Correction: Investigation of common, low-frequency and rare genome-wide variation in anorexia nervosa.

The fortieth author's name was listed incorrectly. The correct presentation is A Keski-Rahkonen.

Investigation of common, low-frequency and rare genome-wide variation in anorexia nervosa.

Anorexia nervosa (AN) is a complex neuropsychiatric disorder presenting with dangerously low body weight, and a deep and persistent fear of gaining weight. To date, only one genome-wide significant locus associated with AN has been identified. We performed an exome-chip based genome-wide association studies (GWAS) in 2158 cases from nine populations of European origin and 15 485 ancestrally matched controls. Unlike previous studies, this GWAS also probed association in low-frequency and rare variants. Sixteen independent variants were taken forward for in silico and de novo replication (11 common and 5 rare). No findings reached genome-wide significance. Two notable common variants were identified: rs10791286, an intronic variant in OPCML (P=9.89 × 10-6), and rs7700147, an intergenic variant (P=2.93 × 10-5). No low-frequency variant associations were identified at genome-wide significance, although the study was well-powered to detect low-frequency variants with large effect sizes, suggesting that there may be no AN loci in this genomic search space with large effect sizes.

A genome-wide association study of anorexia nervosa.

Anorexia nervosa (AN) is a complex and heritable eating disorder characterized by dangerously low body weight. Neither candidate gene studies nor an initial genome-wide association study (GWAS) have yielded significant and replicated results. We performed a GWAS in 2907 cases with AN from 14 countries (15 sites) and 14 860 ancestrally matched controls as part of the Genetic Consortium for AN (GCAN) and the Wellcome Trust Case Control Consortium 3 (WTCCC3). Individual association analyses were conducted in each stratum and meta-analyzed across all 15 discovery data sets. Seventy-six (72 independent) single nucleotide polymorphisms were taken forward for in silico (two data sets) or de novo (13 data sets) replication genotyping in 2677 independent AN cases and 8629 European ancestry controls along with 458 AN cases and 421 controls from Japan. The final global meta-analysis across discovery and replication data sets comprised 5551 AN cases and 21 080 controls. AN subtype analyses (1606 AN restricting; 1445 AN binge-purge) were performed. No findings reached genome-wide significance. Two intronic variants were suggestively associated: rs9839776 (P=3.01 × 10(-7)) in SOX2OT and rs17030795 (P=5.84 × 10(-6)) in PPP3CA. Two additional signals were specific to Europeans: rs1523921 (P=5.76 × 10(-)(6)) between CUL3 and FAM124B and rs1886797 (P=8.05 × 10(-)(6)) near SPATA13. Comparing discovery with replication results, 76% of the effects were in the same direction, an observation highly unlikely to be due to chance (P=4 × 10(-6)), strongly suggesting that true findings exist but our sample, the largest yet reported, was underpowered for their detection. The accrual of large genotyped AN case-control samples should be an immediate priority for the field.

Theory of Mind and social functioning among neuropsychiatric disorders: A transdiagnostic study.

Social dysfunction is commonly present in neuropsychiatric disorders of schizophrenia (SZ) and Alzheimer's disease (AD). Theory of Mind (ToM) deficits have been linked to social dysfunction in disease-specific studies. Nevertheless, it remains unclear how ToM is related to social functioning across these disorders, and which factors contribute to this relationship. We investigated transdiagnostic associations between ToM and social functioning among SZ/AD patients and healthy controls, and explored to what extent these associations relate to information processing speed or facial emotion recognition capacity. A total of 163 participants were included (SZ: n=56, AD: n=50 and age-matched controls: n=57). Social functioning was assessed with the Social Functioning Scale (SFS) and the De Jong-Gierveld Loneliness Scale (LON). ToM was measured with the Hinting Task. Information processing speed was measured by the Digit Symbol Substitution Test (DSST) and facial emotion recognition capacity by the facial emotion recognition task (FERT). Case-control deficits in Hinting Task performance were larger in AD (rrb = -0.57) compared to SZ (rrb = -0.35). Poorer Hinting Task performance was transdiagnostically associated with the SFS (ßHinting-Task = 1.20, p<0.01) and LON (ßHinting-Task = -0.27, p<0.05). DSST, but not FERT, reduced the association between the SFS and Hinting Task performance, however the association remained significant (ßHinting-Task = 0.95, p<0.05). DSST and FERT performances did not change the association between LON and Hinting Task performance. Taken together, ToM deficits are transdiagnostically associated with social dysfunction and this is partly related to reduced information processing speed.

