The Collaborative Cross, a community resource for the genetic analysis of complex traits.

The goal of the Complex Trait Consortium is to promote the development of resources that can be used to understand, treat and ultimately prevent pervasive human diseases. Existing and proposed mouse resources that are optimized to study the actions of isolated genetic loci on a fixed background are less effective for studying intact polygenic networks and interactions among genes, environments, pathogens and other factors. The Collaborative Cross will provide a common reference panel specifically designed for the integrative analysis of complex systems and will change the way we approach human health and disease.

Suicide candidate genes associated with bipolar disorder and schizophrenia: an exploratory gene expression profiling analysis of post-mortem prefrontal cortex.

BACKGROUND: Suicide is an important and potentially preventable consequence of serious mental disorders of unknown etiology. Gene expression profiling technology provides an unbiased approach to identifying candidate genes for mental disorders. Microarray studies with post-mortem prefrontal cortex (Brodmann's Area 46/10) tissue require larger sample sizes. This study poses the question: to what extent are differentially expressed genes for suicide a diagnostic specific set of genes (bipolar disorder vs. schizophrenia) vs. a shared common pathway? RESULTS: In a reanalysis of a large set of Affymetrix Human Genome U133A microarray data, gene expression levels were compared between suicide completers vs. non-suicide groups within a diagnostic group, namely Bipolar disorder (N = 45; 22 suicide completers; 23 non-suicide) or Schizophrenia (N = 45; 10 suicide completers ; 35 non-suicide). Among bipolar samples, 13 genes were found and among schizophrenia samples, 70 genes were found as differentially expressed. Two genes, PLSCR4 (phospholipid scramblase 4) and EMX2 (empty spiracles homolog 2 (Drosophila)) were differentially expressed in suicide groups of both diagnostic groups by microarray analysis. By qRT-PCR, PLSCR4 and EMX2 were significantly down-regulated in the schizophrenia suicide completers, but could not be confirmed in bipolar disorder. CONCLUSION: This molecular level analysis suggests that diagnostic specific genes predominate to shared genes in common among suicide vs. non-suicide groups. These differentially expressed, candidate genes are neural correlates of suicide, not necessarily causal. While suicide is a complex endpoint with many pathways, these candidate genes provide entry points for future studies of molecular mechanisms and genetic association studies to test causality.

Genetic dissection of the tail suspension test: a mouse model of stress vulnerability and antidepressant response.

BACKGROUND: The tail suspension test (TST) is a mouse screening test for antidepressants. METHODS: An F2 intercross was derived from NMRI and 129S6 inbred strains (n = 747). Mice underwent standardized TST with 2 sessions: (1) baseline and (2) imipramine (30 mg/kg, intraperitoneally) TST. RESULTS: A whole genome scan of this intercross mapped significant basal TST quantitative trait loci (QTL) on chromosomes (chr) 5 (peak 61 cM, Lod 5.7), 12 (peak 43 cM, Lod 5.2), and 18 (peak 51 cM, Lod 3.0). A suggestive QTL on chr 4 (peak 62 cM; Lod 3.1) overlapped regions containing previously mapped QTLs. For TST imipramine response, QTL were mapped on chr 1, 4, and 5. The chromosome 5 locus affected basal TST, antidepressant immobility response, and tail suspension-induced hyperthermia (TSIH) behaviors. An outbred NMRI F2 population provided further evidence for a chr 5 QTL. This chr 5 region harbors a cluster of gamma aminobutyric acid (GABA)-A receptor subunits and the human syntenic region includes chr 4p, 1p11, 12q24, and 22q11.24. A significant TSIH QTL (Tsih1) mapped on chr 4 near the Leptin receptor (Lepr). CONCLUSIONS: These QTL provide potential regions of interest for human genetic studies in stress-diathesis models of psychiatric illness and antidepressant responsiveness.

Stress-Induced Anxiety- and Depressive-Like Phenotype Associated with Transient Reduction in Neurogenesis in Adult Nestin-CreERT2/Diphtheria Toxin Fragment A Transgenic Mice.

