Dr Gabriella Juhasz
email: gabriella.juhasz@manchester.ac.uk

Darragh Downey

Neal S Hinvest

Emma Thomas

Diana Chase

Zoltan G Toth

Kathryn Lloyd-Williams

Krisztina Mekli

Hazel Platt

Antony Payton

Gyorgy Bagdy

Rebecca Elliott

Jf William Deakin

Ian M Anderson

Risk-taking behavior in a gambling task associated with variations in the tryptophan hydroxylase 2 gene: relevance to psychiatric disorders

Keywords: risk-taking, TPH2, haplotype analysis, depression, psychiatric disorders

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

G. Juhasz

INTRODUCTION

Decision-making depends on a complex set of processes that are orchestrated in various brain systems to find an optimal outcome [START_REF] Elliott | Role of the orbitofrontal cortex in reinforcement processing and inhibitory control: evidence from functional magnetic resonance imaging studies in healthy human subjects[END_REF][START_REF] Paulus | Decision-making dysfunctions in psychiatry--altered homeostatic processing?[END_REF]. Risktaking refers to the willingness to accept a possible negative outcome in order potentially achieve a desirable outcome, and typically involves assessing the relative probability of winning or losing against the values of the outcomes [START_REF] Anderson | The effect of acute tryptophan depletion on probabilistic choice[END_REF][START_REF] Cardinal | Neural systems implicated in delayed and probabilistic reinforcement[END_REF].

Subjects with depression or anxiety are less likely than controls to take risks to gain reward [START_REF] Elliott | Abnormal response to negative feedback in unipolar depression: evidence for a diagnosis specific impairment[END_REF][START_REF] Forbes | Reward-related decision-making in pediatric major depressive disorder: an fMRI study[END_REF][START_REF] Smoski | Decision-making and risk aversion among depressive adults[END_REF][START_REF] Paulus | Decision-making dysfunctions in psychiatry--altered homeostatic processing?[END_REF]. In contrast, pathological gamblers are more risk-seeking [START_REF] Holt | Is discounting impulsive?. Evidence from temporal and probability discounting in gambling and non-gambling college students[END_REF]. Another aspect of decisionmaking is self control, or the ability to delay gratification, which is more related to impulsiveness and is impaired in patients with substance-use disorder [START_REF] Rogers | Dissociable deficits in the decision-making cognition of chronic amphetamine abusers, opiate abusers, patients with focal damage to prefrontal cortex, and tryptophan-depleted normal volunteers: evidence for monoaminergic mechanisms[END_REF][START_REF] Bechara | Decision-making and addiction (part II): myopia for the future or hypersensitivity to reward?[END_REF][START_REF] Paulus | Decision-making dysfunctions in psychiatry--altered homeostatic processing?[END_REF].

Manipulation of serotonin (5-HT) appears to influence decision-making. Acute tryptophan depletion leads to impulsive choice and impaired decision-making related to different magnitudes of gains, in both animals and humans [START_REF] Rogers | Dissociable deficits in the decision-making cognition of chronic amphetamine abusers, opiate abusers, patients with focal damage to prefrontal cortex, and tryptophan-depleted normal volunteers: evidence for monoaminergic mechanisms[END_REF][START_REF] Mobini | Effects of central 5hydroxytryptamine depletion on sensitivity to delayed and probabilistic reinforcement[END_REF][START_REF] Rogers | Tryptophan depletion alters the decision-making of healthy volunteers through altered processing of reward cues[END_REF][START_REF] Walderhaug | Interactive effects of sex and 5-HTTLPR on mood and impulsivity during tryptophan depletion in healthy people[END_REF]. Furthermore, a recent study showed that tryptophan supplementation significantly decreased loss-aversion [START_REF] Murphy | The Role of Serotonin in Nonnormative Risky Choice: The Effects of Tryptophan Supplements on Loss-aversion in Healthy Adult Volunteers[END_REF]. While 5-HT appears to influence choice involving delayed reinforcement or different magnitudes of gains, it remains unclear whether 5-HT affects choice related to the assessment of probability [START_REF] Mobini | Effects of central 5hydroxytryptamine depletion on sensitivity to delayed and probabilistic reinforcement[END_REF][START_REF] Anderson | The effect of acute tryptophan depletion on probabilistic choice[END_REF][START_REF] Rogers | Tryptophan depletion alters the decision-making of healthy volunteers through altered processing of reward cues[END_REF][START_REF] Cardinal | Neural systems implicated in delayed and probabilistic reinforcement[END_REF], especially at a genetic level.

It has been reported that possession of the less active S allele of the 5-HT transporter 5HTTLPR polymorphism (SLC6A4 gene short/S-long/L promoter variant) increased sensitivity to the probability of winning in a "risky-choice task" [START_REF] Roiser | The effect of polymorphism at the serotonin transporter gene on decision-making, memory and executive function in ecstasy users and controls[END_REF] and promoted disadvantageous choices in the Iowa Gambling Task (IGT) probably due to lack of persistence [START_REF] Must | Major depressive disorder, serotonin transporter, and personality traits: why patients use suboptimal decision-making strategies?[END_REF][START_REF] Homberg | Serotonin transporter dosage modulates long-term decision-making in rat and human[END_REF] or to slower learning of advantageous decision-making [START_REF] Jollant | The influence of four serotonin-related genes on decision-making in suicide attempters[END_REF]. It has been also suggested that 5HTTLPR variations modulate sensitivity to punishment rather than reward, since the LL homozygous individuals are less sensitive to punishment-related information [START_REF] Blair | The role of 5-HTTLPR in choosing the lesser of two evils, the better of two goods: examining the impact of 5-HTTLPR genotype and tryptophan depletion in object choice[END_REF][START_REF] Roiser | A genetically mediated bias in decision making driven by failure of amygdala control[END_REF]. Jollant et al. showed that genetic variations in the TPH1, TPH2 (tryptophan hydroxylase 1 and 2 respectively) and MAOA (monoamine oxydase A) genes that have been associated with suicide were also associated with poorer performance on the IGT in suicide attempters [START_REF] Jollant | The influence of four serotonin-related genes on decision-making in suicide attempters[END_REF]. However, all of the above mentioned studies were carried out in small samples (less than 200 subjects) and require confirmation in larger samples.

In this study the main candidate gene was the TPH2 that codes for the majority of TPH in the human brain [START_REF] Walther | Synthesis of serotonin by a second tryptophan hydroxylase isoform[END_REF]Zill et al, 2007a). Variations in the TPH2 gene have been repeatedly implicated in major depression (MDD), and in suicidal behaviour in MDD patients (Zill et al, 2004a;Zill et al, 2004b;[START_REF] Zhang | Loss-of-function mutation in tryptophan hydroxylase-2 identified in unipolar major depression[END_REF][START_REF] Zhou | Haplotypebased linkage of tryptophan hydroxylase 2 to suicide attempt, major depression, and cerebrospinal fluid 5-hydroxyindoleacetic acid in 4 populations[END_REF]Lopez et al, 2007a;[START_REF] Haghighi | Genetic architecture of the human tryptophan hydroxylase 2 Gene: existence of neural isoforms and relevance for major depression[END_REF]. To cover this gene we used haplotype tagging SNPs similarly to these previous studies.

As the availability of synaptic 5-HT depends on other serotonergic genes we also investigated the well-known 5HTTLPR polymorphism and a functional TPH1 gene variant (A218C, rs1800532). A218C has been shown to alter TPH immunoreactivity in postmortem brain samples of both suicide victims and controls [START_REF] Ono | Tryptophan hydroxylase immunoreactivity is altered by the genetic variation in postmortem brain samples of both suicide victims and controls[END_REF] and has been associated with suicidal behaviour [START_REF] Li | Further clarification of the contribution of the tryptophan hydroxylase (TPH) gene to suicidal behavior using systematic allelic and genotypic meta-analyses[END_REF]. It has been demonstrated that TPH1 is expressed in several human brain areas although at lower levels than TPH2 in the raphe (Zill et al, 2007a). Finally, we genotyped for a functional variant in the 5-HT1A receptor gene (C(-1019)G, rs6295) that modulates the expression of 5-HT1A autoreceptors in raphe cells and that controls 5-HT neuronal firing and this influence synaptic 5-HT release [START_REF] Lemonde | Impaired repression at a 5-hydroxytryptamine 1A receptor gene polymorphism associated with major depression and suicide[END_REF]. This functional polymorphism has previously been associated with depression, suicide and anxiety related traits [START_REF] Lemonde | Impaired repression at a 5-hydroxytryptamine 1A receptor gene polymorphism associated with major depression and suicide[END_REF][START_REF] Strobel | Allelic variation in 5-HT1A receptor expression is associated with anxiety-and depressionrelated personality traits[END_REF].

In order to investigate risky decision-making behavior in a large population we developed a pencil-and-paper probabilistic gambling task administered remotely with questionnaires assessing personality data and current mood and anxiety symptoms. Based on previous literature we hypothesized that subjects with a self-reported history of depression or with a higher neuroticism, depression and/or anxiety scores would make safer (less risky) choices. Furthermore, we hypothesized that variants in the serotonergic genes and especially in the TPH2 gene that regulates the availability of synaptic 5-HT would influence risky decision-making.

G. Juhasz

6

MATERIALS AND METHODS

Participants

This study was part of the EU funded NewMood (New Molecules for Mood Disorders) research program. We recruited participants aged 18-60 years from Greater Manchester, UK through general practices, and a web-site (http://www.newmood.co.uk); details of our recruitment strategy and responses were published previously [START_REF] Juhasz | CNR1 Gene is Associated with High Neuroticism and Low Agreeableness and Interacts with Recent Negative Life Events to Predict Current Depressive Symptoms[END_REF]. The included participants were of Caucasian European origin and could have a self-reported history of depression or anxiety disorders; we excluded those reporting manic or hypomanic episodes, psychotic symptoms or obsessive-compulsive disorder (all information based on background self-report questionnaire data). Thus the population sample in this study consisted of 1188 participants who fulfilled the inclusion criteria, sent back the questionnaire with a completed gambling task and provided DNA. Population details are shown in Table 1.

The study was approved by the local Ethics Committees and was carried out in accordance with the Declaration of Helsinki.

Phenotype assessments

The NewMood booklet consisted of brief or adapted versions of standard questionnaires that have been validated at a second level of the study in a subset of subjects (n=142) by full versions of the questionnaires and face-to-face interviews. The background questionnaire was adapted from a structured self-rating questionnaire [START_REF] Hunt | The prevalence and associated features of chronic widespread pain in the community using the 'Manchester' definition of chronic widespread pain[END_REF] that consists of 22 items and collects information about medical history including personal psychiatric history (particularly depression), family psychiatric history, and socioeconomic background.

