greater similarity of the unidentified nervous Epo receptors (or receptor complexes) across phyla than between mammalian haematopoietic and neuroprotective receptors. Insects may serve as suitable models to evaluate the specific protective mechanisms mediated by Epo and its variants in non-erythropoietic mammalian tissues.

Purine and pyrimidine metabolism: Convergent evidence on chronic antidepressant treatment response in mice and humans.

Selective Serotonin Reuptake Inhibitors (SSRIs) are commonly used drugs for the treatment of psychiatric diseases including major depressive disorder (MDD). For unknown reasons a substantial number of patients do not show any improvement during or after SSRI treatment. We treated DBA/2J mice for 28 days with paroxetine and assessed their behavioral response with the forced swim test (FST). Paroxetine-treated long-time floating (PLF) and paroxetine-treated short-time floating (PSF) groups were stratified as proxies for drug non-responder and responder mice, respectively. Proteomics and metabolomics profiles of PLF and PSF groups were acquired for the hippocampus and plasma to identify molecular pathways and biosignatures that stratify paroxetine-treated mouse sub-groups. The critical role of purine and pyrimidine metabolisms for chronic paroxetine treatment response in the mouse was further corroborated by pathway protein expression differences in both mice and patients that underwent chronic antidepressant treatment. The integrated -omics data indicate purine and pyrimidine metabolism pathway activity differences between PLF and PSF mice. Furthermore, the pathway protein levels in peripheral specimens strongly correlated with the antidepressant treatment response in patients. Our results suggest that chronic SSRI treatment differentially affects purine and pyrimidine metabolisms, which may explain the heterogeneous antidepressant treatment response and represents a potential biosignature.

Exaggerated Increases in Microglia Proliferation, Brain Inflammatory Response and Sickness Behaviour upon Lipopolysaccharide Stimulation in Non-Obese Diabetic Mice.

The non-obese diabetic (NOD) mouse, an established model for autoimmune diabetes, shows an exaggerated reaction of pancreas macrophages to inflammatory stimuli. NOD mice also display anxiety when immune-stimulated. Chronic mild brain inflammation and a pro-inflammatory microglial activation is critical in psychiatric behaviour. OBJECTIVE: To explore brain/microglial activation and behaviour in NOD mice at steady state and after systemic lipopolysaccharide (LPS) injection. METHODS: Affymetrix analysis on purified microglia of pre-diabetic NOD mice (8-10 weeks) and control mice (C57BL/6 and CD1 mice, the parental non-autoimmune strain) at steady state and after systemic LPS (100 µg/kg) administration. Quantitative PCR was performed on the hypothalamus for immune activation markers (IL-1ß, IFNγ and TNFα) and growth factors (BDNF and PDGF). Behavioural profiling of NOD, CD1, BALB/c and C57BL/6 mice at steady state was conducted and sickness behaviour/anxiety in NOD and CD1 mice was monitored before and after LPS injection. RESULTS: Genome analysis revealed cell cycle/cell death and survival aberrancies of NOD microglia, substantiated as higher proliferation on BrdU staining. Inflammation signs were absent. NOD mice had a hyper-reactive response to novel environments with some signs of anxiety. LPS injection induced a higher expression of microglial activation markers, a higher brain pro-inflammatory set point (IFNγ, IDO) and a reduced expression of BDNF and PDGF after immune stimulation in NOD mice. NOD mice displayed exaggerated and prolonged sickness behaviour after LPS administration. CONCLUSION: After stimulation with LPS, NOD mice display an increased microglial proliferation and an exaggerated inflammatory brain response with reduced BDNF and PDGF expression and increased sickness behaviour as compared to controls.

Proteomic and metabolomic profiling reveals time-dependent changes in hippocampal metabolism upon paroxetine treatment and biomarker candidates.

