Compulsivity in mouse strains homologous with chromosomes 7p and 15q linked to obsessive-compulsive disorder.код для вставкиСкачать
RESEARCH ARTICLE Neuropsychiatric Genetics Compulsivity in Mouse Strains Homologous With Chromosomes 7p and 15q Linked to Obsessive-Compulsive Disorder Martien J.H. Kas,1* Cigdem Gelegen,1,2 Filip van Nieuwerburgh,3 Herman G.M. Westenberg,4 Dieter Deforce,3 and Damiaan Denys5 1 Department of Neuroscience and Pharmacology, Rudolf Magnus Institute of Neuroscience, University Medical Centre Utrecht, Utrecht, The Netherlands 2 Department of Medicine, University College London, London, United Kingdom 3 Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium Department of Psychiatry, Rudolf Magnus Institute of Neuroscience, University Medical Centre Utrecht, Utrecht, The Netherlands 4 5 Department of Psychiatry, Academic Medical Center (AMC), University of Amsterdam, and the Institute for Neuroscience, an institute of the Royal Netherlands Academy of Arts and Sciences, Amsterdam, The Netherlands Received 30 October 2008; Accepted 5 May 2009 Obsessive-compulsive disorder (OCD) is a severe anxiety disorder characterized by obsessions and compulsions. The core symptom of OCD is compulsivity, the inability to stop thinking or acting when you want to, despite being aware of the uselessness of the content or the adverse consequences. To initiate a systematic search for genetic mechanisms underlying the pathophysiology of compulsivity, a panel of chromosome substitution (CS) strains, derived from mice that suppress (C57BL/6J strain) or maintain (A/J strain) high levels of repetitive wheel running during 2 hr of daily limited food access, was screened for this compulsive behavior. Following the genetic screen, we found linkage between compulsive wheel running and mouse chromosomes 2, 6, and 7 that show overlap with recently identified human linkage regions for OCD on chromosomes 7p and 15q. In the overlapping (human/mouse) genomic region, the CRH receptor 2 (CRHR2) gene was tested in a human case–control study. An initial exploration in OCD cases versus controls failed to detect an association between four-candidate CRH2R SNP’s within this homologous linkage region and OCD. Genetic fine mapping of compulsivity in mice provides new opportunities to reveal mechanisms underlying this significant psychiatric trait. 2009 Wiley-Liss, Inc. Key words: psychiatry; animal model; endophenotype; genetics; behavior INTRODUCTION Obsessive-compulsive disorder (OCD) is a severe anxiety disorder characterized by obsessions (intrusive, unwanted thoughts) and compulsions (ritualized behaviors intended to overcome the anxiety resulting from the obsessions). The core symptom of OCD is compulsivity, the feeling of loss of voluntary control, the inability to stop acting when you want to, despite being aware of the uselessness 2009 Wiley-Liss, Inc. How to Cite this Article: Kas MJH, Gelegen C, van Nieuwerburgh F, Westenberg HGM, Deforce D, Denys D. 2010. Compulsivity in Mouse Strains Homologous With Chromosomes 7p and 15q Linked to Obsessive-Compulsive Disorder. Am J Med Genet Part B 153B:252–259. of the content, or the adverse consequences in order to relieve anxiety or stress [Denys, 2006]. Compulsivity is epitomized by OCD, but is also present in substance-related disorders, eating disorders, and impulse-control disorders [Holden, 2001]. Though the neurobiological basis of OCD still is unclear, family and twin studies suggest that genetic factors are important in the manifestation of OCD [Hanna et al., 2002; Shugart et al., 2006; Samuels et al., 2007]. Familial studies of OCD indicate that the risk to first-degree relatives is 3–12 times greater than the general population, and twin studies reveal that concordance for OCD is greater among pairs of monozygotic (80–87%) than dizygotic Grant sponsor: The Netherlands Organization for Scientific Research (NWO); Grant number: 91786327; Grant sponsor: ZonMW VIDI; Grant number 91786327. The authors declare that they have no competing financial interests. *Correspondence to: Dr. Martien J.H. Kas, Rudolf Magnus Institute of Neuroscience, Universiteitsweg 100, 3584CG Utrecht, The Netherlands. E-mail: email@example.com Published online 9 June 2009 in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/ajmg.b.30994 252 KAS ET AL. (47–50%) twins [Pauls, 2008]. No genetic factors have yet been identified as a cause, however, recently, a genome-wide linkage scan for OCD found evidence for susceptibility loci on chromosomes 3q, 7p, 1q, 15q, and 6q in OCD [Shugart et al., 2006]. These regions still contain several hundred genes from which some might be potentially relevant for the development of OCD. Animal models may help to unravel genetic mechanisms related to these genomic regions; however, it is highly unlikely that they will recapitulate the entire OCD spectrum. They are inappropriate for investigations into uniquely human aspects of OCD (e.g., obsessions), but