In this issue of Addiction, Huizink et al.[1] focus on cortisol levels as a risk factor for early initiation of cannabis use. In a large cohort sample of 1441 children they obtained cortisol samples at age 11.1 years and assessed exposure to cannabis at 13.6 years. They provide evidence for higher evening cortisol levels in all cannabis users and lower levels of morning cortisol in early (9–12 years; n = 44) versus later (13–14 years: n = 59) onset of cannabis use. Low morning cortisol levels reflect a largely genetic regulation of HPA axis activity and evening cortisol values reflect mainly environmental influences [2].

This is the first study linking low morning cortisol levels to early initiation of cannabis use. Because at this age group substance-induced neuroendocrinological alterations are an unlikely cause for the findings observed, the results of Huizink et al. suggest a causal relationship of low cortisol levels as a risk factor for early onset cannabis use. In this regard their study is very relevant, as it provides conceptual biological links with psychological research indicating an increased risk of substance use disorders in certain externalizing personality types, such as high sensation-seeking individuals. Thus, a hypoarousal of morning cortisol may be evidence of an increased threshold for stress resulting in sensation-seeking behaviour, including substance use, possibly to increase the arousal effects for physiological comfort [3].

The question evolving in this context is whether low morning cortisol levels are associated with externalizing behavioural phenotypes. In the cohort investigated by Huizink et al. relevant aspects of externalizing behaviours, namely oppositional defiant disorder and conduct disorder, have been found to be more pronounced in cannabis users compared to non-users. However, no analysis of a possible interaction of behavioural, neuroendocrinological and substance use measures has been provided by the authors. Further studies of well-characterized samples may wish to address this question and contribute to exciting research on the biological basis of personality traits and their relation to substance use disorders.

Like most interesting research, the work of Huizink et al. raises more questions than a single study can address and points towards the strategies necessary to answer them. The role of genetics and heritability is one such question, which may aid in recognizing the bigger biological picture linking behaviour, neuroendocrinology and substance use phenotypes. In non-alcohol-dependent sons of alcoholic fathers, Zimmermann et al. [4] found an increased response to psychosocial stress and stronger dampening effects of alcohol, compared to family history-negative subjects. These findings are in keeping with a second set of results of Huizink's report, which measured the adaptive response of the HPA axis to environmental stimuli, and found increased levels of evening cortisol in cannabis users versus controls. These (and other) studies provide biological evidence for a further aspect of HPA axis regulation relevant for substance use, namely an increased responsiveness to environmental stimuli, which may underlie the well-known clinical phenomenon in some individuals of drug taking as an attempt to cope with stress.

However, most findings published so far attribute these features to two distinct groups of individuals, suggesting different mechanisms leading to addictive behaviour. Low basal activity and decreased responsiveness is associated with individuals with aggressive and antisocial personality traits [3], of which oppositional defiant disorder and conduct disorder are precursors. Increased responsiveness with normal basal activity is associated with individuals with negative emotionality and high anxiety levels [5]. In the present study, the authors report the somewhat surprising combination of both low morning cortisol and high evening cortisol in early-onset use of cannabis which—if replicated—may raise the question of a common molecular mechanism for both features.

While the molecular mechanism possibly integrating these features may be unclear, the tools for analysis are not. Molecular genetic dissection of HPA axis components both in animal models [6] and humans [7] have identified the corticotrophin-releasing hormone receptor 1 (CRHR1) as crucial in integrating stress response and substance use (e.g. alcohol drinking) behaviour. Further functional analyses of HPA axis genes will help to identify molecular mechanisms of HPA axis regulation and define their role for substance use disorders.

However, the neuroendocrine profile in early-onset cannabis users described by the authors also raises questions pointing towards an altogether different molecular mechanism of HPA axis regulation. Both bluntedincrease in morning cortisol and blunted decrease in evening cortisol result in flattening of the circadian rhythm of HPA axis activity. Circadian rhythmicity is regulated by a master clock in the suprachiasmatic nucleus consisting of Clock genes, which have been linked recently to substance use behaviour in humans and mouse models. They include period 1 [8], period 2 [9] and Clock[10].