Most of the commonly used antidepressants block monoamine reuptake transporters to enhance serotonergic or noradrenergic neurotransmission. Effects besides or downstream of monoamine reuptake inhibition are poorly understood and yet presumably important for the drugs' mode of action. In the present study we aimed at identifying hippocampal cellular pathway alterations in DBA/2 mice using paroxetine as a representative Selective Serotonin Reuptake Inhibitor (SSRI). Furthermore we identified biomarker candidates for the assessment of antidepressant treatment effects in plasma. Hippocampal protein levels were compared between chronic paroxetine- and vehicle-treated animals using in vivo(15)N metabolic labeling combined with mass spectrometry. We also studied the time course of metabolite level changes in hippocampus and plasma using a targeted polar metabolomics profiling platform. In silico pathway analyses revealed profound alterations related to hippocampal energy metabolism. Glycolytic metabolite levels acutely increased while Krebs cycle metabolite levels decreased upon chronic treatment. Changes in energy metabolism were influenced by altered glycogen metabolism rather than by altered glycolytic or Krebs cycle enzyme levels. Increased energy levels were reflected by an increased ATP/ADP ratio and by increased ratios of high-to-low energy purines and pyrimidines. In the course of our analyses we also identified myo-inositol as a biomarker candidate for the assessment of antidepressant treatment effects in the periphery. This study defines the cellular response to paroxetine treatment at the proteome and metabolome levels in the hippocampus of DBA/2 mice and suggests novel SSRI modes of action that warrant consideration in antidepressant development efforts.

Glutamatergic and dopaminergic neurons mediate anxiogenic and anxiolytic effects of CRHR1.

The corticotropin-releasing hormone receptor 1 (CRHR1) critically controls behavioral adaptation to stress and is causally linked to emotional disorders. Using neurochemical and genetic tools, we determined that CRHR1 is expressed in forebrain glutamatergic and γ-aminobutyric acid-containing (GABAergic) neurons as well as in midbrain dopaminergic neurons. Via specific CRHR1 deletions in glutamatergic, GABAergic, dopaminergic, and serotonergic cells, we found that the lack of CRHR1 in forebrain glutamatergic circuits reduces anxiety and impairs neurotransmission in the amygdala and hippocampus. Selective deletion of CRHR1 in midbrain dopaminergic neurons increases anxiety-like behavior and reduces dopamine release in the prefrontal cortex. These results define a bidirectional model for the role of CRHR1 in anxiety and suggest that an imbalance between CRHR1-controlled anxiogenic glutamatergic and anxiolytic dopaminergic systems might lead to emotional disorders.

Prediction of the risk of comorbid alcoholism in schizophrenia by interaction of common genetic variants in the corticotropin-releasing factor system.

CONTEXT: Stress plays a major role in the development of comorbid alcohol use disorder (AUD). In turn, AUD worsens the outcome of psychiatric patients with respect to global disease severity, social situation, and socioeconomic burden. Prediction of persons at risk for AUD is crucial for future preventive and therapeutic strategies. OBJECTIVE: To investigate whether genetic variants of the corticotropin-releasing factor system or their interaction influence the risk of developing AUD in chronic disease populations. DESIGN: Genotype analysis comprising selected single-nucleotide polymorphisms within the CRHR1 and CRHBP genes in patients with schizophrenia and in a nonschizophrenic psychiatric disease control sample should allow the extraction of predictors of comorbid AUD. Gene expression (messenger RNA) analysis in peripheral blood mononuclear cells was performed to gain the first mechanistic insight. SETTING: An ideal setup for this study was the Göttingen Research Association for Schizophrenia Data Collection of schizophrenic patients, specifically intended to enable association of genetic information with quantifiable phenotypes in a phenotype-based genetic association study. Patients  A total of 1037 schizophrenic patients (Göttingen Research Association for Schizophrenia sample), 80 nonschizophrenic psychiatric disease controls as a small replicate sample, and a case-control study including 1141 healthy subjects. MAIN OUTCOME MEASURES: Association of CRHR1 and CRHBP genotypes with the following: (1) AUD; (2) a newly developed alcoholism severity score comprising 5 AUD-relevant variables; and (3) quantitative CRHR1 and CRHBP messenger RNA expression. RESULTS: An interaction of CRHR1 rs110402 and CRHBP rs3811939 predicts high risk of comorbid AUD in schizophrenic patients (odds ratio = 2.27; 95% confidence interval, 1.56-3.30; P < .001) as well as psychiatric disease controls (odds ratio = 4.02; 95% confidence interval, 0.95-17.05; P = .06) and leads to the highest CRHR1/CRHBP messenger RNA ratio (P = .02; dysbalanced stress axis). CONCLUSIONS: The high predictive value of a genetic interaction within the stress axis for the risk of comorbid AUD may be used for novel preventive and individualized therapeutic approaches.