seem more than adequate for studying different forms of compulsivity. Some genetic mouse models provide face validity for compulsive behavior and may have some further translation value. For example, 5-HT2C receptor knockout (KO) mice display compulsive-like behavior such as compulsive chewing of non-nutritive clay and plastic-mesh screens [Chou-Green et al., 2003]. Furthermore, a genetic deletion of the SAPAP3 gene resulted in excessive grooming behavior in mice, a feature that mimics quite closely the human characteristics of compulsivity [Welch et al., 2007]. To initiate a systematic search for genetic mechanisms underlying the pathophysiology of compulsivity, the current study examined a panel of chromosome substitution (CS) strains derived from C57BL/6J (host) and A/J (donor) strains. A previous study has shown that these mice either suppress (C57BL/6J strain) or maintain (A/J strain) high levels of repetitive wheel running during 2 hr of daily limited food access [Gelegen et al., 2008]. Mouse models are widely used to dissect the genetic basis for biological traits and to characterize their functional consequences, not only because of their genetic and physiological similarity to humans, but also because an extraordinary variety of genetic resources enable rigorous functional studies. CS strains are a powerful complement for studying multigenic traits. By partitioning the genome into a panel of new inbred strains with single CSs, (one strain for each chromosome), unique experimental designs and considerable statistical power are possible to accelerate the detection of quantitative trait loci (QTLs) with small effect sizes [Singer et al., 2004]. Several published studies demonstrate the considerable utility of these strains and new applications for CS strains continue to be discovered [Singer et al., 2004; Hill et al., 2006; Kas et al., 2008]. Compulsivity was assumed to be mimicked by continuous and repetitive wheel running during the 2 hr of food availability as it expresses the inability to stop wheel running despite the more appealing food availability. In addition to its face validity, it has been shown that fluoxetine suppresses compulsive wheel running activity in rodents during a daily scheduled feeding paradigm [Altemus et al., 1992]. The behavioral screen of the CS strains revealed that three individual substitution strains carrying A/J chromosomes 2, 6, and 7, have significantly higher levels of wheel running during 2 hr of food access when compared to the C57BL/6J control strain. Interestingly, two of the aforementioned human chromosome regions harboring the OCD susceptibility loci, namely 7p and 15q, are homologous with regions of mouse chromosomes that we identified in the present study. In the overlapping (human/mouse) genomic region, several potential candidate genes are located of which one was tested, the corticotrophinreleasing hormone receptor 2 (CRHR2) gene. Corticotrophin- 253 releasing hormone (CRH) is a neuropeptide known to be a regulator of the hypothalamus–pituitary–adrenal (HPA) axis. Currently there are two known classes of CRH receptors, termed type 1 and type 2, that have been cloned from a number of vertebrate species and are encoded by separate genes. In the brain the highest densities are in the parvoventricular nucleus of the hypothalamus, the amygdala, and the lateral septum [Lovenberg et al., 1995]. The CRH2R gene has previously been associated with anxiety-related behavior [Bale and Vale, 2003; Henry et al., 2006] and, interestingly, the mouse CRH2R gene contains a nonsynonymous coding single nucleotide polymorphism (SNP) in exon 2 (rs30120293) that is polymorphic between A/J and C57BL/6J mice. In an initial case–control study four non-synonymous SNPs in the coding region of the CRHR2 gene were investigated in 156 unrelated patients with OCD. MATERIALS AND METHODS The Chromosome Substitution Strain Panel in Mice A CS strains panel (C57BL/6J-Chr 1A/NaJ to C57BL/6J-Chr 19A/ NaJ, C57BL/6J-Chr XA/NaJ; referred to as CS strains; except for CS strains 13, 16, and Y, due to low availability) [Singer et al., 2004] and their parental strains, C57BL/6J (host strain) and A/J (donor strain), were studied in this experiment. Original CS-strain, C57BL/ 6J and A/J breeding pairs were obtained from the Jackson Laboratory (Bar Harbor, ME) and used in our internal breeding program. In total, 321 mice were generated and all tested in the 11 days lasting behavioral paradigm (described below). This concerned female mice of the C57BL/6J strain (n ¼ 53), A/J strain (n ¼ 24), and of the 18 tested CS strains (n ¼ 244; on average, 13–14 mice per CSstrain). Four weeks after birth, mice were weaned and socially housed (2–4 same sex littermates per cage) with ad libitum access to food and water. The animals were maintained in a 12-hr light/12-hr dark cycle (light intensity of 60 lx; lights off at 13.00 hr). Room temperature was 21.0 1.0 C. At the age of 3–4 months mice