Neuroanatomical studies in rats have identified both direct and indirect pathways by which the suprachiasmatic nucleus controls the circadian rhythm of cortisol release. These include projections from the suprachiasmatic nucleus to corticotrophin-releasing factor immunoreactive cells in the paraventricular nucleus, which in turn contains fibres projecting to the adrenal gland [11]. In addition, projections to the dorsomedial hypothalamus inhibit corticosterone release caused by endogenous vasopressin release and mediated by intermediate GABAergic neurones [12,13]. Interestingly, activation of CB1 receptors by cannabinoids results in suppression of GABA release [14], possibly counteracting an inhibitory effect of circadian rhythm genes on corticosterone levels. This molecular mechanism may influence cannabis use in sensation-seeking individuals.

Whatever the answers may be to the questions raised by this study, it becomes increasingly clear that we will only learn about the biological mechanisms underlying personality and addictive behaviour by pursuing an integrated approach, which combines phenotypic characterization along behavioural and biological lines with genetic experimentation in animal models and human subjects. The good news is: we can do it.

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METHODS: In silico analysis of the biological effects of allelic differences suggested the target single-nucleotide polymorphisms (SNPs) to be analyzed in a sample of 189 patients and 189 matched controls. The most relevant SNP in each gene was identified for the interaction analysis and included in the multivariate assessment of the combined effects of all three SNPs on the disease risk. RESULTS: SAD was associated with variations in each of the three genes in gene-wise logistic regression analysis. In combination analysis of variations of Per2, Arntl, and Npas2, we found additive effects and identified a genetic risk profile for the disorder. Carriers of the risk genotype combination had the odds ratio of 4.43 of developing SAD as compared with the remaining genotypes, and of 10.67 as compared with the most protective genotype combination. CONCLUSION: Variations in the three circadian clock genes Per2, Arntl, and Npas2 are associated with the disease, supporting the hypothesis that the circadian clock mechanisms contribute to winter depression. [less]

Molecular components of the clock include circadian clock genes such as period (Per) 1, 2, and 3. Previously, our lab demonstrated the involvement of mouse Per1 (mPer1) and Per2 (mPer2) in modulating cocaine sensitization and reward. What is more, we investigated voluntary alcohol consumption in Per2 ( Brdm1 ) mice with the results suggesting a relationship between this circadian clock gene and ethanol consumption. Objective To further complement the mPer2 study, our lab proceeded to assess mPer1's possible role on alcohol intake using operant and free choice two bottle paradigms.

METHODS: Using operant conditions, Per1 ( Brdm1 ) and wild type mice were trained to self-administer ethanol (10%) under a fixed ratio 1 (FR1) paradigm. This was ensued by a progressive ratio (PR) schedule. Furthermore, extinction sessions were introduced, followed by reinstatement measures of ethanol-seeking behavior. In another set of animals, the mice were exposed to voluntary long-term alcohol consumption, ensued by a 2-month deprivation phase, after which the alcohol deprivation effect (ADE) was measured.

RESULTS: Mutant mice did not display a significantly divergent number of reinforced lever presses (FR1 and PR) than wild type animals. Furthermore, no significant differences between groups were obtained regarding reinstatement of ethanol-seeking behavior. Similar results were obtained in the two bottle free choice paradigm. Specifically, no genotype differences concerning consumption and preference were observed over a broad range of different ethanol concentrations. Moreover, after the deprivation phase, both groups exhibited significant ADEs, yet no genotype differences.

CONCLUSIONS: Contrary to the mPer2 data, the present findings do not suggest a relationship between the circadian clock gene mPer1 and ethanol reinforcement, seeking, and relapse behavior.