Gene expression profiling in the stress control brain region hypothalamic paraventricular nucleus reveals a novel gene network including amyloid beta precursor protein.

BACKGROUND: The pivotal role of stress in the precipitation of psychiatric diseases such as depression is generally accepted. This study aims at the identification of genes that are directly or indirectly responding to stress. Inbred mouse strains that had been evidenced to differ in their stress response as well as in their response to antidepressant treatment were chosen for RNA profiling after stress exposure. Gene expression and regulation was determined by microarray analyses and further evaluated by bioinformatics tools including pathway and cluster analyses. RESULTS: Forced swimming as acute stressor was applied to C57BL/6J and DBA/2J mice and resulted in sets of regulated genes in the paraventricular nucleus of the hypothalamus (PVN), 4 h or 8 h after stress. Although the expression changes between the mouse strains were quite different, they unfolded in phases over time in both strains. Our search for connections between the regulated genes resulted in potential novel signalling pathways in stress. In particular, Guanine nucleotide binding protein, alpha inhibiting 2 (GNAi2) and amyloid ß (A4) precursor protein (APP) were detected as stress-regulated genes, and together with other genes, seem to be integrated into stress-responsive pathways and gene networks in the PVN. CONCLUSIONS: This search for stress-regulated genes in the PVN revealed its impact on interesting genes (GNAi2 and APP) and a novel gene network. In particular the expression of APP in the PVN that is governing stress hormone balance, is of great interest. The reported neuroprotective role of this molecule in the CNS supports the idea that a short acute stress can elicit positive adaptational effects in the brain.

Chronic mild stress (CMS) in mice: of anhedonia, 'anomalous anxiolysis' and activity.

BACKGROUND: In a substantial proportion of depressed patients, stressful life events play a role in triggering the evolution of the illness. Exposure to stress has effects on different levels in laboratory animals as well and for the rat it has been shown that chronic mild stress (CMS) can cause antidepressant-reversible depressive-like effects. The adoption of the model to the mouse seems to be problematic, depending on the strain used and behavioural endpoint defined. Our aim was to evaluate the applicability of CMS to mice in order to induce behavioural alterations suggested to reflect depression-like symptoms. METHODOLOGY/PRINCIPAL FINDINGS: A weekly CMS protocol was applied to male mice of different mouse strains (D2Ola, BL/6J and BL/6N) and its impact on stress-sensitive behavioural measures (anhedonia-, anxiety- and depression-related parameters) and body weight was assessed. Overnight illumination as commonly used stressor in CMS protocols was particularly investigated in terms of its effect on general activity and subsequently derived saccharin intake. CMS application yielded strain-dependent behavioural and physiological responses including 'paradox' anxiolytic-like effects. Overnight illumination was found to be sufficient to mimic anhedonic-like behaviour in BL/6J mice when being applied as sole stressor. CONCLUSIONS/SIGNIFICANCE: The CMS procedure induced some behavioural changes that are compatible with the common expectations, i.e. 'anhedonic' behaviour, but in parallel behavioural alterations were observed which would be described as 'anomalous' (e.g. decreased anxiety). The results suggest that a shift in the pattern of circadian activity has a particular high impact on the anhedonic profile. Changes in activity in response to novelty seem to drive the 'anomalous' behavioural alterations as well.

Profiling of behavioral changes and hippocampal gene expression in mice chronically treated with the SSRI paroxetine.