were tested in the scheduled feeding paradigm. All experimental procedures were approved by the ethical committee for animal experimentation of the Utrecht University, The Netherlands. The Scheduled Feeding Paradigm in Mice The scheduled feeding paradigm is similar to that used in previous studies [Gelegen et al., 2007]. Briefly, to adapt the animals to running wheel cages, all mice were individually housed in cages with running wheels for 1 week before the start of the experiment. The running wheel activity was registered by a small magnet and a counter that was activated by the magnet when it passed the counter during a revolution of the running wheel. The first week of adaptation to the wheel running cages is termed ‘‘baseline conditions.’’ Under baseline conditions, mice had unrestricted access to food, water, and the running wheel. Body weight and food intake were measured daily just before the beginning of the dark phase. Individual wheel running revolutions were continuously registered using Cage Registration Software (Department of Biomedical Engineering, UMC Utrecht, The Netherlands). Following the first week of baseline, mice were placed on a restricted feeding schedule for 4 consecutive days (2 hr of daily access to food) 254 and this period is defined as ‘‘restriction conditions.’’ During the restriction period, the food was given during the first 2 hr of the dark phase (the habitual activity phase of this nocturnal species). Body weight and food intake were measured before and after food access and running wheel revolutions were registered continuously. Statistics of the Mouse Wheel Running Mouse wheel running revolutions were expressed in the mean standard error of the means for each strain. Differences in wheel running revolutions during the 2 hr of food availability were assessed by a repeated measures ANOVA procedure (SPSS, version 12.0.1. for windows Chicago, IL), using a between subject factor (strain) and within subject factor (days) (a ¼ 0.05). In case of a significant difference between the strains, a one-way ANOVA was performed on each day (with a ¼ 0.05) to identify on which days C57BL/6J mice differ from A/J mice regarding wheel running levels during the 2 hr of food availability. Differences in food intake during ad libitum and 2 hr food access conditions in A/J mice were analyzed using a paired t-test (a ¼ 0.05). For the CS strains to C57BL/6J comparisons (average wheel running activity during the 2 hr of food access on days 3 and 4), significance levels (a ¼ 0.05) were corrected using Dunnett’s method to account for the multiple strain comparison [Belknap, 2003]. The Human OCD Sample The patient sample consists of 156 unrelated patients with OCD from consecutive referrals to the Anxiety Research Unit of the Department of Psychiatry at the University Medical Centre Utrecht, who gave written informed consent for participation in this study that had been approved by the University of Utrecht Medical Ethical Review committee (Utrecht, The Netherlands). All patients were diagnosed with OCD according to DSM-IV criteria and the MiniInternational Neuropsychiatric Interview (MINI), a clinical and structured interview, was used to confirm the diagnosis [Sheehan et al., 1998]. Severity of obsessive-compulsive symptoms was rated with the Y-BOCS, depression with the Hamilton depression scale (HAM-D), and anxiety with the Hamilton anxiety scale (HAM-A) [Hamilton, 1959, 1960; Goodman et al., 1989]. Information on family history was obtained by direct interviews with the patients and the presence of vocal and/or motor tics was assessed during the clinical interview (Table I). The control sample was composed of 156 ethnically matched and unrelated Caucasian subjects from the Netherlands, selected among healthy volunteers. Genotyping of the CRH Receptor 2 (CRHR2) Gene in a Case–Control Association Study In an initial case–control study four non-synonymous polymorphisms in the coding region of the CRHR2 gene were investigated. Blood samples were collected from each subject and frozen at 80 . DNA was extracted from 10 ml of peripheral blood according to standard procedures. The samples were genotyped using TaqMan SNP Genotyping Assays from Applied Biosystems for the NCBI dbSNP identification numbers rs8192492, rs8192495, rs8192498, and rs34625936. At this time, these are the only four validated non- AMERICAN JOURNAL OF MEDICAL GENETICS PART B TABLE I. Demographic and Clinical Characteristics of the Patients Sample Gender (male/female) Age on admission Positive family history Mean age of onset 15 years age of onset >20 years age of onset Duration of illness Symptom dimensions Contamination fear and washing Aggressive, sexual, and religious obsessions Somatic obsessions and checking Symmetry and exactness High-risk assessment and checking Y-BOCS HAM-D HAM-A Comorbid depressive disorder Comorbid anxiety disorder Total sample (n ¼ 156) 56/100 36.6 11.5 43 17.7 8.3 60 45 18.7 11.6 23 