Impulsivity was measured by the Impulsiveness, Venturesome and Empathy Questionnaire (IVE) Impulsiveness subscale [START_REF] Eysenck | Impulsiveness and venturesomeness: their position in a dimensional system of personality description[END_REF]. For the analysis, a continuous weighted dimension score (sum of item scores divided by the number of items completed) was calculated. The IVE Impulsivity score showed significant correlation with the NEO PI-R [START_REF] Ptjr | Revised NEO Personality Inventory (NEO-PI-R) and NEO Five-Factor Inventory (NEO-FFI) professional manual[END_REF]) Impulsiveness (N5) facet score (Pearson correlation: Neuroticism R=0.47, p<0.001, n=142) in our validation sample. To assess personality we used the Big Five Inventory (BFI-44) [START_REF] John | The Big Five Inventory-Versions 4a and 54[END_REF] and a continuous weighted dimension score was calculated for Neuroticism. The 53-item Brief Symptom Inventory (BSI) [START_REF] Derogatis | BSI: Brief Symptom Inventory: Administration, Scoring, and Procedures Manual[END_REF] was used to measure anxiety symptoms (using the anxiety subscale), and depressive symptoms (using the depression subscale plus additional items); a continuous weighted dimension score was calculated for Depression and Anxiety. Validation data of neuroticism and symptom scores were published previously [START_REF] Juhasz | CNR1 Gene is Associated with High Neuroticism and Low Agreeableness and Interacts with Recent Negative Life Events to Predict Current Depressive Symptoms[END_REF].

The Recent Negative Life Events questionnaire was based on the validated List of Life Threatening Experiences (LTE) [START_REF] Brugha | The List of Threatening Experiences: a subset of 12 life event categories with considerable long-term contextual threat[END_REF][START_REF] Rijsdijk | Life events and depression in a community sample of siblings[END_REF] and the sum of life event items was used in the analysis. Questions based on the Childhood Trauma Questionnaire (CTQ) [START_REF] Bernstein | Initial reliability and validity of a new retrospective measure of child abuse and neglect[END_REF] were used to determine Childhood Adversity related to emotional and physical abuse, and emotional and physical neglect. An additional question asked about parental loss during childhood. The sum of item scores was used in the analysis. Summary scores from the full version of CTQ significantly correlated with scores derived from our short questionnaire (Pearson correlation: R=0.75, p<0.001, n=142).

Gambling task

This task was derived from that of Rachlin et al [START_REF] Juhasz Rachlin | Cognition and behavior in studies of choice[END_REF]. Subjects were presented with a picture of two "wheel of fortune"-like spinners each with a pointer at the top. Each spinner was divided into a white and a grey segment and the subject was told to imagine they would 'win' if the spinner stopped with the white segment under the pointer, but not with the grey (Figure 1). Subjects were instructed to indicate which spinner they would choose to maximize their winnings. Dial 'A' provided a smaller (£1) but more likely 'win', while Dial 'B' gave a 'win' twice the amount but with a lower probability that was systematically varied. As our reward task was imaginary, subjects did not gain real monetary reward.

The probability of winning on 'A' (pA) was set to P=0.95, P=0.5, P=0.33 and P=0.25 while probability of winning on 'B' was varied from P=0.80 to P=0.025 but was always lower than pA. As the probability of winning on 'B' increased, an indifference point is reached where the participant values both option equally, and may select 'B' as it offers the potential for a larger 'win'. Odds against winning (Ө) were calculated as [1/p]-1 giving four ӨA values (0.05, 1, 2, 3). The odds against winning on 'B' (ӨB) at which the participant switched from dial 'A' to dial 'B' (the indifference point) was calculated for each value of 'A' by linear interpolation. To assess an individual's overall tendency to take a safer vs. more risky choice we plotted ӨB against ӨA and calculated a global area under the curve measure (AUCӨ) using the trapezoid method requiring all four indifference points; a smaller value indicating safer choice.

Using AUCӨ gives weight to choices where wins are unlikely and is sensitive to how much the individual is willing to take a risk for a greater reward under conditions where both outcomes are uncertain [START_REF] Ho | Theory and method in the quantitative analysis of "impulsive choice" behaviour: implications for psychopharmacology[END_REF].

Given the study's constraints, we were not able to determine directly whether participants understood the task and so we included two trials where one of the choices was so disadvantageous that its selection was irrational and if chosen the individual was excluded.

We also required choices on 'B' to follow the same rank order as their corresponding value on 'A' (allowing one occurrence of the same value for contiguous choices on 'B'). This excluded participants who, for example, chose a smaller indifference probability on 'B' for p=0.33 than for p=0.25 on 'A'. Using these rules 87.1% (n=1035) of participants were included in the final analysis.

From these subjects, 72 completed the computer version of a similar decision-making task and 69 provided calculable data. During this task the participants were shown two 'wheel of fortune' spinners -A and B -that had a green (win) and a red (lose) segment. Alternative A had a reward of 10p; alternative B had a reward of 20p but a lower probability chance of winning compared to A (2 blocks of 41 trials; ӨA: Ө =0.00, Ө=0.33, Ө=1 and Ө=3; ӨB: from Ө=0.00 to Ө=∞). Participants pressed A or B on the keyboard depending on which alternative they preferred. The outcome (which had been decided in a pseudorandom manner) and was then shown to the subject. The reward was imaginary again, so the subjects did not get real monetary reward. A global area under the curve measure (AUCӨ) was calculated from these data similarly to the paper version of the task. Results from the two tasks showed significant correlation (Pearson R=0.30, p=0.012) in this population.

Genotyping

Buccal mucosa cells were collected from the participants and genomic DNA was extracted according to a previously described method [START_REF] Freeman | DNA from buccal swabs recruited by mail: evaluation of storage effects on long-term stability and G. Juhasz suitability for multiplex polymerase chain reaction genotyping[END_REF].

HaploView software (www.broad.mit.edu/personal/jcbarret/haploview/) was employed to identify haplotype tagging SNPs (htSNPs) using the confidence interval method [START_REF] Barrett | Haploview: analysis and visualization of LD and haplotype maps[END_REF][START_REF] Gabriel | The structure of haplotype blocks in the human genome[END_REF]. The tagging was based on the CEPH (Utah residents with ancestry from northern and western Europe) data that were available at the International HapMap Project Phase I. June 2005 release (www.hapmap.org), thus SNPs (functional or tagging) that were identified later were not included in this study. We also genotyped a further three functionally relevant serotonergic polymorphisms: 5HTTLPR, rs6295, and rs1800532.

The chosen SNPs were genotyped using the Sequenom® MassARRAY technology (Sequenom®, San Diego, http://www.sequenom.com) using 25ng of DNA. Determination of the 5HTTLPR genotype was performed in a Thermo-Fast® 384 PCR plate (ABgene Ltd, UK) on a PTC-220 Dyad TM thermocycler (Bio-Rad, Hercules, CA). Polymerase chain reaction products were analyzed using an ABI3100 Genetic Analyzer and GeneScan analysis software (Applied Biosystems, Netherlands). Genotyping was performed blinded with regard to phenotype. All laboratory work was performed under the ISO 9001:2000 quality management requirements. The genotyping methods in more details can be seen in Supplementary Document 1.

Statistical Analysis

Statistical analyses were performed with SPSS for Windows Statistical Analysis Software, Version 15.0. A p value of < 0.05 was adopted for all statistical testing and all reported p values were two-tailed.

We used the HaploView program to explore the haplotype structure of TPH2 gene [START_REF] Gabriel | The structure of haplotype blocks in the human genome[END_REF][START_REF] Barrett | Haploview: analysis and visualization of LD and haplotype maps[END_REF] Haplotype analyses were performed with HelixTree TM 6.4.1 (Golden Helix, USA) using haplotype trend regression. Only haplotypes with a frequency greater than 5% were G. Juhasz 10 used in the linear regression analysis, similar to the method described previously [START_REF] Juhasz | CNR1 Gene is Associated with High Neuroticism and Low Agreeableness and Interacts with Recent Negative Life Events to Predict Current Depressive Symptoms[END_REF]. In all cases, data were adjusted for age and sex. To reduce the influence of multiple testing we used a permutation test, randomly grouping the sample 10000 times.

For both single marker association and haplotypic association analyses we used false discovery rate calculation at a level of 5% (FDR; Qvalue:

http://genomics.princeton.edu/storeylab/qvalue/) to adjust p values according to the number of hypothesis tested [START_REF] Storey | Statistical significance for genomewide studies[END_REF]. We report q value which is a measure of significance of each test of many tests performed simultaneously.

RESULTS

Decision-making task

Women were less likely to make risky decisions than

Genotyping results

HaploView analysis of the TPH2 gene -based on the HapMap Project Phase I. (June 2005) release CEPH population data -resulted in 7 haplotype tagging SNPs that were genotyped in our study. Details about the TPH2 htSNPs and the genotyped 3 functional serotonergic polymorphisms can be seen in Table 2. Although no genomic control by random marker genotyping was performed in the study population we attempted to reduce ethnic variation and stratification effects by including only independent Caucasian subjects of European origin in the analysis. Indeed, all of the genotyped polymorphisms were in Hardy-Weinberg equilibrium both in the total population and separately for those who reported depression and who did not (data not shown). TPH2 linkage disequilibrium (LD) block structure is similar in our population and in the CEPH population (data from the HapMap Project Phase II.

(November 2008) release; see Figure 2). Although in this study we did not genotype functional SNPs in the TPH2 gene, we do show the LD data between our htSNPs and rs4570625 (-703G/A) (Chen et al, 2008) and rs7305115 [START_REF] Lim | Tryptophan hydroxylase 2 (TPH2) haplotypes predict levels of TPH2 mRNA expression in human pons[END_REF] in the CEPH population in order to interpret our results. There are no available data in the CEPH population about other functional SNPs (rs11178997: -473T/A; rs11178998: 90A/G) (Chen et al, 2008;[START_REF] Lin | Association of functional polymorphisms of the human tryptophan hydroxylase 2 gene with risk for bipolar disorder in Han Chinese[END_REF][START_REF] Scheuch | Characterization of a functional promoter polymorphism of the human tryptophan hydroxylase 2 gene in serotonergic raphe neurons[END_REF].

Single marker associations

We found significant association of the AUCӨ and TPH2 htSNPs and after FDR correction the association remained significant for rs6582078 and rs1352250 (Table 3). The minor alleles were positively associated with increased risk-taking in all htSNPs. There was no significant marker-specific association between 5HTTLPR, rs6295 or rs1800532 and risktaking. None of the SNPs showed association with symptom or personality scores after correction for multiple testing (Table S1).

Because AUCӨ showed significant difference between males and females we tested the interaction of SNPs and sex. None of the SNPs showed significant interaction with sex on risk-taking AUCӨ after correction for multiple testing (Table S2a). Furthermore, TPH2 htSNPs did not show significant interaction (epistasis) with 5HTTLPR, rs6295 or rs1800532 on AUCӨ (Table S2b).

Assuming a continuous trait such as AUCӨ data, using an independent-individual study design and assuming an additive linear model relating the phenotype to genotype, we had a 90-93% power in our population (N=1035 and N=1188) to detect a polymorphism (with a minor allele frequency≥10%) that could explain 1% of the variance of the trait at the 5% twotailed significance level. To detect gene x sex interaction in the same model as above and assuming that sex explains 5% variance we had a 91-95% power in our population.

Furthermore, for epistasis we had a 90-94% power in our population if the other gene also explains 1% variance and the gene x gene interaction explains further 1% variance.

Using the computer task data single marker associations showed similar results with higher effect sizes to the pencil-and paper tasks results (see Table 4) .