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These genes are the catechol-O-methyltransferase gene (COMT Val158Met) and the regulatory region (5-HTTLPR) of the serotonin transporter gene. Recently, it has been proposed that 5-HTT expression is not only affected by the common S/L variant of 5-HTTLPR but also by an A to G substitution. Using functional magnetic resonance imaging, we assessed the effects of COMT Val158Met and both 5-HTT genotypes on brain activation by standardized affective visual stimuli (unpleasant, pleasant, and neutral) in 48 healthy subjects. Based on previous studies, the analysis of genotype effects was restricted to limbic brain areas. To determine allele-dose effects, the number of COMT Met158 alleles (i.e., lower activity of COMT) and the number of 5-HTT low expressing alleles (S and G) was correlated with the blood oxygen level-dependent (BOLD) response to pleasant or unpleasant stimuli compared to neutral stimuli. We observed an additive effect of COMT and both 5-HTT polymorphisms, accounting for 40% of the inter-individual variance in the averaged BOLD response of amygdala, hippocampal and limbic cortical regions elicited by unpleasant stimuli. Effects of 5-HTT and COMT genotypes did not affect brain processing of pleasant stimuli. These data indicate that functional brain imaging may be used to assess the interaction of multiple genes on the function of neuronal networks.
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The aim of this lecture is to develop a model of integrated translational addiction research which may result in the establishment of individualized therapeutic approaches for patients with substance use disorders. Methods The genetic basis of substance use disorders is characterized by a contribution of multiple genes to the clinical phenotype. This genetic complexity is based on poly/oligogenicity and genetic heterogeneity, two parallel mechanisms which are present to varying extents in different substance use disorders. To disentangle the complexity and to identify the genetic and neurobiological basis of addictions an integrated, translational approach involving (functional) genetic analyses, animal behavioural experimentation and neuroimaging studies is proposed. Results Examples of this approach are provided by describing a line of work which identified the relevance of circadian rhythm genes in regulating alcohol drinking behaviour in animal models and humans, as well as a complementary approach using endophenotypes in human gene-neuroimaging studies where the effect of single and combined genetic variations on processing of aversive emotional stimuli in the limbic system was demonstrated. Discussion While the combination of genetic, behavioural and neuroimaging analyses are shown to be useful tools to address oligogenicity and genetic heterogeneity in substance use disorders, the clinical relevance of this approach needs to be developed further. Thus, two current major research projects, the European integrated project 'IMAGEN' and the NIHR-Biomedical Research Centre 'Mental Health' in the United Kingdom, which potentially integrate our proposed research strategy with clinically relevant outcomes, will be discussed. [less]

METHODS: Data were available from the Mannheim Study of Children at Risk, an ongoing cohort study of the long-term outcome of early risk factors followed since birth. At age 15 years, 280 participants (135 males, 145 females) completed a self-report questionnaire measuring alcohol use and were genotyped for two SNPs (rs242938, rs1876831) of CRHR1. Assessment of negative life events over the past three years was obtained by a standardized interview with the parents.
RESULTS: Adolescents homozygous for the C allele of rs1876831 drank higher maximum amounts of alcohol per occasion and had greater lifetime rates of heavy drinking in relation to negative life events than individuals carrying the T allele. No gene x environment interactions were found for regular drinking and between rs242938 and stressful life events.
CONCLUSIONS: These findings provide first evidence in humans that the CRHR1 gene interacts with exposure to stressful life events to predict heavy alcohol use in adolescents. [less]

Alcohol-related diseases, especially alcoholism, are the result of cumulative responses to alcohol exposure, the genetic make-up of an individual, and the environmental perturbations over time. This complex gene × environment interaction, which has to be seen in a life-span perspective, leads to a large heterogeneity among alcohol-dependent patients, in terms of both the symptom dimensions and the severity of this disorder. Therefore, a reductionistic approach is not very practical if a better understanding of the pathological processes leading to an addictive behavior is to be achieved. Instead, a systems-oriented perspective in which the interactions and dynamics of all endogenous and environmental factors involved are centrally integrated, will lead to further progress in alcohol research. This review adheres to a systems biology perspective such that the interaction of alcohol with primary and secondary targets within the brain is described in relation to the behavioral consequences. As a result of the interaction of alcohol with these targets, alterations in gene expression and synaptic plasticity take place that lead to long-lasting alteration in neuronal network activity. As a subsequent consequence, alcohol-seeking responses ensue that can finally lead via complex environmental interactions to an addictive behavior.  [less]