14 17 54 47 24.9 5.7 9.5 5.8 1.6 6.7 24 13 synonymous SNPs located in the exons of the CRHR2 gene that are reported in the NCBI SNP database. The association between the distribution of the genotypes and allele frequencies with the subjects and expected frequencies to assess Hardy–Weinberg equilibrium, were ascertained by cross-tabulation and c2 analysis. RESULTS The Scheduled Feeding Paradigm in Mice During ad libitum food availability, both C57BL/6J and A/J mice showed marked day–night differences in voluntary wheel running levels (with high levels during the dark phase, the habitual activity phase of this nocturnal species). When food was only given during the first 2 hr of the dark phase, C57BL/6J mice developed high levels of wheel running in the hours prior to food access. This socalled food anticipatory activity [FAA, Mistlberger, 1994] was not observed in A/J mice (Fig. 1). Furthermore, as scheduled food restriction prolonged over days, C57BL/6J mice showed a strong suppression of wheel running levels during the 2 hr of food availability (Fig. 2a) (F ¼ 105; P < 0.0001). In contrast, A/J mice had similar wheel running levels during the 2 hr of food availability (F ¼ 0.9; P ¼ 0.5), and these wheel running levels were lower during the initial phase of scheduled feeding (days 1 and 2), but significantly higher after extended scheduled feeding (on days 3 and 4) when compared to C57BL/6J (day 1: t ¼ 7.2; P < 0.001, day 2: t ¼ 4.2; P < 0.0001, day 3: t ¼ 2.7; P ¼ 0.008, day 4: t ¼ 7.8; P < 0.001). During the ad libitum period A/J mice ate significantly more food than during the 2 hr restriction period [4.4 0.1 g (ad libitum) vs. 1.5 0.1 g (average food intake per day during food restriction days 1–4) (t ¼ 23.8; P < 0.0001)]. These data indicate KAS ET AL. FIG. 1. Six days of circadian wheel running patterns of C57BL/6J and A/J mice during ad libitum and 2-hr scheduled food access. In contrast to A/J mice (lower panel), C57BL/6J mice (upper panel) exhibitedhighamplitudewheelrunning levelsduringtheirhabitual active phase (note the different scales of the y-axis), showed high levels of wheel running activity in the hours prior to the 2 hr of food access, and showed a strong suppression of their wheel running levels during prolonged scheduled feeding [see arrow (upper panel) and also see Figure 2 (upper panel)]. Black bars below the x-axis indicate the episodes that food was made available. that the similar levels of wheel running in A/J mice during the ad libitum and the food restricted period did not result from equal levels of food intake during these two conditions (e.g., less motivation to reduce wheel running), and further support the notion that A/J mice showed compulsive wheel running during daily scheduled limited food access. To initiate a search for genetic loci underlying this behavioral trait, a panel of CS strains derived from the C57BL/6J and A/J parental strains were tested using the same scheduled feeding paradigm. This behavioral screen revealed that three strains carrying A/J chromosomes 2, 6, and 7, have significant higher levels of wheel running during 2 hr of food access when compared to the C57BL/6J genetic background control strain (Fig. 2b) (F ¼ 3.0; P < 0.0001, using Dunnett’s method for multiple strain comparison [Belknap, 2003]). Homology Between Mouse and Men Recent human genome-wide linkage studies using OCD patient populations revealed several susceptibility loci for this disorder [Hanna et al., 2002; Shugart et al., 2006; Samuels et al., 2007]. 255 FIG. 2. During prolonged scheduled food restriction (days 3 and 4), A/J mice showed significant higher wheel running levels during the 2 hr that food is available when compared to C57BL/6J mice (upper panel). Testing a panel of chromosome substitutions strains derived from these two parental strains revealed that the individual A/J chromosomes 2, 6, and 7 carry at least one genetic locus that contributes to this behavioral trait (lower panel). *Indicates significant differences between the corresponding CS strains (CSS) and the C57BL/6J genetic background controls (average wheel running activity during the 2 hr of food access on days 3 and 4). Interestingly, two of those human linkage regions (7p and 15q) are homologous with regions of the mouse chromosomes that we identified in the present study. The human linkage region for OCD on 7p [Shugart et al., 2006] is homologous with mouse chromosome 6 (regions 8–13 M bp; 49–57 M bp; 57,700–57,800 K bp). Furthermore, the human peak marker for the linkage region on 15q [Shugart et al., 2006] is homologous with mouse chromosome 7. In the overlapping (human/mouse) genomic region, several potential candidate genes are located of which one was tested, namely the CRH2R gene, in a human case–control study. CRH is a neuropeptide known to be a regulator of the HPA axis. The CRH2R gene (on mouse chromosome 6 (55,040,081–55,082,966 bp) and on human chromosomal region 7p15.1) has