TPH2 haplotype association with AUCӨ

The overall genetic effect of TPH2 haplotypes for the AUCӨ data was significant and remained significant after permutation tests and correction for multiple testing (Table 5). The TPH2 haplotypes explained 1.9% variance in risk-taking. The haplotypic association did not change after adding BSI Depression and Anxiety scores (permutated FDR corrected q= 0.018) or BFI neuroticism scores (permutated FDR corrected p= 0.018) as covariates in the analysis. These results suggest that there is a genetic effect on this measure of decision-making, which is independent of current mood and anxiety state or neuroticism. Indeed, the TPH2 gene showed no significant haplotypic association with BSI depression (permutated FDR corrected q=0.337), BSI anxiety (permutated FDR corrected q=0.172), BFI neuroticism (permutated FDR corrected q=0.314) or IVE impulsivity (permutated FDR corrected q=0.174) scores.

The most common haplotype, T,C,T,C,G,T,A was found in 49% of study participants.

Beta values suggest that carriers were relatively risk avoidant and made significantly less risky decisions than the other haplotype carriers (permutated FDR corrected q=0.022). This effect was also significant for the risk taking AUCӨ calculated from the computer task (beta=-2.619, SE=1.271, t=-2.06, permutated p=0.044, explained variance 5.9%, n=69, frequency of the T,C,T,C,G,T,A haplotype=45%).

DISCUSSION

Using haplotypic association we found significant differences in risk-taking behavior on a performance gambling task between the most prevalent haplotype and the other haplotypes of the TPH2 gene. Subjects carrying the most prevalent T,C,T,C,G,T,A haplotype had lower AUCӨ parameters suggesting that they are less likely to make risky decisions. Thus, our finding implies involvement of the serotonergic system, namely the TPH2 gene, in decisionmaking through altering willingness to take risks for reward. In addition, we tested whether differences in risk-taking behavior measured by our task were influenced by other serotonergic genes. We genotyped a further three serotonergic functional polymorphisms (5HTTLPR in serotonin transporter, rs6295 in 5HT1A receptor and rs1800532 in TPH1 genes) but could not demonstrate significant main effects of these polymorphisms or interaction with the TPH2 htSNPs on risk-taking. However, we could not exclude that these genes modulate other aspects of decision-making that were not measured by our task, such as punishment or feedback related processing, as previous studies reported [START_REF] Jollant | The influence of four serotonin-related genes on decision-making in suicide attempters[END_REF][START_REF] Must | Major depressive disorder, serotonin transporter, and personality traits: why patients use suboptimal decision-making strategies?[END_REF][START_REF] Blair | The role of 5-HTTLPR in choosing the lesser of two evils, the better of two goods: examining the impact of 5-HTTLPR genotype and tryptophan depletion in object choice[END_REF][START_REF] Homberg | Serotonin transporter dosage modulates long-term decision-making in rat and human[END_REF].

Accumulating evidence suggest that the more common allele/haplotype variants in the TPH2 gene are associated with lower cerebrospinal fluid 5-hydroxyindoleacetic acid concentrations [START_REF] Zhou | Haplotypebased linkage of tryptophan hydroxylase 2 to suicide attempt, major depression, and cerebrospinal fluid 5-hydroxyindoleacetic acid in 4 populations[END_REF] and with decreased TPH2 mRNA expression (Chen et al, 2008;Chen and Miller, 2008;[START_REF] Haghighi | Genetic architecture of the human tryptophan hydroxylase 2 Gene: existence of neural isoforms and relevance for major depression[END_REF][START_REF] Lim | Tryptophan hydroxylase 2 (TPH2) haplotypes predict levels of TPH2 mRNA expression in human pons[END_REF]. Thus, a considerable proportion of the population, who carry the wild-type (most ancient) variation of the TPH2 gene, express TPH2 at low level that results in decreased serotonin availability at the raphe nucleus and its projections and also show risk aversion based on our results. This situation is somewhat parallel to the acute tryptophan depletion (ATD), which has been shown to reduce 5-HT levels to a functionally significant degree. However, human studies have not demonstrated a consistent effect of reducing 5-HT function using dietary ATD on probabilistic choice behavior: Rogers et al reported that following ATD subjects chose the most likely option less often [START_REF] Rogers | Dissociable deficits in the decision-making cognition of chronic amphetamine abusers, opiate abusers, patients with focal damage to prefrontal cortex, and tryptophan-depleted normal volunteers: evidence for monoaminergic mechanisms[END_REF], while Talbot demonstrated the opposite [START_REF] Talbot | Rapid tryptophan depletion improves decision-making cognition in healthy humans without affecting reversal learning or set shifting[END_REF] and our group could not find any significant difference [START_REF] Anderson | The effect of acute tryptophan depletion on probabilistic choice[END_REF]. The most plausible explanation for these discrepancies is that these studies used different tasks to measure risk-taking and probably were not adequately powered to detect the effect of ATD on probabilistic choice. Indeed, based on our results, variability in serotonin synthesis caused by the TPH2 gene only explained 1.9% (paper-and-pencil task) -5.9% (computer task) of the variance in risk-taking. Another possible explanation is that the genetic make-up of the participants interfered with the effect of the ATD, as has been suggested in relation of the 5HTTLPR variant [START_REF] Blair | The role of 5-HTTLPR in choosing the lesser of two evils, the better of two goods: examining the impact of 5-HTTLPR genotype and tryptophan depletion in object choice[END_REF].

Our results suggest that there is a biological distinction to be made between impulsive and safe/risky decision-making as measured with our task. Although impulsivity has usually been associated with an increased tendency to take risks, such as in DSM-IV-defined impulse control disorders (American Psychiatric Association, 2000), it only appears to predict some risky behaviors while others are more linked to neuroticism and extraversion [START_REF] Cooper | A motivational perspective on risky behaviors: the role of personality and affect regulatory processes[END_REF]. Impulsivity may be more related to inability to delay gratification and [START_REF] Crean | Reward discounting as a measure of impulsive behavior in a psychiatric outpatient population[END_REF] demonstrated that delay discounting has a closer relationship to self reported impulsivity measurements than probability discounting. Low 5-HT function has been associated with increased impulsivity (e.g. [START_REF] Dolan | Relationship between 5-HT function and impulsivity and aggression in male offenders with personality disorders[END_REF] in contrast to our finding that the common TPH2 variant, likely to be associated with reduced 5-HT function, is associated with risk aversion rather than risk-taking when faced with a probabilistic choice. This highlights the complex role of 5-HT in behavior and mood and the need to carefully specify neuropsychological function when investigating the neurobiology of behavior.

Our task has face validity when considered in relation to mood in our subjects.

Depression is characterized by reduced motivation to obtain reward and reduced enjoyment of it [START_REF] Elliott | Abnormal response to negative feedback in unipolar depression: evidence for a diagnosis specific impairment[END_REF][START_REF] Forbes | Reward-related decision-making in pediatric major depressive disorder: an fMRI study[END_REF] while anxiety is associated with intolerance of uncertainty [START_REF] Paulus | Increased activation in the right insula during risk-taking decision making is related to harm avoidance and neuroticism[END_REF][START_REF] Paulus | Decision-making dysfunctions in psychiatry--altered homeostatic processing?[END_REF]. Hence subjects with depression and anxiety, which are frequently co-morbid disorders, would be expected to be less motivated, indeed averse, to take more risks for a greater uncertain reward compared to controls [START_REF] Smoski | Decision-making and risk aversion among depressive adults[END_REF]. This is consistent with the findings in our sample in which higher depression, anxiety and neuroticism ratings were associated with a lower tendency to take risks (AUCӨ) and explained 1.6%-2.8% variance in this phenotype. However, our study also demonstrated that the effect of TPH2 on risk-taking is independent of depression and anxiety symptom scores and also independent of neuroticism, thus indicating a separate mode of action. Furthermore, we could not demonstrate any association between haplotype variants of the TPH2 gene and BSI depression, BSI anxiety, BFI neuroticism or IVE impulsiveness scores. This is in agreement with recent studies which failed to find any association between the TPH2 gene and depression/suicidal behavior [START_REF] Mann | No association between a TPH2 promoter polymorphism and mood disorders or monoamine turnover[END_REF][START_REF] Must | Major depressive disorder, serotonin transporter, and personality traits: why patients use suboptimal decision-making strategies?[END_REF]Zill et al, 2007b;[START_REF] De Luca | The interaction between TPH2 promoter haplotypes and clinical-demographic risk factors in suicide victims with major psychoses[END_REF]Lopez et al, 2007b), but in contrast to earlier findings reporting such an involvement (Zill et al, 2004a;Zill et al, 2004b;[START_REF] Zhou | Haplotypebased linkage of tryptophan hydroxylase 2 to suicide attempt, major depression, and cerebrospinal fluid 5-hydroxyindoleacetic acid in 4 populations[END_REF][START_REF] Van Den Bogaert | Association of brain-specific tryptophan hydroxylase, TPH2, with unipolar and bipolar disorder in a Northern Swedish, isolated population[END_REF][START_REF] Haghighi | Genetic architecture of the human tryptophan hydroxylase 2 Gene: existence of neural isoforms and relevance for major depression[END_REF]Lopez et al, 2007a;[START_REF] Ke | Effect of SNP at position 40237 in exon 7 of the TPH2 gene on susceptibility to suicide[END_REF]. Therefore the interpretation we favor is that the variations in the TPH2 gene might be linked to an endophenotype influencing risktaking behavior that could be an additive or in some cases a vulnerability factor, for psychiatric disorders, for example affective disorders or disturbed mood in impulse control or substance misuse disorders. As previous studies have investigated severe clinical populations (hospitalized major depressive patients, completed suicides with major depression) it is likely they represented extreme cases in which TPH2 haplotypes may have contributed to the phenotypic picture through altered risk-taking behavior, potentially leading to patient selection bias and an apparent association between depression/suicide and TPH2 haplotypes. A modulatory rather than causative role of serotonin and TPH2 in psychiatric disorders was suggested by TPH2 and TPH1/TPH2 double knock-out (KO) animal data.

TPH2 KO animals did not show apparent neuronal developmental abnormalities [START_REF] Gutknecht | Deficiency of brain 5-HT synthesis but serotonergic neuron formation in Tph2 knockout mice[END_REF] and even double KO-s showed only mildly increased anxiety-like behavior [START_REF] Savelieva | Genetic disruption of both tryptophan hydroxylase genes dramatically reduces serotonin and affects behavior in models sensitive to antidepressants[END_REF].

The main strength of our study is that this is the first attempt to investigate the role of serotonergic genes in decision-making behavior in a large sample using a performance measure plus personality and symptom questionnaires. Furthermore, we were able to validate the questionnaire findings in a subgroup using a more extensive assessment including questionnaire and observer ratings and a computerized task. We have also recently reported that there is no difference in risky choice behavior between receiving real and hypothetical rewards when using this computerized task [START_REF] Hinvest | The effects of real vs. hypothetical reward on delay and probability discounting[END_REF]. The findings in the subgroup largely replicated the findings in the main sample. The computerized task showed only a modest, although significant, correlation with the pencil-and-paper task which is likely to be explained by methodological differences (amount to be won and different probabilities)

and sampling error as each indifference point was only assessed on one occasion in the pencil-and-paper task but on two occasions in the computerized version. Differences in genetic effect sizes (small effect sizes for the paper-and-pencil task and medium-large effect sizes for the computerized task, see Table 3 and4) probably are related to these methodological differences as the computerized task is likely to be a more sensitive tool to measure decision making differences.