The dopaminergic system has long featured in alcoholism research; hitherto disappointing results from clinical studies could improve following the discovery that dopamine D3 receptor antagonism produces consistent and robust results in preclinical studies. Corticotropin-releasing factor signalling and the endocannabinoid system integrate stress-related events and thereby mediate relapse behaviour. Overall, these new targets have yielded several compounds that are undergoing clinical testing. However, the heterogeneity in treatment response makes it necessary to characterize genetic and protein markers and endophenotypes for individualized pharmacotherapy. [less]

Gene–environment interactions have been reviewed by focusing on one of the most relevant environmental risk factors for alcoholism, stress. This is examined in more detail by reviewing the functioning of the hypothalamic-pituitary-adrenal (HPA) axis and its genetic and molecular components in this disorder. Recent evidence from animal and human studies have shown that the effects of stress on alcohol drinking are mediated by core HPA axis genes and are associated with genetic variations in those genes. The findings of the studies discussed here suggest that the collaborations of neuroscience, psychobiology and molecular genetics provide a promising framework to elucidate the exact mechanisms of gene–environment interactions as seen to convene upon the HPA axis and effect phenotypes of addiction. [less]

These are the NMDA, GABA_A, glycine, 5-hydroxytryptamine 3 (serotonin) and nicotinic ACh receptors as well as L-type Ca²⁺ channels and G-protein-activated inwardly rectifying K⁺ channels. Following this first hit of alcohol on specific targets in the brain, a second wave of indirect effects on a variety of neurotransmitter/neuropeptide systems is initiated that leads subsequently to the typical acute behavioural effects of alcohol, ranging from disinhibition to sedation and even hypnosis, with increasing concentrations of alcohol. Besides these acute pharmacodynamic aspects of alcohol, we discuss the neurochemical substrates that are involved in the initiation and maintenance phase of an alcohol drinking behaviour. Finally, addictive behaviour towards alcohol as measured by alcohol-seeking and relapse behaviour is reviewed in the context of specific neurotransmitter/neuropeptide systems and their signalling pathways. The activity of the mesolimbic dopaminergic system plays a crucial role during the initiation phase of alcohol consumption. Following long-term, chronic alcohol consumption virtually all brain neurotransmission seems to be affected, making it difficult to define which of the systems contributes the most to the transition from controlled to compulsive alcohol use. However, compulsive alcohol drinking is characterized by a decrease in the function of the reward neurocircuitry and a recruitment of antireward/stress mechanisms comes into place, with a hypertrophic corticotropin-releasing factor system and a hyperfunctional glutamatergic system being the most important ones. [less]

Methodology/Principal Findings:To investigate whether PI3K contributes to patterns of risky alcohol drinking in human, we investigated genetic variations in PIK3R1, encoding the 85 kD regulatory subunit of PIK, in 145 family trios consisting of 15–16 year old adolescents and their parents. Screening for mutations in exons, exon-intron boundaries and regulatory sequences, we identified 14 single nucleotide polymorphisms (SNPs) in the PIK3R1 gene region from exon 1 to the beginning of the 3' untranslated region (UTR). These SNPs defined haplotypes for the respective PIK3R1 region. Four haplotype tagging (ht)SNPs (rs706713, rs2302975, rs171649 and rs1043526), discriminating all haplotypes with a frequency =4.5% were identified. These htSNPs were used to genotype adolescents from the “Mannheim Study of Risk Children” (MARC). Transmission disequilibrium tests in these adolescents and their parents demonstrated sex-specific association of two SNPs, rs2302975 and rs1043526, with patterns of risky alcohol consumption in male adolescents, including lifetime prevalence of drunkenness (p = 0.0019 and 0.0379, respectively) and elevated maximum amount of drinking (p = 0.0020 and 0.0494, respectively), as a measure for binge drinking pattern.
Conclusions/Significance: Our findings highlight a previously unknown relevance of PIK3R1 genotypes for alcohol use disorders and might help discriminate individuals at risk for alcoholism. [less]