INTRODUCTION: Monoamine-based antidepressants inhibit neurotransmitter reuptake within short time. However, it commonly takes several weeks until clinical symptoms start to resolve--indicating the involvement of effects distant from reuptake inhibition. OBJECTIVE: To unravel other mechanisms involved in drug action, a "reverse" pharmacological approach was applied to determine antidepressant-induced alterations of hippocampal gene expression. MATERIALS AND METHODS: The behavioral response to long-term paroxetine administration of male DBA/2Ola mice was assessed by the forced swim test (FST), the modified hole board (mHB), and the dark/light box. Hippocampi of test-naive mice were dissected, and changes in gene expression by paroxetine treatment were investigated by means of microarray technology. RESULTS AND DISCUSSION: Robust effects of paroxetine on passive stress-coping behavior in the FST were observed. Furthermore, anxiolytic properties of long-term antidepressant treatment could be identified in DBA mice in both, the mHB and dark/light box. Analysis of microarray results revealed a list of 60 genes differentially regulated by chronic paroxetine treatment. Preproenkephalin 1 and inhibin beta-A showed the highest level of transcriptional change. Furthermore, a number of candidates involved in neuroplasticity/neurogenesis emerged (e.g., Bdnf, Gfap, Vim, Sox11, Egr1, Stat3). Seven selected candidates were confirmed by in situ hybridization. Additional immunofluorescence colocalization studies of GFAP and vimentin showed more positive cells to be detected in long-term paroxetine-treated DBA mice. CONCLUSION: Candidate genes identified in the current study using a mouse strain validated for its responsiveness to long-term paroxetine treatment add, in our opinion, to unraveling the mechanism of action of paroxetine as a representative for SSRIs.

The temporal dynamics of intrahippocampal corticosterone in response to stress-related stimuli with different emotional and physical load: an in vivo microdialysis study in C57BL/6 and DBA/2 inbred mice.

There is strong evidence for a pivotal interaction of corticosteroid signalling and behavioral adaptation to stress. To further elucidate this relation, we monitored the dynamics of free corticosterone in the murine hippocampus of two inbred mouse strains using in vivo microdialysis. C57BL/6JOlaHsd (C57BL/6) and DBA/2OlaHsd (DBA/2) inbred mouse strains have been shown to differ in their anxiety-related and depression-like behavior and provide, thus, an interesting animal model to study the stimulus-response profile of the hypothalamus-pituitary-adrenocortical (HPA) system as a function of emotional and physical load. We, first, compared peripheral and intracerebral concentration patterns of corticosterone by simultaneous microdialysis of the jugular vein and the hippocampus in anesthetized mice and found strain differences in blood versus intracerebral free corticosterone concentrations. C57BL/6 showed almost the same steroid levels in either compartment, whereas DBA/2 mice displayed higher glucocorticoid levels in the circulation than in the hippocampus. This data suggest a strain difference in the tissue environment influencing the amount of biological active corticosterone at the receptor site. Measurements of intrahippocampal corticosterone in freely moving mice revealed that DBA/2 display a prolonged glucocorticoid increase in response to a single forced swimming stress (FST), as compared to C57BL/6 mice indicating a reduced inhibitory HPA axis feedback. Exposure to a novel environment (NE) induced a desensitization of the HPA system in DBA/2 animals as they show an attenuated intracerebral corticosterone dynamics after a subsequent FST. Testing animals in an elevated plus-maze (EPM), however, did not significantly stimulate coriticosterone release in either strain. The analysis of the area under the curve revealed a high amount of corticosterone released through FST and a low glucocorticoid release after NE or EPM exposure that are independent of the strain. This data indicate a strong stimulus dependency of corticosterone secretion that is strain independent, whereas the dynamics and feedback of the HPA axis is different between both inbred strains. Behavioral phenotyping of animals revealed a strong impact of microdialysis procedure on FST and EPM performance. Innate emotionality differences of both strains, however, were not affected. Though descriptive in nature, the present results suggest an altered corticosteroid signalling in the DBA/2 strain compared to C57BL/6 mice. Whether this observation causally underlies the differences in anxiety-related and depression-like behavior has to be further experimentally validated. In addition, our study highlights the use of in vivo microdialysis to assess the neuroendocrine endophenotype of animal models via profiling of stimulus-response patterns of stress hormones.