Depression and anxiety involve hippocampal dysfunction, but the specific relationship between these mood disorders and adult hippocampal dentate gyrus neurogenesis remains unclear. In both humans with MDD and rodent models of depression, administration of antidepressants increases DG progenitor and granule cell number, yet rodents with induced ablation of DG neurogenesis typically do not demonstrate depressive- or anxiety-like behaviors. The conflicting data may be explained by the varied duration and degree to which adult neurogenesis is reduced in different rodent neurogenesis ablation models. In order to test this hypothesis we examined how a transient-rather than permanent-inducible reduction in neurogenesis would alter depressive- and anxiety-like behaviors. Transgenic Nestin-CreERT2/floxed diphtheria toxin fragment A (DTA) mice (Cre+DTA+) and littermates (Cre+DTA-; control) were given tamoxifen (TAM) to induce recombination and decrease nestin-expressing stem cells and their progeny. The decreased neurogenesis was transient: 12 days post-TAM Cre+DTA+ mice had fewer DG proliferating Ki67+ cells and fewer DCX+ neuroblasts/immature neurons relative to control, but 30 days post-TAM Cre+DTA+ mice had the same DCX+ cell number as control. This ability of DG neurogenesis to recover after partial ablation also correlated with changes in behavior. Relative to control, Cre+DTA+ mice tested between 12-30 days post-TAM displayed indices of a stress-induced anxiety phenotype-longer latency to consume highly palatable food in the unfamiliar cage in the novelty-induced hypophagia test, and a depression phenotype-longer time of immobility in the tail suspension test, but Cre+DTA+ mice tested after 30 days post-TAM did not. These findings suggest a functional association between adult neurogenesis and stress induced anxiety- and depressive-like behaviors, where induced reduction in DCX+ cells at the time of behavioral testing is coupled with stress-induced anxiety and a depressive phenotype, and recovery of DCX+ cell number corresponds to normalization of these behaviors.

Tail-suspension induced hyperthermia: a new measure of stress reactivity.

The tail suspension test (TST), an antidepressant screening paradigm, uses the uncontrollable, inescapable stressor of tail suspension to elicit immobility. As hyperthermia occurs following numerous stressors, hyperthermia might exist following the TST. We tested whether tail suspension induced hyperthermia (TSIH) was a distinct variable for TST. Hyperthermia was measured by two methods: a rectal probe and a subcutaneously implanted microchip (ELAMS()). In outbred ICR male mice, TSIH was robustly demonstrated compared to control (No-TST) mice. TSIH peaked after TST and remained elevated at 120 min. Among five (129/SvEvTac, A/J, C57BL/6J, NMRI and ICR) strains examined for TSIH, significant strain variations were detected. NMRI showed the highest temperature rise (2.3 degrees C) and A/J mice showed the lowest (0.6 degrees C). Sex differences were found for the C57BL/6J and NMRI strains on TSIH. TSIH and duration of immobility were not significantly correlated (r=0.22, P=0.17) in outbred mice. Both duration of TST immobility and TSIH were measured when ICR male mice were administered diazepam, imipramine (a TCA antidepressant), venlafaxine (a SNRI antidepressant), sertraline and paroxetine (SSRI antidepressants), propranolol and nadolol (beta-adrenergic receptor blockers), CP-154,526 (a CRF(1) receptor antagonist), and indomethacin (a cyclo-oxygenase inhibitor). Diazepam dose-dependently increased immobility and decreased TSIH. Propranolol blocked TSIH, but nadolol had no effect. Antidepressants showed more complex patterns of effects with venlafaxine, sertraline, and paroxetine inhibiting TSIH. TSIH demonstrated inter-strain variability, sex differences and a distinct pharmacology, suggesting that TSIH provides an independent, robust physiologic parameter to supplement the TST paradigm. This TSIH method may prove useful for pharmacologic, transgenic, and mechanistic studies.

An exploratory factor analysis of the Tail Suspension Test in 12 inbred strains of mice and an F2 intercross.