Our TPH2 finding has limitations as we did not genotype recently identified functional variants in this gene. Therefore we compared the linkage disequilibrium pattern in our population and in the CEPH population, using our haplotype tagging SNPs (based on the International HapMap Project Phase I. June 2005 release) and recently genotyped functional SNPs (CEPH population, HapMap Project Phase II. November 2008 release), to interpret our results (Figure 2). We can conclude that our study satisfactorily captured the possible influence of functional variants that resides in the region around intron 5 to intron 8 and previously have been implicated in regulation of splicing, and therefore might influence the level of the active enzyme [START_REF] Haghighi | Genetic architecture of the human tryptophan hydroxylase 2 Gene: existence of neural isoforms and relevance for major depression[END_REF][START_REF] Lim | Tryptophan hydroxylase 2 (TPH2) haplotypes predict levels of TPH2 mRNA expression in human pons[END_REF]. Nonetheless, we have limited information about the promoter region of the TPH2 gene, which exhibits only moderate LD with our two most significant SNPs (rs6582078 and rs1352250) (Figure 2b) (Chen et al, 2008;[START_REF] Lin | Association of functional polymorphisms of the human tryptophan hydroxylase 2 gene with risk for bipolar disorder in Han Chinese[END_REF][START_REF] Scheuch | Characterization of a functional promoter polymorphism of the human tryptophan hydroxylase 2 gene in serotonergic raphe neurons[END_REF]. Associations have been demonstrated with the TPH2 promoter region and traits related to emotional dysregulation, such as harm avoidance [START_REF] Gutknecht | Tryptophan hydroxylase-2 gene variation influences personality traits and disorders related to emotional dysregulation[END_REF][START_REF] Reuter | Association between a polymorphism in the promoter region of the TPH2 gene and the personality trait of harm avoidance[END_REF][START_REF] Strobel | Genetic Variation of Serotonin Function and Cognitive Control[END_REF] and amygdala activation during emotional processing [START_REF] Brown | A regulatory variant of the human tryptophan hydroxylase-2 gene biases amygdala reactivity[END_REF][START_REF] Canli | Amygdala responsiveness is modulated by tryptophan hydroxylase-2 gene variation[END_REF]. However, we did not find any association between haplotype variants of the TPH2 gene and depression, anxiety, neuroticism or impulsiveness scores that might be influenced by functional variations in the TPH2 promoter region. Using denser htSNP cover on the full length of the TPH2 gene and including independent replication populations into the study would help us to answer these questions and support our new findings. The lack of independent replication population in the present study is a major limitation and warrants further research in this field.

We genotyped functional polymorphisms in other serotonergic genes but could not replicate previous positive findings [START_REF] Ono | Tryptophan hydroxylase immunoreactivity is altered by the genetic variation in postmortem brain samples of both suicide victims and controls[END_REF][START_REF] Lesch | Association of anxiety-related traits with a polymorphism in the serotonin transporter gene regulatory region[END_REF][START_REF] Lemonde | Impaired repression at a 5-hydroxytryptamine 1A receptor gene polymorphism associated with major depression and suicide[END_REF] despite our study being sufficiently powered to identify genetic effect that explain 1% variance in personality and symptom scores [START_REF] Juhasz | CNR1 Gene is Associated with High Neuroticism and Low Agreeableness and Interacts with Recent Negative Life Events to Predict Current Depressive Symptoms[END_REF]. Our findings are however consistent with the recent scientific literature as a large meta-analysis showed that several studies have failed to show association between anxiety-related traits and 5HTTLPR, except neuroticism measured by NEO [START_REF] Munafo | 5-HTTLPR genotype and anxiety-related personality traits: a meta-analysis and new data[END_REF]: it has been argued that genetic effects on neuroticism might be complex and smaller than 1% per genetic variants [START_REF] Shifman | A whole genome association study of neuroticism using DNA pooling[END_REF].

In conclusion, our study is the first to support a role for the TPH2 gene as a trait factor in determining risky choice behavior as tested in a probabilistic choice task. We also demonstrated that risk-taking behavior is modified by state dependent elements, such depression and anxiety, independently of the TPH2 gene effect. Thus, this biological marker may contribute to the expression of psychiatric phenotypes and possibly to a vulnerability to develop specific disorders. Our results support the importance of identifying quantitative traits, biological markers or endophenotypes that might be more informative for genetic studies than heterogenic, complex disorders such as depression or complex traits such as impulsivity. (1) Genotyping success rate was above 93% for all SNPs (5) HWE: Hardy-Weinberg Equilibrium (6) No CEPH data, based on (Lesch et al, 1996) (7) No CEPH data, based on [START_REF] Strobel | Allelic variation in 5-HT1A receptor expression is associated with anxiety-and depressionrelated personality traits[END_REF]

( 2 )

 2 Position according to National Center for Biotechnology Information (NCBI, March 2007) (3) CEPH: collection consists of 30 mother-father-child trios with northern and western Europe ancestry, data based on the HapMap Project Phase II. (November 2008) release (4) MAF: minor allele frequency

Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 .

 2 Figure 2. LD (linkage disequilibrium) R square data of the TPH2 gene in the CEPH population (HapMap Project Phase II. November 2008 release) and in our study. Blocks of LD have been identified based on the method published by Gabriel et al. (Gabriel et al, 2002) using Haploview program.

  ; Quanto 1.2 version (http://hydra.usc.edu/gxe) to

	calculate	the	power	of	the	recruited	populations;	and	PLINK	v1.06
	(http://pngu.mgh.harvard.edu/purcell/plink/) for testing association of different genetic models
	(dominant, recessive and additive; linear regression model co-variation with age and sex),
	interactions between SNPs (epistasis) and with sex, and for calculating Hardy-Weinberg
	analysis.

Table 1 .

 1 Population details for those who fulfilled the inclusion criteria for the study (n=1188).

	BFI: Big Five Inventory; BSI: Brief Symptom Inventory; IVE: Impulsiveness, Venturesome and
	Empathy Questionnaire
	Demographics
	Sex
	female	816 (69%)
	male	372 (31%)
	Age (mean ± SEM)	33.79 ± 0.29
	Education (A levels or higher)	771 (65%)
	Financial situation:
	Living comfortably	672 (57%)
	Just getting by	376 (32%)
	Finding it difficult to make ends meet	140 (11%)
	Personal psychiatric history
	Reported depression	630 (53%)
	Single episode	171 (14%)
	Recurrent episodes	459 (39%)
	Reported suicide attempt	181 (15%)
	Reported anxiety	338 (28%)
	Reported substance use disorder	80 (7%)
	Family psychiatric history
	Reported depression in immediate blood
		421 (35%)
	relatives
	Personality scores
	BFI Neuroticism (mean ± SEM; range 1-5)	3.30 ± 0.03
	IVE Impulsiveness (mean ± SEM; range 0-1)	0.36 ± 0.01
	Symptom scores
	BSI Depression (mean ± SEM; range 0-4)	0.98 ± 0.03

Table 2 .

 2 Summary of the genotyped SNPs

	SNP				Allele	Manchester	CEPH (3)
	no	DbSNP (1) no Position (2) Location	(minor/major)	MAF (4)	HWE (5) p	MAF (4)
	TPH2 (Chr12q21.1)
	1	rs1843809	70634965	intron5	G/T	16%	0.118	12%
	2	rs1386493	70641446	intron5	T/C	18%	0.420	15%
	3	rs6582078	70661158	intron7	G/T	43%	0.305	34%
	4	rs10506645	70671767	intron7	T/C	24%	0.125	22%
	5	rs1352250	70684051	intron8	A/G	43%	0.856	38%
	6	rs1487275	70696559	intron8	G/T	27%	0.759	25%
	7	rs1386485	70698634	intron8	C/A	34%	0.690	30%
	SLC6A4 (Chr17q11.1-q12)
	8	5HTTLPR	25588244-	promoter	S/L	44%	0.676	43% (6)
			25588593
	HTR1A (Chr5q11.2-q13)
	9	rs6295	63294321 promoter	G/C	48%	0.125	54% (7)
	TPH1 (Chr11p15.3-p14)
	11	rs1800532	18004392	intron5	A/C	40%	0.195	40%

Table 3 .

 3 Single marker association data for risk-taking (AUCӨ) using the paper-and-pencil task (n=1035). In all calculations, age and sex were covariates. t: t-test statistics, Cohen's d: standardized effect sizes, p: uncorrected significance value , FDR q: false discovery rate

		significance value.
		Bold: significance value <0.05
	No	SNP	A1	test	beta	SE	95% CI	95% CI	t	Cohen's	p	FDR q
							lower	upper		d
	1	rs1843809	G	ADD	0.883	0.411	0.077	1.690	2.148	0.14	0.032	0.239
			G DOM 0.987	0.454	0.097	1.877	2.173	0.14	0.030	0.239
			G	REC	1.004	1.520	-1.974	3.982	0.661	0.04	0.509	0.839
	2	rs1386493	T	ADD	0.850	0.388	0.090	1.610	2.193	0.14	0.029	0.239
			T	DOM 0.867	0.440	0.004	1.729	1.970	0.12	0.049	0.318
			T	REC	1.911	1.277	-0.592	4.413	1.496	0.09	0.135	0.589
	3	rs6582078	G	ADD	1.173	0.300	0.584	1.761	3.904	0.25	0.0001	0.005
			G DOM 1.272	0.445	0.399	2.145	2.855	0.18	0.004	0.119
			G	REC	1.926	0.543	0.861	2.991	3.545	0.22	0.0004	0.014
	4	rs10506645	T	ADD	0.747	0.352	0.056	1.438	2.119	0.13	0.034	0.239
			T	DOM 0.618	0.422	-0.209	1.446	1.465	0.09	0.143	0.589
			T	REC	2.267	0.946	0.414	4.120	2.398	0.15	0.017	0.239
	5	rs1352250	A	ADD	1.223	0.300	0.636	1.810	4.082	0.26	0.00005	0.005
			A DOM 1.243	0.446	0.370	2.117	2.789	0.18	0.005	0.122
			A	REC	2.139	0.541	1.079	3.200	3.954	0.25	0.00008	0.005
	6	rs1487275	G	ADD	0.804	0.342	0.134	1.474	2.352	0.15	0.019	0.239
			G DOM 0.947	0.426	0.112	1.781	2.224	0.14	0.026	0.239
			G	REC	1.169	0.844	-0.484	2.822	1.386	0.09	0.166	0.627
	7	rs1386485	C	ADD	0.837	0.316	0.217	1.456	2.648	0.17	0.008	0.160
			C DOM 0.904	0.427	0.068	1.741	2.118	0.14	0.034	0.239
			C	REC	1.496	0.663	0.196	2.796	2.255	0.14	0.024	0.239

Table 4 .

 4 Single marker association data for risk-taking (AUCӨ) using the computer task (n=69). In all calculations, age and sex were covariates. t: t-test statistics, Cohen's d: standardized effect sizes, p: uncorrected significance value. Bold: p<0.05.