Evidence for epigenetic interactions for loci on mouse chromosome 1 regulating open field activity.

The expression of motor activity levels in response to novel situations is under complex genetic and environmental control. Several genetic loci have been implicated in the regulation of this behavioral phenotype, but their relationship to epigenetic and epistatic interactions is relatively unknown. Here, we report on a quantitative trait locus (QTL) on mouse chromosome 1 for novelty-induced motor activity in the open field, using chromosome substitution strains derived from a high active host strain (C57BL/6J) and a low active donor strain (A/J). The QTL for open field (horizontal distance moved) peaked at the location of Kcnj9, however, QTL detection was initially masked by an interplay of both grandparent genetic origin and genetic co-factors influencing behavior on chromosome 1. Our findings indicate that epigenetic interactions can play an important role in the identification of behavioral QTLs and must be taken into consideration when applying behavioral genetic strategies.

Leptin's effect on hyperactivity: potential downstream effector mechanisms.

Up to 80% of patients with Anorexia Nervosa (AN) demonstrate hyperactivity. Hyperactivity counteracts weight gain during treatment and is associated with poor outcome of the disease. We hypothesized that hyperactivity in AN patients has a neurobiological basis and used an animal model-based translational approach to gain insight in mechanisms underlying this hyperactivity. Previously we and others showed that leptin treatment attenuates hyperactivity in the rat activity-based anorexia (ABA) model. The mechanisms involved in this process are, however, unknown. Here we describe potential downstream effector mechanisms involved in the attenuation of hyperactivity by leptin treatment in ABA rats.

The impact of hyperactivity and leptin on recovery from anorexia nervosa.

In anorexia nervosa (AN), hyperactivity is observed in about 80% of patients and has been associated with low leptin levels in the acute stage of AN and in anorexia animal models. To further understand the importance of this correlation in AN, we investigated the relationship between hypoleptinaemia and hyperactivity in AN patients longitudinally and assessed their predictive value for recovery. Body weight, activity levels, and serum leptin levels were assessed in adolescents and adult AN patient groups at the start and during treatment, up to a year. In the adolescent group, initial leptin and activity levels were correlated. This negative correlation changes over time into a positive correlation with physiological recovery. Treatment outcome in both groups could be predicted by initial BMI and leptin levels but not by activity levels. No major relationship of activity with the course of recovery was detected, suggesting that in contrast to the acute stage of the disease, leptin and activity levels during the recovery process are dissociated.

Novel approach to the behavioural characterization of inbred mice: automated home cage observations.

Here we present a newly developed tool for continuous recordings and analysis of novelty-induced and baseline behaviour of mice in a home cage-like environment. Aim of this study was to demonstrate the strength of this method by characterizing four inbred strains of mice, C57BL/6, DBA/2, C3H and 129S2/Sv, on locomotor activity. Strains differed in circadian rhythmicity, novelty-induced activity and the time-course of specific behavioural elements. For instance, C57BL/6 and DBA/2 mice showed a much faster decrease in activity over time than C3H and 129S2/Sv mice. Principal component analysis revealed two major factors within locomotor activity, which were defined as 'level of activity' and 'velocity/stops'. These factors were able to distinguish strains. Interestingly, mice that displayed high levels of activity in the initial phase of the home cage test were also highly active during an open-field test. Velocity and the number of stops during movement correlated positively with anxiety-related behaviour in the elevated plus maze. The use of an automated home cage observation system yields temporal changes in elements of locomotor activity with an advanced level of spatial resolution. Moreover, it avoids the confounding influence of human intervention and saves time-consuming human observations.

The appetite suppressant d-fenfluramine reduces water intake, but not food intake, in activity-based anorexia.