previously been associated with anxiety-related behavior [Bale and Vale, 2003; Henry et al., 2006] and, interestingly, the mouse CRH2R gene contains a non- 256 AMERICAN JOURNAL OF MEDICAL GENETICS PART B TABLE II. Allele Frequencies and Genotype Distribution of the rs8192492, rs8192495, rs8192498, and rs34625936 Polymorphisms Allele frequencies Rs8192492 OCD patients Controls Genotypes n T C TT T/C CC 156 156 0.00% 0.00% 100.00% 100.00% 0 (0.00%) 0 (0.00%) 0 (0.00%) 0 (0.00%) 156 (100.00%) 156 (100.00%) Allele frequencies Rs8192495 OCD patients Controls Genotypes n G A GG G/A AA 156 156 100.00% 100.00% 0.00% 0.00% 156 (100.00%) 156 (100.00%) 0 (0.00%) 0 (0.00%) 0 (0.00%) 0 (0.00%) Allele frequencies n Rs8192498 OCD patients Controls 156 156 Genotypes G A GG G/A AA 98.72% 98.72% 1.28% 1.28% 152 (97.44%) 152 (97.44%) 4 (2.56%) 4 (2.56%) 0 (0.00%) 0 (0.00%) Allele frequencies Rs34625936 OCD patients Controls Genotypes n C G CC C/G GG 156 156 99.04% 99.36% 0.96% 0.64% 153 (98.08%) 154 (98.72%) 3 (1.92%) 2 (1.28%) 0 (0.00%) 0 (0.00%) synonymous coding SNP in exon 2 (rs30120293) that is polymorphic between A/J and C57BL/6J mice (the donor and host strains, respectively, for the CS strains used in this study). Results of the CRH Receptor 2 (CRHR2) Gene Association The genotypic pattern of distribution and the allele frequencies of the four polymorphisms of the CRHR2 gene and OCD are shown in Table II. The allele frequencies of the patients group and the control group are almost identical. The measured allele frequencies are in accordance with the allele frequencies reported by the HapMap project and the Applera Genome Resequencing Initiative (AGI). The HapMap European allele frequencies for rs8192492, rs8192495, and rs34625936 are 100.0% C-allele, 100.0% G-allele, and 100.0% C-allele, respectively. The Caucasian AGI allele frequencies for rs8192498 and rs34625936 are 97% G-allele and 97% C-allele, respectively. DISCUSSION In the present study, we have analyzed compulsive wheel running behavior in CS strains of mice. This study showed that three A/J chromosomes (namely chromosomes 2, 6, and 7) contributed to the significant higher levels of wheel running during 2 hr of food access when compared to the C57BL/6J genetic background control strain. Interestingly, the identified mouse chromosomes overlap with two previously observed human OCD chromosomal regions, 7p and 15q [Shugart et al., 2006]. Several candidate genes are located in these homologous chromosomal regions of which the CRH2R gene was tested in a human case–control study. Here, no association was found between four polymorphisms of the CRHR2 gene and OCD. However, the observed homology at the genome level between human OCD and compulsive wheel running in mice provides, in addition to face and predictive validity [Altemus et al., 1992], a good starting point for further genetic fine mapping of mouse candidate genes for this neurobehavioral trait. Additional genetic fine mapping of the mouse chromosomes linked to compulsive wheel running will also further contribute to determine the level of synteny between the human loci for OCD and the mouse loci found for compulsive wheel running behavior. The CS strains of mice have been proven a sensitive detection method for QTL identification [Singer et al., 2004; Kas et al., 2009a]. In addition to the identification of neurobiological mechanisms for compulsivity, valid animal models will be required to functionally test potential biological substrates and novel pharmacological therapies for this neurobehavioral trait that is relevant to a wide KAS ET AL. variety of psychiatric disorders. Indeed, several animal models for compulsivity have been proposed [see Joel, 2006; Korff and Harvey, 2006 for review], and a recent study on the SAPAP3 gene mutation in mice provided novel insights in the potential mechanisms underlying excessive grooming behavior [Welch et al., 2007], a feature reminiscent to compulsive behavior in trichotillomania and skin picking in humans. While most of these models show face and (sometimes) predictive validity, construct validity is the most difficult to provide for animal models of neuropsychiatric disorders, simply because the lack of knowledge about the underlying etiological mechanisms of these complex disorders. Therefore, the question remains how these various animal models will translate to compulsivity observed in a wide variety of psychiatric disorders, such as in OCD, drug addiction, and eating disorders. Recently, it was proposed that certain behavioral domains, such as compulsivity, are present across the spectrum of psychiatric disorders [Kas et al., 2007]. These domains may represent intermediate phenotypes related to a particular disorder that mark the pathway between the genotype and the endophenotype of interest [Gottesmann and Gould, 2003]. The observed homologous regions between human OCD and mouse compulsive running in the present study, therefore, may provide a