P2RX7, a gene coding for a purinergic ligand-gated ion channel, is associated with major depressive disorder.

The P2RX7 gene is located within a region on chromosome 12q24.31 that has been identified as a susceptibility locus for affective disorders by linkage and association studies. P2RX7 is a purinergic ATP-binding calcium channel expressed in neurons as well as in microglial cells in various brain regions. We investigated 29 single nucleotide polymorphisms (SNPs) within the P2RX7 gene and adjacent genes in a sample of 1000 German Caucasian patients suffering from recurrent major depressive disorder (MDD). These were contrasted with diagnosed healthy Caucasian controls from the same population (n=1029). A non-synonymous coding SNP in the P2RX7 gene (rs2230912), previously found to be associated with bipolar disorder, was significantly associated (P=0.0019) with MDD. This polymorphism results in an amino acid exchange in the C-terminal cytosolic domain of the P2RX7 channel protein, suggesting that the observed P2RX7 polymorphism might play a causal role in the development of depression.

Genetic identification of AChE as a positive modulator of addiction to the psychostimulant D-amphetamine in zebrafish.

Addiction is a complex maladaptive behavior involving alterations in several neurotransmitter networks. In mammals, psychostimulants trigger elevated extracellular levels of dopamine, which can be modulated by central cholinergic transmission. Which elements of the cholinergic system might be targeted for drug addiction therapies remains unknown. The rewarding properties of drugs of abuse are central for the development of addictive behavior and are most commonly measured by means of the conditioned place preference (CPP) paradigm. We demonstrate here that adult zebrafish show robust CPP induced by the psychostimulant D-amphetamine. We further show that this behavior is dramatically reduced upon genetic impairment of acetylcholinesterase (AChE) function in ache/+ mutants, without involvement of concomitant defects in exploratory activity, learning, and visual performance. Our observations demonstrate that the cholinergic system modulates drug-induced reward in zebrafish, and identify genetically AChE as a promising target for systemic therapies against addiction to psychostimulants. More generally, they validate the zebrafish model to study the effect of developmental mutations on the molecular neurobiology of addiction in vertebrates.

Corticotropin-releasing hormone activates ERK1/2 MAPK in specific brain areas.

Corticotropin-releasing hormone (CRH) coordinates hormonal and behavioral responses to stress. The mitogen-activated protein kinase extracellular signal-related kinase 1/2 (ERK1/2) mediates several functions in different forebrain structures and recently has been implicated in CRH signaling in cultured cells. To study in vivo CRH-mediated activation of central ERK1/2, we investigated the expression pattern of the phosphorylated ERK1/2(p-ERK1/2) in the mouse brain after intracerebroventricular CRH injections. As shown by immunohistochemistry and confocal microscopy analysis, CRH administration increased p-ERK1/2 levels specifically in the CA3 and CA1 hippocampal subfields and basolateral complex of the amygdala, both structures related to external environmental information processing and behavioral aspects of stress. Other regions such as hypothalamic nuclei and the central nucleus of the amygdala, also related to central CRH system but involved in the processing of the ascending visceral information and neuroendocrine-autonomic response to stress, did not show CRH-mediated ERK1/2 activation. To dissect the involvement of CRH receptor 1 (CRHR1) and CRHR2, we used conditional knockout mice in which Crhr1 is inactivated in the anterior forebrain and limbic structures. The conditional genetic ablation of Crhr1 inhibited the p-ERK1/2 increase, underlining the involvement of CRHR1 in the CRH-mediated activation. These findings underscore the fact that CRH activates p-ERK1/2 through CRHR1 only in selected brain regions, pointing to a specific role of this pathway in mediating behavioral adaptation to stress.

Identification of molecules potentially involved in mediating the in vivo actions of the corticotropin-releasing hormone receptor 1 antagonist, NBI30775 (R121919).