Biochemical, genetic and imaging studies support the involvement of the serotonin (5-HT) system in anorexia nervosa. Activity-based anorexia (ABA) is considered an animal model of anorexia nervosa, and combines scheduled feeding with voluntary running wheel activity (RWA). We investigated the effect of d-fenfluramine (d-FEN) treatment on development and propagation of ABA. d-FEN is an appetite suppressant and acts on 5-HT(2C) receptors that are located on pro-opiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus. Since stimulation activation of the melanocortin system stimulates ABA, we hypothesized that d-FEN treatment enhances the development and propagation of ABA. Rats were exposed to the ABA model and chronically infused with d-FEN. Unexpectedly, d-FEN-treated ABA rats did not reduce food intake or increase wheel running as compared with vehicle-treated ABA rats. Furthermore d-FEN treatment did not affect body weight loss, hypothalamus-pituitary-adrenal axis activation, or starvation-induced hypothermia in ABA rats. POMC mRNA levels in d-FEN-treated rats were not different from vehicle-treated rats after one week of exposure to the ABA paradigm. However, d-FEN-treated ABA rats showed hypodypsia and increased plasma osmolality and arginine-vasopressin expression levels in the hypothalamus. We conclude that d-FEN treatment does not enhance ABA under the experimental conditions of this study, but strongly reduces water intake in ABA rats.

The MC4 receptor and control of appetite.

Mutations in the human melanocortin (MC)4 receptor have been associated with obesity, which underscores the relevance of this receptor as a drug target to treat obesity. Infusion of MC4R agonists decreases food intake, whereas inhibition of MC receptor activity by infusion of an MC receptor antagonist or with the inverse agonist AgRP results in increased food intake. This review addresses the role of the MC system in different aspects of feeding behaviour. MC4R activity affects meal size and meal choice, but not meal frequency, and the type of diet affects the efficacy of MC4R agonists to reduce food intake. The central sites involved in the different aspects of feeding behaviour that are affected by MC4R signalling are being unravelled. The paraventricular nucleus plays an important role in food intake per se, whereas MC signalling in the lateral hypothalamus is associated with the response to a high fat diet. MC4R signalling in the brainstem has been shown to affect meal size. Further genetic, behavioural and brain-region specific studies need to clarify how the MC4R agonists affect feeding behaviour in order to determine which obese individuals would benefit most from treatment with these drugs. Application of MCR agonists in humans has already revealed side effects, such as penile erections, which may complicate introduction of these drugs in the treatment of obesity.

Crepuscular rhythms of EEG sleep-wake in a hystricomorph rodent, Octodon degus.

Sleep-wake circadian rhythms are well documented for nocturnal rodents, but little is known about sleep regulation in diurnal or crepuscular rodent species. This study examined the circadian sleep-wake rhythms in Octodon degus by means of electroencephalogram (EEG) analysis. Recordings were made from animals housed with or without running wheels in the cages. In a 24-h light-dark (LD) cycle (LD 12:12), sleep and wake patterns were highly fragmented under both conditions except for crepuscular timed episodes of waking. Without running wheels, sleep bout lengths averaged 3.7 +/- 0.1 min, and total sleep time was 37.6 +/- 3.7% per 24 h. Although the percentage of total wakefulness was similar during the light phase (63.4 +/- 2.4%) and dark phase (61.5 +/- 2.8%), measures of locomotor activity (LMA) and body temperature were generally greater during the day than during the night. Without running wheels, EEG slow wave activity (SWA) in nonrapid eye movement (NREM) sleep exhibited a circadian waveform that was elevated only during the light phase. SWA peaked at Zeitgeber Time 7 (ZT 7) (several hours after the dominant waking episode at ZT 23), then declined across the later half of the light phase and into the dark phase. Voluntary wheel running did not alter daily total sleep time, the duration of average sleep bouts, or maximum sleep bouts, but it increased the episode of waking, LMA, and body temperature at ZTs 11-12. Under these conditions, NREM sleep and SWA exhibited crepuscular patterns, with elevated SWA during the day and night. Although Octodon degus exhibited no strong preference for sleep during the light or dark phase, these data suggest that in this species homeostatic sleep responses (indicated by SWA) are gated by the dominant crepuscular episode(s) of waking and can be influenced by wheel running.