	No	SNP	A1	test	beta	SE	95% CI	95% CI	t	Cohen's	p
							lower	upper		d
	1 rs1843809	G	ADD	1.297	0.801	-0.273	2.867	1.619	0.41	0.111
			G DOM	1.341	0.863	-0.350	3.032	1.555	0.39	0.125
			G	REC	2.372	3.395	-4.283	9.027	0.699	0.18	0.488
	2 rs1386493	T	ADD	1.138	0.705	-0.243	2.520	1.616	0.41	0.111
			T DOM	1.033	0.847	-0.627	2.693	1.220	0.31	0.227
			T	REC	3.162	1.956	-0.672	6.995	1.617	0.41	0.111
	3 rs6582078	G	ADD	1.045	0.700	-0.327	2.417	1.493	0.37	0.141
			G DOM	2.012	1.111	-0.166	4.191	1.811	0.45	0.075
			G	REC	0.649	1.123	-1.553	2.850	0.577	0.14	0.566
	4 rs10506645 T	ADD -0.107 0.762	-1.601	1.386	-0.141	-0.04	0.889
			T DOM	0.011	0.972	-1.894	1.917	0.012	0.00	0.991
			T	REC -0.603 1.750	-4.032	2.827	-0.345	-0.09	0.732
	5 rs1352250	A	ADD	1.498	0.598	0.326	2.670	2.505	0.63	0.015
			A DOM	2.507	0.911	0.721	4.293	2.750	0.69	0.008
			A	REC	1.231	1.039	-0.805	3.268	1.185	0.30	0.241
	6 rs1487275	G	ADD	1.392	0.721	-0.020	2.805	1.932	0.49	0.058
			G DOM	1.913	0.893	0.163	3.662	2.143	0.54	0.036
			G	REC	0.939	1.784	-2.558	4.436	0.526	0.13	0.601
	7 rs1386485	C	ADD	1.642	0.565	0.535	2.750	2.906	0.73	0.005
			C DOM	2.109	0.819	0.503	3.715	2.574	0.65	0.013
			C	REC	2.275	1.125	0.070	4.479	2.022	0.51	0.048
	8 5HTTLPR	A	ADD -0.040 0.685	-1.383	1.302	-0.059	-0.01	0.953
			A DOM -0.379 1.071	-2.477	1.720	-0.354	-0.09	0.725
			A	REC	0.333	1.168	-1.957	2.623	0.285	0.07	0.777

Table 5 .

 5 TPH2 haplotypic regression results for risk-taking measured by AUCӨ. Age and sex were covariate in the calculations and the order of the htSNPs in the haplotypes corresponds to the SNP order in Table2. t: t-test statistics, p: uncorrected significance value, FDR q: false discovery rate significance value.

	Haplotypes	Frequencies	beta	SE	t	p	FDR q
	1 T,C,T,C,G,T,A	49%	-1.239	0.551	-2.248	0.025	0.022
	2 G,T,G,C,A,G,C	14%	3.194	1.855	1.722	0.085	0.058
	3 T,C,G,T,A,T,A	11%	4.790	1.699	2.819	0.005	0.008
	4 T,C,G,T,A,G,C	10%	0.228	1.913	0.119	0.905	0.348
	5 Rare haplotypes	16%		left out regressor
	Full vs Reduced Model				0.0004	0.003
	P-Value:
	Permuted P-Value:					0.014	0.016

ACKNOWLEDGEMENTS

The authors are grateful to Heaton Mersey Medical Practice and Cheadle Medical Practice for their assistance in the recruitment and to Dr. Xiayi Ke for statistical advices. The study was supported by the Sixth Framework Program of the EU, NewMood, LSHM- CT-2004-503474.

This work was presented in preliminary form at the BAP 2007 Summer Meeting of the British Association for Psychopharmacology, Harrogate, UK, 22-25 July 2007, and at the 20th ECNP Congress, Vienna, Austria, 13 -17 October 2007.

DISCLOSURE/ CONFLICT OF INTEREST

The authors declare that, except for income received from their primary employer, no financial support or compensation has been received from any individual or corporate entity over the past three years for research or professional service and there are no personal financial holdings that could be perceived as constituting a potential conflict of interest.


MD, PhD Vibe G Frokjaer

MD, PhD Maj Vinberg

MD, PhD David Erritzoe

MSc William Baaré

MSc Klaus Kähler Holst

Professor Erik Lykke Mortensen

MD Haroon Arfan

MSc, PhD Jacob Madsen

Professor Terry L Jernigan

Professor Lars Vedel Kessing

Professor Gitte Moos Knudsen

Familial risk for mood disorder and the personality risk factor, neuroticism, interact in their association with frontolimbic serotonin 2A receptor binding

Keywords: Neuroticism, familial risk and 5-HT2A 5-HT, 5-HT2A, depression, personality, neuroticism, stress, PET

Life stress is a robust risk factor for later development of mood disorders, particularly for individuals at familial risk. Likewise, scoring high on the personality trait neuroticism is associated with an increased risk for mood disorders. Neuroticism partly reflects stress vulnerability and is positively correlated to frontolimbic serotonin 2A (5-HT 2A ) receptor binding. Here we investigate whether neuroticism interacts with familial risk in relation to frontolimbic 5-HT 2A receptor binding.

Twenty-one healthy twins with a co-twin history of mood disorder and 16 healthy twins without a co-twin history of mood disorder were included. They answered selfreport personality questionnaires and underwent [ 18 F]altanserin positron emission tomography (PET).

We found a significant interaction between neuroticism and familial risk in predicting the frontolimbic 5-HT 2A receptor binding (p=0.026) in an analysis adjusting for age and body mass index. Within the high-risk group only, neuroticism and frontolimbic 5-HT 2A receptor binding was positively associated (p=0.0037).

In conclusion our data indicate that familial risk and neuroticism interact in their relation to frontolimbic 5-HT 2A receptor binding. These findings point at a plausible neurobiological link between genetic and personality risk factors and vulnerability to developing mood disorders. It contributes to our understanding of why some people at high risk develop mood disorders while others do not. We speculate that an increased stress reactivity in individuals at high familial risk for mood disorders might enhance the effect of neuroticism in shaping the impact of potential environmental stress and thereby influence serotonergic neurotransmission.

Introduction

Stress is a robust risk factor for later development of mood disorders, particularly for individuals at familial risk. Seminal studies by Kendler et al. have demonstrated interactions between familial risk and severe life stress in the risk of developing major depression [START_REF] Kendler | Stressful life events, genetic liability, and onset of an episode of major depression in women[END_REF]. The background for a heritable stress vulnerability is most likely to be genetically complex in nature [START_REF] Levinson | The genetics of depression: a review[END_REF] even though, Caspi et al. have demonstrated a link between stress vulnerability and the low expressing variant of a polymorphism in the promoter region of the serotonin transporter gene [START_REF] Caspi | Influence of life stress on depression: moderation by a polymorphism in the 5-HTT gene[END_REF], a finding that has been replicated in 15 independent studies so far [START_REF] Uher | The moderation by the serotonin transporter gene of environmental adversity in the aetiology of mental illness: review and methodological analysis[END_REF]. In a study by our group we have, again, linked stress vulnerability and serotonergic neurotransmission by demonstrating a coupling between the personality trait neuroticism and frontolimbic serotonin 2A (5-HT 2A ) receptor binding in a large sample of healthy volunteers [START_REF] Frokjaer | Frontolimbic serotonin 2A receptor binding in healthy subjects is associated with personality risk factors for affective disorder[END_REF]. Repeatedly, studies have shown that environmental experience moderates genes' effect on mental health. As such, identifying genetic vulnerability factors depends on focusing on relevant interactions with additional factors, e.g. environmental factors, that promote penetration of the genetic effects [START_REF] Moffitt | Strategy for investigating interactions between measured genes and measured environments[END_REF]. One such additional factor to be taken into consideration is personality or general emotional response tendencies of an individual. The personality trait, neuroticism, reflects an individuals' tendency to experience negative emotions and ability to cope with stress, factors that are critical in the development of depression. The level of neuroticism will shape a persons' perception of potential environmental stress and hence modulate the impact of a stressful environment [START_REF] Jacobs | Stress-related negative affectivity and genetically altered serotonin transporter function: evidence of synergism in shaping risk of depression[END_REF]. It is well documented that neuroticism is a risk factor for developing depression [START_REF] Fanous | A longitudinal study of personality and major depression in a population-based sample of male twins[END_REF]Kendler et al, 1993a). The genetic factors influencing the development of the personality trait neuroticism only moderately overlap with genetic risk for developing major depression. In a large twin population of 20.692 members of same-sex twin pairs the genetic correlation between neuroticism and depression was estimated to be +0.46 (Kendler et al, 2006b); this correlation does not seem to be gender specific [START_REF] Fanous | Neuroticism, major depression and gender: a population-based twin study[END_REF]. As such, a substantial proportion of the genetic vulnerability to major depression is not reflected in neuroticism and, therefore, may independently modulate the risk for mood disorders, and possibly also relate to serotonergic neurotransmission.

Serotonergic neurotransmission is a key component of the pathophysiology of mood disorders and, also, neuroticism is linked to serotonergic neurotransmission independent on familial risk for mood disorders or psychopathology [START_REF] Frokjaer | Frontolimbic serotonin 2A receptor binding in healthy subjects is associated with personality risk factors for affective disorder[END_REF]. Also, serotonergic neurotransmission is critical in the mechanisms of action of antidepressants. Although the major focus has been on the serotonin transporter, also 5-HT 2A receptor inhibition and down-regulation, e.g. as seen with SSRIs, mediate antidepressant treatment effects (14). Additional evidence for 5-HT 2A receptor changes in depression comes from post mortem studies in the subset of depressed patients that commit suicide of which the majority report that 5-HT 2A receptor binding is increased in dorsolateral prefrontal cortex [START_REF] Stockmeier | Involvement of serotonin in depression: evidence from postmortem and imaging studies of serotonin receptors and the serotonin transporter[END_REF]. Although initial findings of in vivo receptor imaging studies were contradictory, two recent studies have confirmed that frontal cortex 5-HT 2A receptor binding is increased, both in recovered, un-medicated patients with a history of major depression [START_REF] Bhagwagar | Increased 5-HT(2A) receptor binding in euthymic, medication-free patients recovered from depression: a positron emission study with [(11)C]MDL 100,907[END_REF], and in un-medicated patients with severe depression [START_REF] Meyer | Dysfunctional attitudes and 5-HT2 receptors during depression and self-harm[END_REF]. Interestingly, negativistic thinking and 5-HT 2A receptor binding is correlated both in depressed patients [START_REF] Bhagwagar | Increased 5-HT(2A) receptor binding in euthymic, medication-free patients recovered from depression: a positron emission study with [(11)C]MDL 100,907[END_REF][START_REF] Meyer | Dysfunctional attitudes and 5-HT2 receptors during depression and self-harm[END_REF] and in healthy individuals [START_REF] Frokjaer | Frontolimbic serotonin 2A receptor binding in healthy subjects is associated with personality risk factors for affective disorder[END_REF] in terms of dysfunctional attitudes and neuroticism scores, respectively. So far, no studies have investigated whether this coupling is a trait or state feature of depression. Here we test whether the coupling between neuroticism and frontolimbic 5-HT 2A receptor binding is stronger in individuals at high familial risk as compared to individuals at low risk.

Materials and methods

Twenty-three healthy high-risk twins with a co-twin diagnosed with mood disorder and 18 low-risk twins whose co-twin did not have the diagnosis were initially included. They received brain MRI and [ 18 F]altanserin PET, were administered personality assessments, and were screened for psychiatric symptoms. Four subjects were excluded from analyses.