RATIONALE: The neuropeptide corticotropin-releasing hormone (CRH) plays a central role in the regulation of the hypothalamo-pituitary-adrenocortical (HPA) axis. The view that CRH hypersecretion underlies anxiety and mood disorders was recently supported by preclinical and clinical data obtained after application of the CRH receptor (CRH-R1) antagonist NBI30775 (R121919). Despite its therapeutic efficacy, there is only little information about its mechanisms of action on cellular and molecular targets. OBJECTIVE: To identify some of the intracellular substrates mediating the actions of NBI30775 after its acute administration in a stress-independent animal model. RESULTS: Of the different doses of NBI30775 tested (0.5, 1, 5 and 30 mg/kg), the 1-mg/kg dose proved behaviorally active insofar that it reduced anxiety-like behavior in mice under basal conditions. Subsequent analysis of brain tissues revealed NBI30775-induced increases in the nuclear translocation of glucocorticoid receptors (GR) and BAG-1, an upregulation of mRNA transcripts encoding GR, mineralocorticoid receptors (MR) and CRH-R1, and a suppression of the DNA-binding activity of the transcription factor AP-1. These changes were significant at a dose of 1 mg/kg of NBI30775. CONCLUSION: NBI30775 reduces levels of anxiety in mice (under basal conditions) with a steep dose-response curve. Molecules such as GR, MR, BAG-1 and AP-1 have been identified as some of the drug's intracellular targets; interestingly, changes in these molecules have also been seen in response to conventional antidepressants, showing that structurally and mechanistically unrelated anxiolytic and antidepressant drugs can influence common downstream pathways.

Importance of NO/cGMP signalling via cGMP-dependent protein kinase II for controlling emotionality and neurobehavioural effects of alcohol.

Cyclic GMP is a second messenger for nitric oxide (NO) that acts as a mediator for many different physiological functions. The cGMP-dependent protein kinases (cGKs) mediate cellular signalling induced by NO and cGMP. Here, we explored the localization of cGMP-dependent protein kinase type II (cGKII) in the mouse brain. In situ hybridization revealed high levels of cGKII mRNA in cerebral cortex, thalamic nuclei, hypothalamic nuclei, and in several basal forebrain regions including medial septum, striatum and amygdala. The close link to NO and the distribution pattern of cGKII suggested that this enzyme might be involved in emotional reactions and responses to drugs of abuse. Therefore, cGKII knockout animals (cGKII-/-) were compared with littermate controls in behavioural tests (i) for emotion-linked and (ii) for acute and chronic ethanol responses. Deletion of cGKII did not influence aggressive behaviour but led to enhanced anxiety-like behaviour. In terms of acute responses to ethanol, cGKII-/- mice were hyposensitive to hypnotic doses of ethanol as measured by the loss of righting reflex, without an alteration in their blood alcohol elimination. In a two-bottle free choice test, cGKII-/- mice showed elevated alcohol consumption. No taste differences to sweet solutions were observed compared to control animals. In summary, our data show that cGKII activity modulates anxiety-like behaviour and neurobehavioural effects of alcohol.

Stress and alcohol drinking.

The concept of stress-relief by alcohol has led to many investigations in order to elucidate the mechanisms of interactions of stress and alcohol, and the stress-reducing effect of alcohol as a motivation for alcohol consumption. The hypothalamo-pituitary-adrenocortical (HPA) system is one of the biological systems affected by both stress and alcohol. However, there is a high individual variation in the response of the HPA axis to either stress or alcohol. Factors like quality, severity and duration of stress, dose of alcohol and frequency of stress or alcohol exposure add to the individual response to stress or alcohol. The individual response is determined by interactions of genetic, environmental and experiential factors. Facing that complexity, with even more factors to be named, the often reported inconsistencies in both human and animal studies are not only attributable to methodological differences. Nevertheless, there are studies showing an influence of stress on alcohol consumption which most likely depends on the sample of probands examined. To our view, the concept of stress-relief by alcohol as a basic motivation for developing alcohol drinking habits is only applicable to subgroups of drinkers. Individuals with a dysfunctional HPA axis, inherited and/or acquired, might represent such a subgroup of stress-motivated drinkers.

Limbic corticotropin-releasing hormone receptor 1 mediates anxiety-related behavior and hormonal adaptation to stress.

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.