To explore the genetic dimensions of the stress response in rodents, we tested 12 inbred strains of mice and an F2 intercross (n=745) on the Tail Suspension Test (TST) and the Tail Suspension-Induced Hyperthermia (TSIH) paradigm. These selected 12 strains provide a representative sampling of the genetic heterogeneity of mousedom. An F2 intercross was derived from NMRI and 129S6 strains, which differ in their responses on the TST. Both inbred strains and F2 mice underwent a standardized protocol of automated TST with two sessions: (1) baseline and (2) imipramine TST. The duration of immobility and the body temperature after TST were recorded. The inbred strains were also tested in the Light-Dark Transition (LDT) test and in the Open Field Test (OFT), measuring the distance traveled, vertical movements, and center time as independent variables. The F2 mice were measured for core temperature after TST (TSIH). High intercorrelations among strain means were found for the LDT and OFT measures. Principal components analysis extracted four factors: "exploratory fear," body weight, imipramine response on immobility, and "stress reactivity." These dimensions were largely confirmed in the F2 population with one additional factor: imipramine response on TSIH. The results support a distinction between "stress reactivity" as measured by the TST and "exploratory fear" behavior as measured by the LDT and OFT.

Preventing depressive relapse and recurrence in higher-risk cognitive therapy responders: a randomized trial of continuation phase cognitive therapy, fluoxetine, or matched pill placebo.

IMPORTANCE: Strategies to improve the course of recurrent major depressive disorder have great public health relevance. To reduce the risk of relapse/recurrence after acute phase cognitive therapy (CT), a continuation phase model of therapy may improve outcomes. OBJECTIVES: To test the efficacy of continuation phase CT (C-CT) and fluoxetine for relapse prevention in a pill placebo (PBO)-controlled randomized trial and compare the durability of prophylaxis after discontinuation of treatments. DESIGN: A sequential, 3-stage design with an acute phase (all patients received 12 weeks of CT); 8-month experimental phase (responders at higher risk were randomized to C-CT, fluoxetine, or PBO); and 24 months of longitudinal, posttreatment follow-up. SETTING: Two university-based specialty clinics. PATIENTS: A total of 523 adults with recurrent major depressive disorder began acute phase CT, of which 241 higher-risk responders were randomized and 181 subsequently entered the follow-up. INTERVENTIONS: Cognitive therapy responders at higher risk for relapse were randomized to receive 8 months of C-CT (n = 86), fluoxetine (n = 86), or PBO (n = 69). MAIN OUTCOMES AND MEASURES: Survival analyses of relapse/recurrence rates, as determined by blinded evaluators using DSM-IV criteria and the Longitudinal Interval Follow-up Evaluation. RESULTS As predicted, the C-CT or fluoxetine groups were significantly less likely to relapse than the PBO group across 8 months. Relapse/recurrence rates for C-CT and fluoxetine were nearly identical during the 8 months of treatment, although C-CT patients were more likely to accept randomization, stayed in treatment longer, and attended more sessions than those in the fluoxetine and PBO groups. Contrary to prediction, relapse/recurrence rates following the discontinuation of C-CT and fluoxetine did not differ. CONCLUSIONS AND RELEVANCE: Relapse risk was reduced by both C-CT and fluoxetine in an enriched randomization sampling only CT responders. The preventive effects of C-CT were not significantly more durable than those of fluoxetine after treatment was stopped, suggesting that some higher-risk patients may require alternate longer-term interventions. TRIAL REGISTRATION: clinicaltrials.gov Identifiers: NCT00118404, NCT00183664, and NCT00218764.

Dysfunction of astrocyte connexins 30 and 43 in dorsal lateral prefrontal cortex of suicide completers.

BACKGROUND: Suicide is an important public health problem that results from the interaction of both psychosocial and biological factors. Although it is known that particular neurobiological processes underlie suicidal ideation and behavior, there still remains limited knowledge about the specific factors involved. METHODS: To explore the neurobiology of suicide we generated microarray data from dorsal lateral prefrontal cortex (DLPFC) in each of 28 male French-Canadian subjects (20 suicide completers). These results were followed up in a larger French-Canadian sample (n = 47, 38 suicide completers) and in microarray data available from the Stanley Foundation (n = 100, 36 suicide completers). To investigate the molecular mechanisms of this finding, we performed RNA interference and electrophoretic mobility shift assays. Animal behavioral experiments were done to control for drug and alcohol effects. RESULTS: We found reduced expression of Cx30 and Cx43 in DLPFC of suicide completers. We identified a previously unknown function for Sox9 as a transcription factor affecting expression of Cx30 in brain. CONCLUSIONS: These results suggest that alterations of astrocyte connexins might be involved in the suicide process and provide further evidence implicating astrocytes in psychopathology.