good starting point to identify genetic factors for compulsivity and to provide genetic validity [Kas et al., 2009b] of compulsive wheel running activity in mice for compulsive behavior in humans. Though these results indicate that compulsive running may be a valid animal model for OCD, the face validity of the running wheel experiment is weakened by the fact that compulsive running/ exercise occurs in association with daily scheduled food restriction. Indeed, this paradigm has also been indicated as an animal model for pathophysiological processes of anorexia nervosa [Kas et al., 2003; Gelegen et al., 2007]. On the other hand, there is quite some degree of co-morbidity between OCD and anorexia nervosa, indicating some overlap between these different classified disorders [Kaye et al., 2004; Swinbourne and Touyz, 2007]. Furthermore, compulsivity is thought to be driven by stressful events and food restriction is generally considered as such. It may therefore be possible that certain mouse strains have, depending on their genetic background, high levels of compulsivity under various stressful conditions and that wheel running behavior is one form of compulsive act to deal with these conditions. CRH is the principal regulator of the HPA axis and an activator of the sympathoadrenal (SA) and systemic sympathetic (SS) systems. A number of psychiatric disorders, including major depression and post-traumatic stress disorder have been associated with dysregulation of the HPA axis [Barden et al., 1995; de Kloet et al., 2008; Heim et al., 2008]. This study is, to our knowledge, the first report on CRHR2 gene polymorphisms in OCD. The four non-synonymous SNPs located in the exons of the CRHR2 gene that are currently reported as validated in the NCBI SNP database, were analyzed because these four SNPs have the highest chance of giving a causal association with OCD. Although the CRHR2 gene is a plausible functional candidate gene influencing the reactivity of the HPA axis and the development of anxiety disorders, our findings provide no evidence for an association between the four investigated polymorphisms of the CRHR2 gene and OCD. Because not all SNPs in the CRHR2 region were covered in this study, it 257 cannot be concluded that the CRHR2 gene is not involved in the etiology of OCD. It can only be concluded that the four analyzed SNPs do not have a major effect on the risk of OCD. Though our results should be interpreted with caution given the limited sample sizes, and given the limited number of analyzed SNPs, they are in accordance with Tharmalingam et al.  who genotyped three polymorphisms of the CRHR2 gene in 183 patients with DSM-IV panic disorder and 75 case–controls and failed to find an association with panic disorder. Still, some studies support an association between CRHR2 genes and anxiety disorders. In animal experiments, CRHR2-deficient mice display stress-sensitive and increased anxiety-like behavior [Bale et al., 2000; Coste et al., 2000; Kishimoto et al., 2000]. Recently, SNPs in the CRH gene have been found to be associated with behavioral inhibition, a childhood risk factor for panic disorder and social phobia [Smoller et al., 2003, 2005]. These findings suggest that the CRHR2 gene is a plausible functional candidate gene influencing the reactivity of the HPA axis and the development of anxiety disorders. On the other hand, in comparison with findings in depression patients, relatively little is known of putative CRH system deregulation in OCD and anxiety disorders. Except for Altemus et al. who reported significantly elevated cerebrospinal fluid (CSF) CRH levels in patients with OCD compared with controls, the HPA axis has been studied scarcely in OCD. Initial investigations of CRH deregulation in anxiety disorders have been mostly negative, with generalized anxiety disorder, panic disorder, and OCD patients exhibiting no difference from controls [Jolkkonen et al., 1993; Chappell et al., 1996; Fossey et al., 1996]. These negative findings are based on using a single lumbar puncture technique for CSF sampling. This technique is stressful and increases CRH release in all subjects, which may mask baseline differences in CRH concentrations between anxiety disorder and control populations [Geracioti et al., 1992, 1997]. Serial CRH sampling techniques, in which CRH is sampled over longer periods of time, may be a more sensitive method to detect baseline CRH abnormalities [Risbrough and Stein, 2006]. Thus, this study showed that mouse chromosomes linked to compulsive wheel running during scheduled feeding are homologous with human genome linkage regions recently identified for OCD. These findings provide novel opportunities to obtain genetic validity for an animal model of human compulsivity and to identify neurobiological mechanisms underlying this behavioral trait that is relevant to a wide variety of psychiatric disorders, including OCD, drug addiction, and eating disorders. ACKNOWLEDGMENTS This research was supported by The Netherlands Organization for Scientific Research (NWO), ZonMW VIDI grant 91786327 to Dr. M.J. Kas. REFERENCES Altemus M, Pigott T, Kalogeras KT, Demitrack M, Dubbert B, Murphy DL, Gold PW. 1992. Abnormalities in the regulation of vasopressin and corticotropin releasing-factor secretion in obsessive-compulsive disorder. Arch Gen Psychiatry 49(1):9–20. 