In two cases the measurement of plasma parent compound of [ 18 F]altanserin failed and in one case the radiochemistry production was too small. Finally, one low-risk participant was excluded since he post inclusion was diagnosed with possible Tourette's syndrome post inclusion. His twin brother and his nephew had severe Tourette's syndrome and this predisposition had not been noted in the initial psychiatric screening. Thus, 37 participants were available for analyses: 21 high-risk (12 dizygotic (DZ), 9 monozygotic (MZ)) twins, with familial predisposition through unipolar (17), and bipolar (4) co-twins, mean age 39.3 years (22.1 to 60.9), and 16 low-risk twins (10 DZ, 6 MZ), mean age 38.6 (25.1 to 61.6) years. Female:male ratio was 13:8 (high-risk) and 12:4 (low-risk) reflecting the usual gender distribution in depression. The high-risk individuals' co-twin received their diagnosis at a mean age of 32±8.8 years. All participants were life-time naive for antidepressants and antipsychotics. Five women used hormonal contraception (2 highand 3 low-risk). One used non-sedative antihistamine.

Healthy high-risk and low-risk individuals were identified by linking information from the Danish Twin Registry, the Danish Psychiatric Central Research Register and the Danish Civil Registration System. This linkage identified same-sex twin pairs in which one twin had been treated in a psychiatric hospital setting for an affective episode and discharged with a diagnosis of depression or recurrent depression or a first diagnosis of manic mixed episode or bipolar affective disorder (from 1968-1993: ICD-8-codes 296.09, 296.29, 296.89, 296.99 and296.19, 296.39 and from 1994: ICD-10-codes: F32-33.9 and F30-31.6, F34.0 F38.00) and the other twin had not (yet) been diagnosed with an affective disorder, the high-risk healthy co-twin. Low-risk twins were recruited from twin pairs where both twins did not have a registered diagnosis or personal history of affective disorder. All subjects gave written informed consent.

The participants were part of a larger cohort enrolled in a high-risk study [START_REF] Christensen | Subclinical psychopathology and socio-economic status in unaffected twins discordant for affective disorder[END_REF][START_REF] Vinberg | Personality traits in unaffected twins discordant for affective disorder[END_REF]. Of the total cohort of 234 individuals, 100 were below 60 yrs of age and able to undergo an MR-scan. Of these 100 eligible individuals, 45% from the high-risk group and 37% of the low-risk group participated, which did not represent a significant difference (p=0.42, Fischer's Exact Test)".

Twelve of the participants from the low-risk group were also included in a study linking neuroticism and frontolimbic 5-HT 2A in 83 healthy volunteers [START_REF] Frokjaer | Frontolimbic serotonin 2A receptor binding in healthy subjects is associated with personality risk factors for affective disorder[END_REF].

Clinical data, symptom scores, and personality assessment. Participants were rated in a face-to-face interview by a trained clinician (MV). Persons with a lifetime diagnosis of mood disorder, schizoaffective disorder or schizophrenia according to Schedules for Clinical Assessment in Neuropsychiatry (SCAN version 2.1) were excluded from the study. The Hamilton Depression Scale (HAM-D 17-item) was used to assess depressive symptoms. Self-rating of psychopathology was assessed with the 21-item Beck Depression Inventory (BDI 21), and the 14-item Anxiety Subscale (BDI 14) as described elsewhere [START_REF] Christensen | Subclinical psychopathology and socio-economic status in unaffected twins discordant for affective disorder[END_REF]. Participants were interviewed about stressful life events (SLE) in the year prior to inclusion (recent life events), as described elsewhere [START_REF] Vinberg | Personality traits in unaffected twins discordant for affective disorder[END_REF].

Participants also completed the Danish version of the 240 item NEO-PI-R (NEO Personality Inventory Revised) self-report personality questionnaire [START_REF] Skovdahl-Hansen | Dokumentation for den danske udgave af NEO PI-R og NEO PI-R Kort Version[END_REF]) on the same day as the PET scanning. NEO-PI-R evaluates the broad personality dimensions of Neuroticism, Extraversion, Openness, Agreeableness, and Conscientiousness. Each dimension score is derived by adding the scores from assessment of six constituent personality traits (facets), and each trait score is derived by adding the scores on 8 items. The overall neuroticism score was the outcome measure.

Post hoc, the contribution from the constituent traits of neuroticism (anxiety, depression, self-conscientiousness, vulnerability, impulsiveness, and angry hostility) was evaluated.

Imaging. The 5-HT 2A receptor binding was imaged with [ 18 F]altanserin PET according to Pinborg et al. [START_REF] Pinborg | Quantification of 5-HT2A receptors in the human brain using [18F]altanserin-PET and the bolus/infusion approach[END_REF] using a maximum dose of 3.7 MBq/kg bodyweight.

After bolus-infusion of tracer for 2 hours to attain tracer steady state conditions, 40 minutes emission scans were acquired as a sequence of 5 frames of each 8 minutes, with an eighteen-ring GE-Advance scanner (GE, Milwaukee, Wisconsin, USA) operating in 3D-acquisition mode. The total axial field of view was 15.2 cm with an approximate inplane resolution of 6 mm. During scanning, the fraction of un-metabolized tracer in venous plasma was determined at 3 time points using high performance liquid chromatography analysis (HPLC). Reconstruction, including attenuation correction, and scatter correction are described in detail elsewhere [START_REF] Pinborg | Quantification of 5-HT2A receptors in the human brain using [18F]altanserin-PET and the bolus/infusion approach[END_REF]. The 5 frames of PET data were aligned using the AIR algorithm (Woods et al, 1998). Structural magnetic resonance (MR) brain imaging was conducted in all subjects; MPRAGE sequences were acquired on a 3T scanner (Trio, Siemens, Erlangen, Germany).

MR/PET co-registration. PET and MR images were co-registered through manual translation and rotation of the PET image with subsequent visual inspection in three planes, as described in Adams et al. [START_REF] Adams | A database of[END_REF]. Data analyses were carried out by the same person.

Volumes of interest (VOIs) and partial volume correction. Based on the method by

Svarer et al. [START_REF] Svarer | MR-based automatic delineation of volumes of interest in human brain PET images using probability maps[END_REF] a frontolimbic volume was automatically delineated on each individual's transaxial MRI slices in a strictly user-independent fashion. This method utilizes a VOI probability map based on a template set of ten MRIs, where VOIs have been defined manually. The frontolimbic volume was the volume of interest based on our earlier findings of a coupling between neuroticism and frontolimbic 5-HT 2A receptor binding in a large sample of healthy volunteers [START_REF] Frokjaer | Frontolimbic serotonin 2A receptor binding in healthy subjects is associated with personality risk factors for affective disorder[END_REF]. The frontolimbic volume included orbitofrontal, medial inferior frontal, superior frontal, anterior cingulate, posterior cingulate, hippocampus, and entorhinal cortices. as defined in [START_REF] Quarantelli | Integrated Software for the Analysis of Brain PET/SPECT Studies with Partial-Volume-Effect Correction[END_REF].

Quantification of the 5-HT 2A receptor binding. The outcome parameter was the binding potential of specific tracer binding (BP P ). Cerebellum was used as a reference region since it represents non-specific binding only [START_REF] Pinborg | Quantification of 5-HT2A receptors in the human brain using [18F]altanserin-PET and the bolus/infusion approach[END_REF]. BP P is defined as follows:

BP P = (C VOI -C Reference ) / C Plasma = f p * (B max /K d ) (mL/mL)
where C VOI and C Reference are mean counts in steady state in the VOI and in the reference region, respectively, C Plasma is the steady state activity of non-metabolized tracer in plasma, f P is the free fraction of radiotracer, B max is the density of receptor sites available for tracer binding, and K d is the affinity constant of the radiotracer to the receptor [START_REF] Pinborg | Quantification of 5-HT2A receptors in the human brain using [18F]altanserin-PET and the bolus/infusion approach[END_REF]. The non-specific binding was calculated as the partial-volume corrected gray-matter cerebellar regional concentration (counts/(sec*mL)) normalized by the plasma concentration, C Plasma , (counts/(sec*mL)).

[ 18 F]altanserin gives rise to a radiolabelled metabolite that crosses the blood brain barrier, however, with the bolus-infusion study design the non-specific binding is subtracted and thus the BP P is independent of the metabolite fraction.

Statistics. Group comparisons of clinical data were performed using unpaired t-tests.

Differences in proportions were tested with Fischer's Exact Test. Uncorrected p-values are reported.

Main effects of neuroticism, risk, gender, smoking, symptom scores, and SLE on frontolimbic 5-HT 2A receptor binding were tested by multiple linear regression analysis in a model adjusting for age and body mass index (BMI), known to influence 5-HT 2A receptor binding [START_REF] Erritzoe | Brain serotonin 2A receptor binding: Relations to body mass index, tobacco and alcohol use[END_REF].

To explore whether the association between frontolimbic 5-HT 2A receptor binding and neuroticism was strongest in the group at high familial risk, we tested the interaction between neuroticism and risk in a multiple linear regression analysis with frontolimbic 5-HT 2A receptor binding as the outcome parameter and age, BMI, neuroticism, risk-status, and "neuroticism by risk" as covariates.

The effect of "zygosity by risk" was also tested assuming that a genetic effect would be most prominent in MZ high-risk subjects followed by DZ high-risk, and the low-risk subjects. Post hoc, the effect of "gender by risk" interaction was tested since some genetic risk factors are likely to be sex-specific in their effect (Kendler et al, 2006a). Linearity of quantitative variables was confirmed by including second order terms in the models. Variance homogeneity and normality were assured by graphical evaluation. Model assumptions were met. P-values, as estimated by two sided tests, parameter estimates with standard errors (SE) and 95% confidence limits, and degrees of freedom (DF) are reported when appropriate. P-values < 0.05 were considered statistically significant. The analyses were performed in R 2.5.1 (http://www.R-project.org).

Results

Group differences and main effects

Demographical data and symptom scores for high-and low-risk individuals are given in Table 1. The only statistically significant difference between the total high-and low-risk groups was that the high-risk group smoked more (p=0.035). At the subgroup level, the low-risk group differed from the high-risk DZ group in proportion of smokers (p=0.038), and in cigarettes per day (p=0.008) only. Imaging outcomes are reported in Table 2.

There were no significant group differences in frontolimbic BP P , non-specific binding, C Plasma or f p even at the un-corrected level.

Neuroticism tended to correlate with frontolimbic 5-HT 2A receptor binding in a simple model adjusting for age and BMI only (slope: 0.010, confidence limits: [-0.00013 ; 0.021] BP P per neuroticism unit, p=0.053). In this model, age had a significant negative (slope: -0.017, confidence limits: [-0.033 ; -0.0016] BP P per year, p=0.032), and BMI a positive (slope: 0.090, confidence limits: [0.025 ; 0.15] BP P per BMI unit, p=0.0080), effect on binding as expected [START_REF] Erritzoe | Brain serotonin 2A receptor binding: Relations to body mass index, tobacco and alcohol use[END_REF]. We saw no main effects of familial risk, gender, smoking, symptom scores, or SLE on frontolimbic 5-HT 2A receptor binding when adjusting for age and BMI (table 2). Further, we observed no effects of bi-versus uni-polar co-twin history, years passed from age of onset/diagnosis of co-twin, or age of onset/diagnosis of the co-twin (table 2).