An E3 ubiquitin ligase, Really Interesting New Gene (RING) Finger 41, is a candidate gene for anxiety-like behavior and beta-carboline-induced seizures.

BACKGROUND: Identification of the genes underlying psychiatric illness remains a thorny problem. Previously, Quantitative Trait Loci (QTL) for anxiety-like behaviors and beta-carboline-induced seizure vulnerability have been mapped to the distal portion of mouse chromosome 10, with crosses of A/J and C57BL6 mice. METHODS: An interval specific congenic strain for this chromosomal 10 region facilitated the genetic dissection of novelty-induced exploratory behaviors. RESULTS: By microarray studies, an unsuspected E3 ubiquitin ligase, Really Interesting New Gene (RING) Finger 41 (Rnf41) was differentially expressed in the region of interest, being upregulated in the hippocampi of B6 compared with A/J as well as congenic A.B6(chr10) versus A/J. By quantitative real-time polymerase chain reaction (qRT-PCR), Rnf41 expression levels were significantly increased 1.5- and 1.3-fold in the hippocampi of C57BL6/J and A.B6(chr10) mice compared with A/J mice, respectively. Protein levels of Rnf41 were increased in hippocampi of B6 mice compared with A/J mice across postnatal development with a 5.5-fold difference at P56. Yeast two-hybrid studies searching for Rnf41 binding partners in fetal hippocampus identified several potential targets. An interaction between Rnf41 and NogoA was validated by glutathionine-S-transferase-Rnf41 pulldown experiments. Re-analysis of a microarray database of human postmortem prefrontal cortex (Brodmann's Area 46/10) found that RNF41 messenger RNA expression levels were reduced significantly in patients with major depression and bipolar disorder compared with unaffected control subjects and confirmed by qRT-PCR. CONCLUSIONS: Overall, Rnf41 is nominated as a candidate gene for anxiety-like behaviors, depression, and vulnerability to seizures. The RNF41 and its binding partners suggest molecular pathways underlying behavior, highlighting a potential role for the ubiquitin proteasome system in psychiatric illness.

Molecular adaptations underlying susceptibility and resistance to social defeat in brain reward regions.

While stressful life events are an important cause of psychopathology, most individuals exposed to adversity maintain normal psychological functioning. The molecular mechanisms underlying such resilience are poorly understood. Here, we demonstrate that an inbred population of mice subjected to social defeat can be separated into susceptible and unsusceptible subpopulations that differ along several behavioral and physiological domains. By a combination of molecular and electrophysiological techniques, we identify signature adaptations within the mesolimbic dopamine circuit that are uniquely associated with vulnerability or insusceptibility. We show that molecular recapitulations of three prototypical adaptations associated with the unsusceptible phenotype are each sufficient to promote resistant behavior. Our results validate a multidisciplinary approach to examine the neurobiological mechanisms of variations in stress resistance, and illustrate the importance of plasticity within the brain's reward circuits in actively maintaining an emotional homeostasis.

Looking forward in geriatric anxiety and depression: implications of basic science for the future.

Major depression and anxiety are common psychiatric illnesses whose etiology remains incompletely understood. This review highlights progress in understanding the etiology of these illnesses through genetic strategies and looks forward to their impact on geriatric psychiatry. We briefly address three broad domains of progress, namely 1) genetic approaches to etiology, including linkage and association studies, pharmacogenetics ("personalized medicine"), and gene x environment interactions; 2) mechanisms of thyroid and testosterone action via nuclear receptors, given these hormones' status as possible augmenters of antidepressants; and 3) the role of the neuroimmune system as a contributor to the stress response. Genetic strategies offer one path for converting correlational findings into causal pathways while complementing studies of a gene's function at the molecular, cellular, network, and whole-organismal levels. Neuroendocrine supplementation (thyroid and testosterone) has a long history and tradition. A molecular understanding of nuclear receptor pathways and their coactivators, the mediator complex proteins, provides a rationale for improved targeting of hormonal action in a tissue-selective manner, yielding drugs with improved safety and efficacy. Neural-immune interactions in psychiatric illness remain tantalizing topics. Research suggests that cytokine pathways may contribute to the maintenance or susceptibility to stress, anxiety, and depressive disorders. The reciprocal and recursive interactions among basic science, drug discovery, and clinical science will continue to provide hopeful themes for improving the lives of patients with treatment-refractive psychiatric illness.