258 Bale TL, Vale WW. 2003. Increased depression-like behaviors in corticotropin-releasing factor receptor-2-deficient mice: Sexually dichotomous responses. J Neurosci 23(12):5295–5301. Bale TL, Contarino AB, Smith GW, Chan R, Gold LH, Sawchenko PE, Koob GF, Vale WW, Lee KF. 2000. Mice deficient for corticotropin-releasing hormone receptor-2 display anxiety-like behaviour and are hypersensitive to stress. Nat Genet 24(4):410–414. Barden N, Reul JM, Holsboer F. 1995. Do antidepressants stabilize mood through actions on the hypothalamic-pituitary-adrenocortical system? Trends Neurosci 18(1):6–11. Belknap JK. 2003. Chromosome substitution strains: Some quantitative considerations for genome scans and fine mapping. Mamm Genome 14(11):723–732. Chappell P, Leckman J, Goodman W, Bissette G, Pauls D, Anderson G, Riddle M, Scahill L, McDougle C, Cohen D. 1996. Elevated cerebrospinal fluid corticotropin-releasing factor in tourette’s syndrome: Comparison to obsessive compulsive disorder and normal controls. Biol Psychiatry 39(9):776–783. Chou-Green JM, Holscher TD, Dallman MF, Akana SF. 2003. Compulsive behavior in the 5-HT2C receptor knockout mouse. Physiol Behav 78(4–5):641–649. Coste SC, Kesterson RA, Heldwein KA, Stevens SL, Heard AD, Hollis JH, Murray SE, Hill JK, Pantely GA, Hohimer AR, Hatton DC, Phillips TJ, Finn DA, Low MJ, Rittenberg MB, Stenzel P, Stenzel-Poore MP. 2000. Abnormal adaptations to stress and impaired cardiovascular function in mice lacking corticotropin-releasing hormone receptor-2. Nat Genet 24(4):403–409. de Kloet CS, Vermetten E, Geuze E, Lentjes EG, Heijnen CJ, Stalla GK, Westenberg HG. 2008. Elevated plasma corticotrophin-releasing hormone levels in veterans with posttraumatic stress disorder. Prog Brain Res 167:287–291. Denys D. 2006. Pharmacotherapy of obsessive-compulsive disorder and obsessive-compulsive spectrum disorders. Psychiatr Clin North Am 29(2):553–584. Fossey MD, Lydiard RB, Ballenger JC, Laraia MT, Bissette G, Nemeroff CB. 1996. Cerebrospinal fluid corticotropin-releasing factor concentrations in patients with anxiety disorders and normal comparison subjects. Biol Psychiatry 39(8):703–707. Gelegen C, Collier DA, Campbell IC, Oppelaar H, van den Heuvel J, Adan RA, Kas MJ. 2007. Difference in susceptibility to activity-based anorexia in two inbred strains of mice. Eur Neuropsychopharmacol 17(3):199–205. Gelegen C, van den Heuvel J, Collier DA, Campbell IC, Oppelaar H, Hessel E, Kas MJ. 2008. Dopaminergic and BDNF signalling in inbred mice exposed to a restricted feeding schedule. Genes Brain Behav 7(5):552–559. Geracioti TD, Orth DN, Ekhator NN, Blumenkopf B, Loosen PT. 1992. Serial cerebrospinal-fluid corticotropin-releasing hormone concentrations in healthy and depressed humans. J Clin Endocrinol Metab 74(6):1325–1330. AMERICAN JOURNAL OF MEDICAL GENETICS PART B Hamilton M. 1959. The assessment of anxiety states by rating. Br J Med Psychol 32:50–55. Hamilton M. 1960. A rating scale for depression. J Neurol Neurosurg Psychiatry 23:56–62. Hanna GL, Veenstra-VanderWeele J, Cox NJ, Boehnke M, Himle JA, Curtis GC, Leventhal BL, Cook EH Jr. 2002. Genome-wide linkage analysis of families with obsessive-compulsive disorder ascertained through pediatric probands. Am J Med Genet 114(5):541–552. Heim C, Newport DJ, Mletzko T, Miller AH, Nemeroff CB. 2008. The link between childhood trauma and depression: Insights from HPA axis studies in humans. Psychoneuroendocrinology 33(6):693–710. Henry B, Vale W, Markou A. 2006. The effect of lateral septum corticotropin-releasing factor receptor 2 activation on anxiety is modulated by stress. J Neurosci 26(36):9142–9152. Hill AE, Lander ES, Nadeau JH. 2006. Chromosome substitution strains: A new way to study genetically complex traits. Methods Mol Med 128:153–172. Holden C. 2001. ‘Behavioral’ addictions: Do they exist? Science 294(5544):980–982. Joel D. 2006. Current animal models of obsessive compulsive disorder: A critical review. Prog Neuropsychopharmacol Biol Psychiatry 30(3):374–388. Jolkkonen J, Lepola U, Bissette G, Nemeroff C, Riekkinen P. 1993. Csf corticotropin-releasing factor is not affected in panic disorder. Biol Psychiatry 33(2):136–138. Kas MJ, van Dijk G, Scheurink AJ, Adan RA. 2003. Agouti-related protein prevents self-starvation. Mol Psychiatry 8:235–240. Kas MJ, Fernandes C, Schalkwyk LC, Collier DA. 2007. Genetics of behavioural domains across the neuropsychiatric spectrum of mice and men. Mol Psychiatry 12:324–330. Kas MJ, de Mooij-van Malsen JG, Olivier B, Spruijt BM, Van Ree JM. 2008. Differential genetic regulation