Effect of familial risk on the association between frontolimbic 5-HT 2A receptor binding and neuroticism

As illustrated in figure 1, we saw an interaction between neuroticism and risk in the expected direction, that is, the association between neuroticism and frontolimbic 5-HT 2A receptor binding was stronger in the high-risk group (difference in slopes: 0.023, confidence limits: [0.0030 ; 0.043] BP P per neuroticism unit, p=0.026, DF:31). Within the high-risk group, neuroticism and frontolimbic 5-HT 2A receptor binding was positively associated (slope: 0.027, confidence limits: [0.0093 ; 0.044] BP P per neuroticism unit, p=0.0037), whereas, this association was not significant in the low-risk group (slope: 0.0037, confidence limits: [-0.0080; 0.015] BP P per neuroticism unit, p=0.52), DF:31.

When adding zygosity to the model, the 2 high-risk groups of increasing risk-load (high-risk DZ < high-risk MZ) showed the expected hierarchy in terms of increasing association between frontolimbic 5-HT 2A and neuroticism (high-risk DZ: slope: 0.019, confidence limits: [-0.005; 0.042] BP P per neuroticism unit, p=0.12, and high-risk MZ: slope: 0.028, confidence limits [0.0007; 0.056] BP P per neuroticism unit, p=0.045, DF:28). This hierarchy represented a trend only, p=0.12. For comparison, the slope estimate for the low-risk group was closer to zero, Figure 2.

The association between neuroticism and 5-HT 2A receptor binding in the high-risk group was primarily driven by the constituent traits self-conscientiousness (p=0.005), anxiety (p=0.020), depression (p=0.025), and less convincingly by vulnerability (p=0.12), whereas little or no association was seen with impulsivity (p=0.24) or angry hostility (p=0.27), DF:17.

Gender by risk interaction

In high-risk females, the association between frontolimbic 5-HT 2A receptor binding and neuroticism was more pronounced than in the remaining group (difference in slope: 0.029 BP P per neuroticism unit, confidence limits: [0.0061 ; 0.051], p=0.014, DF:31). Within the group of 13 high-risk females the effect of neuroticism on frontolimbic 5-HT 2A receptor binding adjusted for age and BMI was 0.033 BP P per neuroticism unit, confidence limits: [0.013 ; 0.054], p=0.0021.

Discussion

We have demonstrated that the association between frontolimbic 5-HT 2A receptor binding and neuroticism, initially observed in healthy volunteers [START_REF] Frokjaer | Frontolimbic serotonin 2A receptor binding in healthy subjects is associated with personality risk factors for affective disorder[END_REF], is stronger in individuals at high familial risk of developing mood disorders than in individuals at low risk. These results are consistent with the hypothesis that familial risk of developing mood disorders modifies the association between frontolimbic 5-HT 2A receptor binding and neuroticism. Since the study design did not include both twins in a pair, a traditional analysis of the relative contribution from genetic versus shared early environmental factors could not be undertaken, but this was not the aim of the study. We did, however, see that the high-risk groups of increasing risk-load tended to show an increasing association between frontolimbic 5-HT 2A and neuroticism suggesting that genetic risk factors play a role in modifying the association.

The interaction between neuroticism and familial risk in predicting frontolimbic 5-HT 2A receptor binding may be genetically based, and/or be related to disturbances in serotonin homeostasis, and to increased stress reactivity which is possibly associated with both neuroticism [START_REF] Portella | Enhanced early morning salivary cortisol in neuroticism[END_REF] and familial risk of developing mood disorder [START_REF] Mannie | Increased waking salivary cortisol levels in young people at familial risk of depression[END_REF][START_REF] Modell | Hormonal response pattern in the combined DEX-CRH test is stable over time in subjects at high familial risk for affective disorders[END_REF]. Such relations may be established in early brain development [START_REF] Gaspar | The developmental role of serotonin: news from mouse molecular genetics[END_REF]. Chronic disturbances in serotonin homeostasis may affect 5-HT 2A receptor levels; Increased synaptic serotonin is suggested to lead to 5-HT 2A receptor down-regulation [START_REF] Meyer | The effect of paroxetine on 5-HT(2A) receptors in depression: an [(18)F]setoperone PET imaging study[END_REF][START_REF] Gunther | Effects of chronic citalopram treatment on 5-HT1A and 5-HT2A receptors in group-and isolation-housed mice[END_REF], whereas, low synaptic serotonin may up-regulate 5-HT 2A receptor levels [START_REF] Cahir | Acute and chronic tryptophan depletion differentially regulate central 5-HT1A and 5-HT 2A receptor binding in the rat[END_REF][START_REF] Heal | Intracerebroventricular administration of 5,7dihydroxytryptamine to mice increases both head-twitch response and the number of cortical 5-HT2 receptors[END_REF].

The latter could be linked to high risk of developing mood disorders. However, our data does not support that familial risk in itself is associated with increased frontolimbic 5-HT 2A receptor binding, since we observed no differences in binding between the low-risk and the high-risk group. Also, there were no between group differences in non-specific binding, plasma concentration or free fraction of tracer indicating that such differences have not driven the study results.

Stress responses may critically interfere with serotonergic neurotransmission and increased stress responsiveness is associated with an increased risk of developing a mood disorder. Animal studies support that chronic stress is associated with up-regulation of frontal 5-HT 2A receptor binding particularly in vulnerable rat strains characterised by anxious [START_REF] Berton | Differential effects of social stress on central serotonergic activity and emotional reactivity in Lewis and spontaneously hypertensive rats[END_REF] and depressive-like behaviours [START_REF] Dwivedi | Differential regulation of serotonin (5HT)2A receptor mRNA and protein levels after single and repeated stress in rat brain: role in learned helplessness behavior[END_REF]. Also in organotypic hippocampal cultures chronic exposure (7 days) to corticosterone increased 5-HT 2A protein levels [START_REF] Trajkovska | Activation of glucocorticoid receptors increases 5-HT2A receptor levels[END_REF]. Furthermore, several studies suggest a link between increased biological stress reactivity and the serotonergic transmitter system in terms of the low expressing polymorphism of the serotonin transporter promoter gene (Barr et al, 2004b;[START_REF] Gotlib | HPA Axis Reactivity: A Mechanism Underlying the Associations Among 5-HTTLPR, Stress, and Depression[END_REF], a gene that is also associated with an increased vulnerability to develop depression in response to life stress [START_REF] Uher | The moderation by the serotonin transporter gene of environmental adversity in the aetiology of mental illness: review and methodological analysis[END_REF]. Apart from genetic factors, stress reactivity also varies with environmental factors including early life stress [START_REF] Kajantie | Fetal origins of stress-related adult disease[END_REF][START_REF] Talge | Antenatal maternal stress and long-term effects on child neurodevelopment: how and why?[END_REF] and emotional response tendencies [START_REF] Mormede | Molecular genetic approaches to investigate individual variations in behavioral and neuroendocrine stress responses[END_REF], e. g., personality, in humans [START_REF] Portella | Enhanced early morning salivary cortisol in neuroticism[END_REF]. Therefore, interestingly, the stronger association between neuroticism and 5-HT 2A receptor binding in individuals at high familial risk of developing mood disorders may reflect an increased sensitivity to environmental stress in those individuals. Since the level of neuroticism will shape a person's perception of potential stressful experiences and as such modify the impact of environmental stress [START_REF] Jacobs | Stress-related negative affectivity and genetically altered serotonin transporter function: evidence of synergism in shaping risk of depression[END_REF], neuroticism and familial risk may interact in predicting 5-HT 2A receptor binding. However, the explanation of possible mechanisms remains speculative.

Unfortunately, we have no measures of physiological stress reactivity, e.g. waking cortisol. Thus, we cannot validate that the high-risk group had enhanced stress reactivity.

Longitudinal studies with clinical follow-up of high risk individuals will be needed to explore whether the combination of high neuroticism and high frontolimbic 5-HT 2A receptor binding would predict later development of mood disorder. However, the involvement of 5-HT 2A receptor binding in major depression has been explored in several cross-sectional postmortem [START_REF] Stockmeier | Involvement of serotonin in depression: evidence from postmortem and imaging studies of serotonin receptors and the serotonin transporter[END_REF] and imaging studies [START_REF] Attar-Levy | The cortical serotonin2 receptors studied with positron-emission tomography and [18F]-setoperone during depressive illness and antidepressant treatment with clomipramine[END_REF][START_REF] Biver | Serotonin 5-HT2 receptor imaging in major depression: focal changes in orbito-insular cortex[END_REF][START_REF] Yatham | Brain serotonin2 receptors in major depression: a positron emission tomography study[END_REF][START_REF] Messa | 5-HT(2A) receptor binding is reduced in drug-naive and unchanged in SSRI-responder depressed patients compared to healthy controls: a PET study[END_REF] of which two studies took into account measures of negativistic thinking. A recent study by Bhagwagar et al. [START_REF] Bhagwagar | Increased 5-HT(2A) receptor binding in euthymic, medication-free patients recovered from depression: a positron emission study with [(11)C]MDL 100,907[END_REF], in un-medicated, euthymic patients recovered from depression, support that a higher frontolimbic 5-HT 2A receptor level may be a trait factor of the susceptibility to develop depression and correlate positively with measures of negativistic thinking. Also, Meyer et al. found that in currently depressed patients, cortical, predominantly frontal, 5-HT 2 receptor binding was correlated to dysfunctional attitudes (pessimistic, negativistic thinking), whereas only the subgroup of patients with the most prominent negativistic thinking had a higher frontal 5-HT 2 receptor binding than healthy controls [START_REF] Meyer | Dysfunctional attitudes and 5-HT2 receptors during depression and self-harm[END_REF]. As such, even in the depressed state, a positive association between negativistic thinking and frontal 5-HT 2A receptor binding seems to exist. In none of these studies, however, was neuroticism directly taken into account.

Neuroticism mean score and variance did not differ significantly between individuals at high versus low risk and hence, the stronger association in the high-risk group is not driven by a larger variance in neuroticism score. This is in accordance with an analysis in a larger cohort of 211 healthy high-risk and low-risk individuals from the same study where no difference in neuroticism score could be identified when correcting for gender, minor psychopathology, and the effect of SLE [START_REF] Vinberg | Personality traits in unaffected twins discordant for affective disorder[END_REF].

In the present study we also replicated the earlier observed association between neuroticism and frontolimbic 5-HT 2A receptor binding [START_REF] Frokjaer | Frontolimbic serotonin 2A receptor binding in healthy subjects is associated with personality risk factors for affective disorder[END_REF] this time, in an independent sample of 21 healthy individuals at high familial risk of developing mood disorders. As observed earlier, some constituent traits of neuroticism contribute more to the association. Whereas we did not see such a strong relationship with vulnerability, as previously [START_REF] Frokjaer | Frontolimbic serotonin 2A receptor binding in healthy subjects is associated with personality risk factors for affective disorder[END_REF], a subset of the neuroticism traits consisting of the facets self-consciousness, depression, anxiety, and vulnerability contributed to the association in subjects at high familial risk. While these traits inherently reflect sensitivity to stress and trauma, the remaining two facets of neuroticism, angry hostility and impulsiveness, clearly reflect a disposition to more active reaction when faced with frustration [START_REF] Skovdahl-Hansen | Dokumentation for den danske udgave af NEO PI-R og NEO PI-R Kort Version[END_REF]. Interestingly, in this independent sample, we confirmed our previous finding that angry hostility and impulsiveness facets of neuroticism do not contribute to the correlation between high frontolimbic 5-HT 2A receptor binding and neuroticism. Therefore, we speculate that angry hostility and impulsiveness may not significantly contribute to the personality related risk of developing depressive disorder and that the subset of neuroticism is a more sensitive risk marker.