Fine mapping of a major locus on chromosome 10 for exploratory and fear-like behavior in mice.

Advanced intercross lines (AIL) and interval-specific congenic strains (ISCS) were used to fine map previously coarsely defined quantitative trait loci (QTL) on Chromosomes 1, 10, and 19, influencing behaviors in the open Field (OF) and light-dark (LD) paradigms in mice. F12(A x B) AIL mice (N = 1130) were phenotyped, genotyped, and mapped. The ISCS were studied only in the telomeric Chromosome 10 region of interest, containing the exploratory and excitability QTL1 (Exq1). The Chromosome 10 Exq1 and Chromosome 19 Exq4 loci mapped robustly in the AIL. The most significant QTL findings (2.0 LOD score intervals; peak; LOD score) came from the TD15 and LD transitions traits, yielding estimated intervals of 2.2 cM for Exq1 (71.3-73.5 cM; peak 72.3 cM; LOD 11.9) and 9.0 cM for Exq4 (29.0-38.2 cM; peak 34 cM; LOD 4.2). The replicated QTLs on Chromosome 1 failed to map in this AIL population. The ISCS data confirmed Exq1 loci in general. However, the ISCS data were complex and less definitive for localizing the Exq1 loci. These exploratory and fear-like behaviors result from inheriting "many small things," namely, QTL explaining 2%-7% of the phenotypic variance. These results highlight the challenges of positionally cloning loci of small effect for complex traits. In particular, fine-mapping success may depend on the genetic architecture underlying complex traits.

Genetic contributions to body weight in mice: relationship of exploratory behavior to weight.

OBJECTIVE: The A/J and C57BL/6J mouse strains differ markedly in their exploratory behavior and their weight gain on a high-fat diet. We examined the genetic contributions of exploratory behavior to body weight and tested for shared, pleiotropic loci influencing energy homeostasis. RESEARCH METHODS AND PROCEDURES: Segregating (AxB6)F2 intercross (n = 514) and (B6AF1xA/J)N2 backcross (N = 223) populations were studied, phenotyping for weight and exploratory behaviors. Relationships among traits were analyzed by correlations. Weight traits were dissected with a genome-wide scan. RESULTS: Modest correlations were found between exploratory behaviors and weight, explaining 2% to 14% of the variance. Quantitative trait loci (QTL) for body weight at 8 weeks (wgt8), 10 weeks (wgt10), and 2-week weight gain (difference between weeks 8 and 10) on a 6% fat diet were mapped. Two QTL on chromosome 1 (peaks at 66 cM and 100 cM; Bw8q1) affected wgt8 [likelihood of the odds ratio (Lod), 3.0 and 4.4] and wgt10 (Lod, 2.2 and 3.4), respectively. In the backcross, a significant QTL on chromosome 4 (peak at 66 cM; Bw8q2) affected wgt 8 (Lod, 3.3) and wgt10 (Lod, 3.1). For 2-week weight gain, suggestive QTL were mapped on chromosomes 4 and 6. The chromosome 6 QTL region overlaps a human 7q locus for obesity. A search for between-strain sequence polymorphisms in the leptin and NPY genes was unrevealing. DISCUSSION: In mice, loci influencing exploratory activity play a modest role in body-weight regulation. Some forms of obesity may emerge from loci regulating normal body weight.

The nature and identification of quantitative trait loci: a community's view.

This white paper by eighty members of the Complex Trait Consortium presents a community's view on the approaches and statistical analyses that are needed for the identification of genetic loci that determine quantitative traits. Quantitative trait loci (QTLs) can be identified in several ways, but is there a definitive test of whether a candidate locus actually corresponds to a specific QTL?