of motor activity and anxiety-related behaviors in mice using an automated home cage task. Behav Neurosci 122(4):769–776. Kas MJ, de Mooij-van Malsen JG, de Krom M, van Gassen KL, van Lith HA, Olivier B, Oppelaar H, Hendriks J, de Wit M, Groot Koerkamp MJ, Holstege FC, van Oost BA, de Graan PN. 2009a. High-resolution genetic mapping of mammalian motor activity levels in mice. Genes Brain Behav 8(1):13–22. Kas MJ, Gelegen C, Schalkwyk LC, Collier DA. 2009b. Interspecies comparisons of functional genetic variations and their implications in neuropsychiatry. Am J Med Genet Part B 150B(3):309–317. Kaye WH, Bulik CM, Thornton L, Barbarich N, Masters K. 2004. Comorbidity of anxiety disorders with anorexia and bulimia nervosa. Am J Psychiatry 161(12):2215–2221. Kishimoto T, Radulovic J, Radulovic M, Lin CR, Schrick C, Hooshmand F, Hermanson O, Rosenfeld MG, Spiess J. 2000. Deletion of crhr2 reveals an anxiolytic role for corticotropin-releasing hormone receptor-2. Nat Genet 24(4):415–419. Geracioti TD, Loosen PT, Orth DN. 1997. Low cerebrospinal fluid corticotropin-releasing hormone concentrations in eucortisolemic depression. Biol Psychiatry 42(3):165–174. Korff S, Harvey BH. 2006. Animal models of obsessive-compulsive disorder: Rationale to understanding psychobiology and pharmacology. Psychiatr Clin North Am 29(2):371–390. Goodman WK, Price LH, Rasmussen SA, Mazure C, Fleischmann RL, Hill CL, Heninger GR, Charney DS. 1989. The yale-brown obsessive compulsive scale. 1. Development, use, and reliability. Arch Gen Psychiatry 46(11):1006–1011. Lovenberg TW, Liaw CW, Grigoriadis DE, Clevenger W, Chalmers DT, De Souza EB, Oltersdorf T. 1995. Cloning and characterization of a functionally distinct corticotropin-releasing factor receptor subtype from rat brain. Proc Natl Acad Sci USA 92(3):836–840 Erratum in Proc Natl Acad Sci USA, 92(12):5759. Gottesman II, Gould TD. 2003. The endophenotype concept in psychiatry: Etymology and strategic intentions. Am J Psychiatry 160(4):636–645. Mistlberger RE. 1994. Circadian food-anticipatory activity: Formal models and physiological mechanisms. Neurosci Biobehav Rev 18(2):171–1195. KAS ET AL. Pauls DL. 2008. The genetics of obsessive compulsive disorder: A review of the evidence. Am J Med Genet Part C 148C(2):133–139. Risbrough VB, Stein MB. 2006. Role of corticotropin releasing factor in anxiety disorders: A translational research perspective. Horm Behav 50(4):550–561. Samuels JF, Bienvenu OJ III, Pinto A, Fyer AJ, McCracken JT, Rauch SL, Murphy DL, Grados MA, Greenberg BD, Knowles JA, Piacentini J, Cannistraro PA, Cullen B, Riddle MA, Rasmussen SA, Pauls DL, Willour VL, Shugart YY, Liang KY, Hoehn-Saric R, Nestadt G. 2007. Hoarding in obsessive-compulsive disorder: Results from the OCD Collaborative Genetics Study. Behav Res Ther 45(4): 673–686. Sheehan DV, Lecrubier Y, Sheehan KH, Amorim P, Janavs J, Weiller E, Hergueta T, Baker R, Dunbar GC. 1998. The mini-international neuropsychiatric interview (mini): The development and validation of a structured diagnostic psychiatric interview for dsm-iv and icd-10. J Clin Psychiatry 59:22–33. Shugart YY, Samuels J, Willour VL, Grados MA, Greenberg BD, Knowles JA, McCracken JT, Rauch SL, Murphy DL, Wang Y, Pinto A, Fyer AJ, Piacentini J, Pauls DL, Cullen B, Page J, Rasmussen SA, Bienvenu OJ, Hoehn-Saric R, Valle D, Liang KY, Riddle MA, Nestadt G. 2006. Genomewide linkage scan for obsessive-compulsive disorder: Evidence for susceptibility loci on chromosomes 3q, 7p, 1q, 15q, and 6q. Mol Psychiatry 11(8):763–770. 259 Singer JB, Hill AE, Burrage LC, Olszens KR, Song J, Justice M, et al. 2004. Genetic dissection of complex traits with chromosome substitution strains of mice. Science 304(5669):445–448. Smoller JW, Rosenbaum JF, Biederman J, Kennedy J, Dai D, Racette SR, Laird NM, Kagan J, Snidman N, Hirshfeld-Becker D, Tsuang MT, Sklar PB, Slaugenhaupt SA. 2003. Association of a genetic marker at the corticotropin-releasing hormone locus with behavioral inhibition. Biol Psychiatry 54(12):1376–1381. Smoller JW, Yamaki LH, Fagerness JA, Biederman J, Racette S, Laird NM, Kagan J, Snidman N, Faraone SV, Hirshfeld-Becker D, Tsuang MT, Slaugenhaupt SA, Rosenbaum JF, Sklar PB. 2005. The corticotropinreleasing hormone gene and behavioral inhibition in children at risk for panic disorder. Biol Psychiatry 57(12):1485–1492. Swinbourne JM, Touyz SW. 2007. The co-morbidity of eating disorders and anxiety disorders: A review. Eur Eat Disord Rev 15:253–274. Tharmalingam S, King N, De Luca V, Rothe C, Koszycki D, Bradwejn J, Macciardi F, Kennedy JL. 2006. Lack of association between the corticotrophin-releasing hormone receptor 2 gene and panic disorder. Psychiatr Genet 16(3):93–97. Welch JM, Lu J, Rodriguiz RM, Trotta NC, Peca J, Ding JD, Feliciano C, Chen M, Adams JP, Luo J, Dudek SM, Weinberg RJ, Calakos N, Wetsel WC, Feng G. 2007. Cortico-striatal synaptic defects and OCD-like behaviours in Sapap3-mutant mice. Nature 448(7156):894–900.