The frequency of current smokers was significantly higher in the high-risk group than in the low-risk group. This is well in line with the observation that people with current or past depression are more likely to have been smokers at some point in their lives, and may even reflect common genetic factors between smoking addiction and risk of developing depression (Kendler et al, 1993b). Smoking inhibits degradation of serotonin by inhibiting the monoamine-oxidase-A and may therefore, potentially, lead to decreased frontal 5-HT 2A receptor binding through adaptation to increased serotonin levels. However, such an effect was not observed in the present sample, Table 3, and has not been demonstrated in a large sample of healthy volunteers [START_REF] Erritzoe | Brain serotonin 2A receptor binding: Relations to body mass index, tobacco and alcohol use[END_REF]. Therefore, we do not expect that this have biased our findings. Nevertheless, if significant, smoking in the high-risk group would bias the interaction between familial risk and neuroticism towards a negative correlation with 5-HT 2A receptor binding, opposite our findings.

Even though gender does not significantly influence the 5-HT 2A receptor binding, we observed a "gender by risk" interaction suggesting a gender-related effect of familial risk factors on the association between frontolimbic 5-HT 2A receptor binding and neuroticism. However, as this study was not designed to detect gender by risk interactions this finding will need to be replicated in a more focused design. If sustained, this may reflect gender-specific differences in genetic vulnerability to environmental stress (Barr et al, 2004a;[START_REF] Brummett | Effects of Environmental Stress and Gender on Associations among Symptoms of Depression and the Serotonin Transporter Gene Linked Polymorphic Region (5-HTTLPR)[END_REF].

Methodological considerations

Our results should be interpreted in the context of some potentially significant methodological considerations.

Firstly, the high-risk group consisted of subjects with variable risk-load and several aspects potentially contributed to reduce the risk: a. A tendency towards a late age of onset of the affected co-twin; The average age of diagnosis of the co-twin was 32.2 ± 8.8 years, with 3 affected co-twins being older than 40 years (43,47,54). The typical age of onset for bipolar disorder is 18 years [START_REF] Pini | Prevalence and burden of bipolar disorders in European countries[END_REF] and for major depression it is 30 years [START_REF] Hasin | Epidemiology of major depressive disorder: results from the National Epidemiologic Survey on Alcoholism and Related Conditions[END_REF]. b. A large variation in the period of time between inclusion in our study and onset of the co-twin's mood disorder; The mean symptom free interval was 7.0±7.6 years, but for 3 subjects more than 10 years passed from the age of onset of the co-twin (10, 20, and 34 years). These subjects, despite their predisposition, did not develop mood disorders for a long time and may therefore be protected. c. Subjects volunteering for an extensive investigation program, including a PETstudy, would possibly tend to be more vigorous. However, they are not expected to differ with regard to neuroticism score [START_REF] Frokjaer | Frontolimbic serotonin 2A receptor binding in healthy subjects is associated with personality risk factors for affective disorder[END_REF].

Consequently, had the group been more selected towards high risk, it is possible that an even stronger association between frontolimbic 5-HT 2A receptor binding and neuroticism would have been present.

Secondly, high-risk subjects with unipolar and bipolar predisposition were pooled (17 unipolar and 4 bipolar). It is possible that unipolar and bipolar disorder constitute a continuous spectrum, rather than being categorically distinct [START_REF] Akiskal | The DSM-IV and ICD-10 categories of recurrent [major] depressive and bipolar II disorders: evidence that they lie on a dimensional spectrum[END_REF]. More importantly, keeping our main goal, to identify high-risk individuals, in mind, there is no difference in the frequency of unipolar depression in relatives of unipolars and bipolars [START_REF] Kutcher | Affective disorders in first-degree relatives of adolescent onset bipolars, unipolars, and normal controls[END_REF].

Thirdly, we studied twins and, thus, the outcome may not be representative of singletons. However, several studies support that both monozygotic and dizygotic twins resemble the background population in their risk for mood disorders [START_REF] Kendler | The treated incidence of psychotic and affective illness in twins compared with population expectation: a study in the Swedish Twin and Psychiatric Registries[END_REF].

Finally, the post hoc analyses on effect of zygosity in the high-risk group and gender by risk effect on the association between neuroticism and frontolimbic 5-HT 2A receptor binding represent trends only. The generated hypotheses need to be confirmed in future studies.

In conclusion, our data suggest that familial risk of developing mood disorders modifies the positive association between frontolimbic 5-HT 2A receptor binding and neuroticism.

The stronger association between neuroticism and frontolimbic 5-HT 2A receptor binding in individuals at high familial risk may reflect an increased stress reactivity that enhances the effect of neuroticism in shaping the impact of potential environmental stress and thereby influences serotonergic neurotransmission. Statistics: For the total high-risk and low-risk groups differences in numerical variables were tested by unpaired t-tests, differences in proportions by Fischer's exact test. P-values are reported with no corrections for multiple comparisons. Data for the high-risk subgroups are included for descriptive purposes. receptor BP P and neuroticism, adjusted for BMI and age. In the high-end of the neuroticism scale, the high-risk group showed an elevated BP P , whereas, in the low-end they showed a decreased BP P as compared to the low-risk group. This reflects the stronger association between neuroticism and frontolimbic 5-HT 2A receptor binding in individuals at high familial risk of developing mood disorders (p=0.026). The high-risk group showed a significant positive association between neuroticism and frontolimbic 5-HT 2A receptor binding, whereas the low-risk group did not. Please see text for estimates.

Table 1. Comparison of clinical data

Titles and legends to figures

Point-wise 95% symmetric confidence bands of the regression lines are displayed.

The regression lines represent the associations given a mean BMI and mean age. show the strongest association followed by the high-risk DZ group, and the low-risk group. However, the differences in slopes between groups represented a trend only (p=0.12).

  To enable partial volume correction of the PET data, MRIs were segmented into gray matter, white matter, and cerebrospinal fluid tissue classes using Statistical Parametric Mapping (SPM2; Wellcome Department of Cognitive Neurology, London, UK). Partial volume correction was performed according to Müller-Gärtner et al (1992),

  BDI depression: Becks Depression Inventory, depression symptoms component (21 items). BDI anxiety: anxiety component (14 items). Hamilton: Hamilton Depression rating Scale score of depressive symptoms (17 items). SLE: the fraction of participants experiencing 3 or more stress-full life events within the last 12 months. Neuroticism: score from 240-item NEO-PI-R, Danish version. Body mass index= weight/ height 2 (kg/m 2 ). Smoking: proportion of smokers. Years without diagnosis: number of years passed from age of onset of mood disorder of co-twin and the PET-scan. MZ: monozygotic, DZ: dizygotic. NA: not applicable.

Figure 1 .

 1 Figure 1. Effect of familial risk on the association between frontolimbic 5-HT 2A

Figure 2 .

 2 Figure 2. Effect of zygosity and familial risk on the association between frontolimbic

Table 2 . Comparison of imaging outcome parameters

 2

		Total groups			Sub-groups
		High-risk	Low-risk	p-value	High-risk MZ	High-risk DZ
		N=21	N=16		N=9	N=12
	Age (years)	39.3±11.9	38.6±12.0	p=0.95	41.3±12.1	38.0±12.1
	Gender (% women)	62%	75%	p=0.49	78%	50%
	Smoking (% smokers)	48%	12%	p=0.035	33%	58%
	Cigarettes per day	6.9±8.0	1.2±3.2	p=0.013	4.4±6.8	8.8±8.6
	Alcohol (units/week)	7.1±8.0	3.6±2.9	p=0.11	5.6±5.9	8.3±9.5
	Body mass index	23.9±3.1	25.4±3.1	p=0.15	24.4±3.2	23.4±3.0
	Education years	12.7±2.8	13.8±2.6	p=0.27	12.9±2.2	12.6±3.3
	Hamilton	2.7±1.3	2.3±1.4	p=0.34	2.3±1.0	3.0±1.5
	BDI depression	2.2±2.5	1.3±1.3	p=0.17	1.7±1.7	2.7±2.9
	BDI anxiety	0.9±1.4	1.2±1.5	p=0.55	0.67±1.0	1.1±1.7
	Neuroticism	71±14	68±25	p=0.67	76±13	68±14
	SLE, 3 or more	40%	27%	p=0.49	44%	33%
	Twin age of onset	32.2±8.8	NA	NA	33.3±8.0	31.6±9.6
	Years without diagnosis 7.0±7.6	NA	NA	7.7±10.3	6.4±5.2

Abbreviations: BP P , binding potential of specific [

18 F

]altanserin binding. C plasma , steady state activity of non-metabolized [

18

Table 3 . Main effects of clinical variables on frontolimbic 5-HT 2A receptor binding in a model adjusting for age and BMI

 3 Smoking: smoker>non-smoker. SLE_3or more: 3 or more>0,1,2 SLE. Risk-status: high-risk>low-risk. The effects of diagnoses, age of onset and "years passed" concern the high-risk group only. Bi or unipolar diagnoses of co-twin: diagnosis at first admission. Age of onset: age of first diagnosis.Years passed: number of years passed since age of onset of mood disorder of co-twin to PET-scan. DF: degrees of freedom.

		Total groups			Sub-groups
		High-risk	Low-risk	p-value	High-risk MZ,	High-risk DZ
		N=21	N=16		N=9	N=12
	Frontolimbic BP P	2.56±0.64	2.76±0.48	p=0.32	2.86±0.61	2.34±0.60
	(unitless)
	Non-specific binding	1.97±0.53	2.10±0.67	p=0.52	2.16±0.65	1.83±0.39
	(unitless)
	C plasma	1.92±0.51	2.02±0.38	p=0.50	1.88±0.52	1.95±0.52
	kBq/mL
	f p	0.31±0.16	0.36±0.13	p=0.38	0.26±0.15	0.35±0.16
	%

F]altanserin in plasma. f p , free (non-protein bound) fraction of [

18 F]

altanserin in plasma. MZ, monozygotic. DZ, dizygotic. Non-specific binding is the volume-weighted average partial volume corrected counts per second in the cerebellum gray-matter divided by C plasma . Statistics: For the total high-risk and low-risk groups differences in numerical variables were tested by unpaired t-tests. Data for the high-risk subgroups are included for descriptive purposes.

ACKNOWLEDGEMENTS

We are grateful to the participants. We wish to thank Karin Stahr, Dorthe Givard and the staff at the PET centre, Rigshospitalet for their superb technical assistance. Thanks to the Department of Psychiatric Demography, University of Aarhus, Psychiatric Hospital, Riskov, Denmark, and to the Danish Twin Registry for cooperation in the study. The John and Birthe Meyer Foundation is thanked for the donation of the Cyclotron and PET scanner. This work was generously supported by Danish Medical Research Council, The Health Science Faculty, University of Copenhagen, the 1991 Pharmacy Foundation, The Lundbeck Foundation, the EU Sixth framework programme (EC-FP6-project DiMI (LSHB-CT-2005-512146), Sawmill owner Jeppe Juhl and Wife Ovita Juhls Foundation, "Laegernes forsikringsforening af 1891", and "Fonden af 1870".

COMPETING INTEREST STATEMENT

The authors declare that, except for income received from their primary employers, no financial support or compensation has been received from any individual or corporate entity over the past three years for research or professional service and there are no personal financial holdings that could be perceived as constituting a potential conflict of interest.