Scheduled voluntary wheel running activity modulates free-running circadian body temperature rhythms in Octodon degus.

Entrainment of the circadian pacemaker to nonphotic stimuli, such as scheduled wheel-running activity, is well characterized in nocturnal rodents, but little is known about activity-dependent entrainment in diurnal or crepuscular species. In the present study, effects of scheduled voluntary wheel-running activity on circadian timekeeping were investigated in Octodon degus, a hystricomorph rodent that exhibits robust crepuscular patterns of wakefulness. When housed in constant darkness, O. degus exhibited circadian rhythms in wheel-running activity and body temperature (Tb) with an average period length (tau) of 23.39 +/- 0.11 h. When wheel running was restricted to a fixed 2-h schedule every 24 h, tau increased on average 0.39 +/- 0.09 h but did not result in steady-state entrainment. Instead, relative coordination between the fixed running schedule and circadian timing was observed. Tau was greatest when scheduled wheel running occurred at CT 20.5 (0.4 h greater than DD baseline tau). Scheduled running activity also influenced Tb waveform symmetry, reflecting concomitant changes in the circadian activity-rest ratio (alpha:rho). Aftereffects of the scheduled wheel-running paradigm were also observed. In 2 animals, tau lengthened from 23.20 and 23.80 h to 24.14 and 24.15 h, respectively, and remained relatively stable for approximately 1 month during the wheel schedule. Although behavioral activity appears to be a weak zeitgeber in this species, these data suggest that nonphotic stimuli can phase delay the circadian pacemaker in O. degus at similar times of the day as in nocturnal hamsters and mice, and in humans.

A candidate syntenic genetic locus is associated with voluntary exercise levels in mice and humans.

Individual levels of physical activity, and especially of voluntary physical exercise, highly contribute to the susceptibility for developing metabolic, cardiovascular diseases, and potentially to psychiatric disorders. Here, we applied a cross-species approach to explore a candidate genetic region for voluntary exercise levels. First, a panel of mouse chromosome substitution strains was used to map a genomic region on mouse chromosome 2 that contributes to voluntary wheel running levels - a behavioral readout considered a model of voluntary exercise in humans. Subsequently, we tested the syntenic region (HSA20: 51,212,545-55,212,986) in a human sample (Saint Thomas Twin Register; n=3038) and found a significant association between voluntary exercise levels (categorized into excessive and non-excessive exercise) and an intergenic SNP rs459465 (adjusted P-value of 0.001). Taking under consideration the methodological challenges embedded in this translational approach in the research of complex phenotypes, we wanted to further test the validity of this finding. Therefore, we repeated the analysis in an independent human population (ALSPAC data set; n=2557). We found a significant association of excessive exercise with two SNPs in the same genomic region (rs6022999, adjusted P-value of P=0.011 and rs6092090, adjusted P-value of 0.012). We explored the locus for possible candidate genes by means of literature search and bioinformatics analysis of gene function and of trans-regulatory elements. We propose three potential human candidate genes for voluntary physical exercise levels (MC3R, CYP24A1, and GRM8). To conclude, the identified genetic variance in the human locus 20q13.2 may affect voluntary exercise levels.

Circadian timed wakefulness at dawn opposes compensatory sleep responses after sleep deprivation in Octodon degus.

The circadian timing system in mammals is thought to promote wakefulness and oppose sleep drive that accumulates across the activity phase in diurnal and nocturnal species. Whether the circadian system actively opposes compensatory sleep responses in mammals with episodes of alertness consolidated at dawn and dusk is unknown. In the present study, an interaction between circadian timed arousal at dawn and compensatory sleep responses after sleep deprivation (SD) was examined in Octodon degus, a hystricomorph rodent with crepuscular episodes of wakefulness. Recovery sleep was compared after 6 hours and 12 hours of SD ending at either CT 21 or 12, just before the dawn, and just after the dusk crepuscular episodes of consolidated wakefulness, respectively. Total sleep time and NREM sleep after SD increased proportionally to the amount of sleep loss; however, compensatory sleep responses after SD were attenuated at CT 23, a circadian time when a crepuscular event of wakefulness occurs in this species. EEG slow-wave activity (SWA) and body temperature levels in the first two hours after 6 and 12 hours of SD ending at CT 12 were similar. However, both were significantly higher than after 12 hours of SD ending at CT 21, suggesting factors other than the amount of prior wake duration can influence SWA levels. This study provides evidence that the circadian arousal system opposes compensatory sleep responses at dawn by actively promoting wakefulness in this species.

The use of mouse models to unravel genetic architecture of physical activity: a review.

The discovery of genetic variants that underlie a complex phenotype is challenging. One possible approach to facilitate this endeavor is to identify quantitative trait loci (QTL) that contribute to the phenotype and consequently unravel the candidate genes within these loci. Each proposed candidate locus contains multiple genes and, therefore, further analysis is required to choose plausible candidate genes. One of such methods is to use comparative genomics in order to narrow down the QTL to a region containing only a few genes. We illustrate this strategy by applying it to genetic findings regarding physical activity (PA) in mice and human. Here, we show that PA is a complex phenotype with a strong biological basis and complex genetic architecture. Furthermore, we provide considerations for the translatability of this phenotype between species. Finally, we review studies which point to candidate genetic regions for PA in humans (genetic association and linkage studies) or use mouse models of PA (QTL studies) and we identify candidate genetic regions that overlap between species. On the basis of a large variety of studies in mice and human, statistical analysis reveals that the number of overlapping regions is not higher than expected on a chance level. We conclude that the discovery of new candidate genes for complex phenotypes, such as PA levels, is hampered by various factors, including genetic background differences, phenotype definition and a wide variety of methodological differences between studies.

Social isolation stress reduces hippocampal long-term potentiation: effect of animal strain and involvement of glucocorticoid receptors.

BACKGROUND: Depressive patients show cognitive impairments that are strongly associated with cortisol levels and hippocampus functioning that interact via unknown mechanisms. In addition, a relation between depression and hippocampal synaptic plasticity was described. METHODS: In the first experiment, strain-dependent effects of 72-h social isolation on long-term potentiation (LTP) in the CA1 area of the in vitro hippocampus, was determined. Extracellular field excitatory postsynaptic potentials were recorded and a brief high-frequency stimulation (100 Hz, 1s) was applied and recording resumed after the high frequency stimulation (HFS) for 30 min to determine the effect of HFS. In the second experiment we investigated the effect of 72 h of corticosterone treatment and the involvement of glucocorticoid receptors (GRs) in the effect of 72 h of social isolation on LTP in the CA1 area of hippocampus, in vitro. RESULTS: Genetic background has a major effect on the level of hippocampal LTP impairment in mice following social isolation. Data showed that the potentiation levels in socially housed (SH) A/J mice were significantly higher than the SH C57BL/6J mice (224.88 ± 16.65, 131.56 ± 6.25% of the baseline values, t(9)=2.648, p=0.026). However, both strains showed depressed induction of potentiation when reared in an isolated environment for 72 h, and no significant difference was recorded between the two (112.88 ± 16.65%, and 117.91 ± 3.23% of the baseline values, respectively, t(10)=0.618, p=0.551). Social isolation increased corticosterone levels significantly and chronic corticosterone infusion in SH phenocopied the LTP impairments observed in socially isolated mice. Infusion of the GR antagonist RU38486 rescued the LTP-impairments following social isolation. CONCLUSIONS: These findings support the notion that increased levels of stress hormone act via the GR on hippocampal functioning and that, in this way, the cognitive deficits in mood disorders may be restored.

Cross-species genetics converge to TLL2 for mouse avoidance behavior and human bipolar disorder.

Interspecies genetic analysis of neurobehavioral traits is critical for identifying neurobiological mechanisms underlying psychiatric disorders, and for developing models for translational research. Recently, after screening a chromosome substitution strain panel in an automated home cage environment, chromosomes 15 and 19 were identified in female mice for carrying genetic loci that contribute to increased avoidance behavior (sheltering preference). Furthermore, we showed that the quantitative trait locus (QTL) for baseline avoidance behavior on chromosome 15 is homologous with a human linkage region for bipolar disorder (8q24). Similarly, we now performed comparative analysis on the QTL for avoidance behavior found on chromosome 19 and correspondingly revealed an overlap of the mouse interval and human homologous region 10q23-24, which has been previously linked to bipolar disorders. By means of a comparative genetic strategy within the human homologous region, we describe an association for TLL2 with bipolar disorder using the genome-wide association study (GWAS) data set generated by the Wellcome Trust Case Control Consortium (WTCCC). On the basis of genetic homology and mood stabilizer sensitivity, our data indicate the intriguing possibility that mouse home cage avoidance behavior may translate to a common biochemical mechanisms underlying bipolar disorder susceptibility. These findings pave new roads for the identification of the molecular mechanisms and novel treatment possibilities for this psychiatric disorder, as well as for the validity of translational research of associated psychiatric endophenotypes.

NPY receptor subtype specification for behavioral adaptive strategies during limited food access.

The neuropeptide Y (NPY) system in the brain regulates a wide variety of behavioral, metabolic and hormonal homeostatic processes required for energy balance control. During times of limited food availability, NPY promotes behavioral hyperactivity necessary to explore and prepare for novel food resources. As NPY can act via 5 different receptor subtypes, we investigated the path through which NPY affects different behavioral components relevant for adaptation to such conditions. We tested NPY Y1 and Y2 receptor knockout mice and their wild-type littermate controls in a daily scheduled limited food access paradigm with unlimited access to running wheel. Here we show that NPY Y1 receptor deficient mice lack the expression of appetitive behavior and that NPY Y2 receptors control the level of hyperactive behavior under these conditions. Thus, receptor specificity determines the differential expression of NPY-mediated behavioral adaptations to overcome a negative energy status.

In search for significant cognitive features in Klinefelter syndrome through cross-species comparison of a supernumerary X chromosome.

The behavioral characterization of animals that carry genetic disorder abnormalities in a controlled genetic and environmental background may be used to identify human deficits that are significant to understand underlying neurobiological mechanisms. Here, we studied whether previously reported object recognition impairments in mice with a supernumerary X chromosome relate to specific cognitive deficits in Klinefelter syndrome (47,XXY). We aimed to optimize face validity by studying temporal object recognition in human cognitive assays. Thirty-four boys with Klinefelter syndrome (mean age 12.01) were compared with 90 age-matched normal controls, on a broad range of visual object memory tasks, including tests for pattern and temporal order discrimination. The results indicate that subjects with Klinefelter syndrome have difficulty in the processing of visual object and pattern information. Visual object patterns seem difficult to discriminate especially when temporal information needs to be processed and reproduced. On the basis of cross-species comparison, we propose that impaired temporal processing of object pattern information is an important deficit in Klinefelter syndrome. The current study shows how cross-species behavioral characterization may be used as a starting point to understand the neurobiology of syndromal phenotypic expression. The features of this study may serve as markers for interventions in Klinefelter syndrome. Similar cross-species evaluations of standard mouse behavioral paradigms in different genetic contexts may be powerful tools to optimize genotype-phenotype relationships.

Neurobiology driving hyperactivity in activity-based anorexia.

Hyperactivity in anorexia nervosa is difficult to control and negatively impacts outcome. Hyperactivity is a key driving force to starvation in an animal model named activity-based anorexia (ABA). Recent research has started unraveling what mechanisms underlie this hyperactivity. Besides a general increase in locomotor activity that may be an expression of foraging behavior and involves frontal brain regions, the increased locomotor activity expressed before food is presented (food anticipatory behavior or FAA) involves hypothalamic neural circuits. Ghrelin plays a role in FAA, whereas decreased leptin signaling is involved in both aspects of increased locomotor activity. We hypothesize that increased ghrelin and decreased leptin signaling drive the activity of dopamine neurons in the ventral tegmental area. In anorexia nervosa patients, this altered activity of the dopamine system may be involved not only in hyperactivity but also in aberrant cognitive processing related to food.