Method: Data are from an ongoing prospective study of the long-term outcome of early risk factors studied since birth. At age 15 years, 220 participants (108 males, 112 females) completed a self-report questionnaire measuring smoking behavior and were genotyped for five dopamine gene variants.
Results: Smoking initiation was related to allelic variation in the dopamine D4 receptor gene (DRD4), whereas smoking continuation and dependence showed association with the dopamine D2 receptor gene (DRD2). Adolescents with the seven-repeat allele of the common DRD4 exon 3 polymorphism had rates of ever smoking that were significantly higher than in those with other genotypes. Once smoking started, carriers of the T allele of a single nucleotide polymorphism of DRD2 (rs4648317) reported higher rates of current smoking and scored higher on nicotine dependence than their allelic counterparts. Among current smokers, intention to quit was significantly lower in adolescents homozygous for the 10-repeat allele of the common dopamine transporter 3' untranslated region polymorphism.
Conclusions: Our results provide preliminary evidence of genetic influences on different stages of smoking and suggest the importance of specific dopamine genes in smoking progression in adolescence.
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induced BS. Here, we investigate further the mechanisms underlying Ro 15-4513-induced behavioural sensitisation in a2(H101R) mice. a2(H101R) mice sensitised to Ro 15-4513 (10 mg/kg) showed an enhanced stimulant response to cocaine (10 mg/kg). In contrast, cocaine (10 mg/kg)-sensitised a2(H101R) mice did not show enhanced sensitivity to the stimulant effects of Ro 15-4513 (1, 3 and 10 mg/kg), suggesting that the neural adaptations underlying Ro 15-4513 induced BS are related to, but not identical with those associated with cocaine-induced plasticity. Secondly, we investigated whether a2-containing receptors are involved in mediating the ability of BZs to facilitate cocaine-induced activity. The non-selective (i.e., a1, a2, a3 and a5 subtype) benzodiazepine GABAA receptor agonist midazolam (10 and 30 mg/kg) potentiated cocaine (10 mg/kg) hyperactivity in wildtype mice, but not in a2(H101R) mice, in which a2-containing receptors are insensitive to benzodiazepines. To determine where a2 receptors are localised we compared BZ-insensitive sites between wildtype (a4 and a6) and a2(H101R) (a2, a4 and a6) mice, using quantitative autoradiography to estimate [3H]Ro 15-4513 binding in the presence of 10 µM diazepam. a2 receptors were found in projection areas of the mesolimbic dopamine pathway including accumbens, central amygdala, and basolateral amygdala as well as CA1 and CA3 areas of the hippocampus. The involvement of the a2-containing receptor in mediating BZ's potentiating effect on cocaine hyperactivity suggests that the locomotor stimulant effects of BZs and psychostimulants may be mediated by a common neural system, but the lack of cross sensitisation to Ro 15-4513 in cocaine-sensitised a2(H101R) mice, suggests that this form of BS may occur downstream of plastic events underlying cocaine sensitisation. [less]

They are characterized by phenotypic and genetic heterogeneity as well as polygenicity, implying a contribution of different neurobiological mechanisms to the clinical diagnosis. Therefore, treatments for most substance use disorders are often only partially effective, with a substantial proportion of patients failing to respond. To address heterogeneity and polygenicity, strategies have been developed to identify more homogeneous subgroups of patients and to characterize genes contributing to their phenotype. These include genetic linkage and association studies as well as functional genetic analysis using endophenotypes and animal behavioural experimentation. Applying these strategies in a translational context aims at improving therapeutic response by the identification of subgroups of addiction patients for individualized, targeted treatment strategies. This article aims to discuss strategies addressing heterogeneity and polygenicity of substance use disorders by presenting results of recent research on genetic and environmental components of addiction. It will also introduce the European IMAGEN study that aims to integrate methodical approaches discussed in order to identify the genetic and neurobiological basis of behavioural traits relevant to the development of addictions.  [less]

Design: Association analysis of alcohol dependence and haplotype-tagging single nucleotide polymorphisms (SNPs) covering 10 glutamatergic genes. Resequencing of functional domains of these genes identified 204 SNPs. Haplotypes with a frequency of 5% or greater could be discriminated by 21 haplotype-tagging SNPs analyzed for association in 2 independent samples of alcohol-dependent adult patients and controls as well as adolescent trios.
Setting: Four university medical centers in the south of Germany.
Participants: One thousand three hundred thirty-seven patients and 1555 controls (study 1: 544 patients, 553 controls; study 2: 793 patients, 1002 controls). One hundred forty-four trios of 15-year-old adolescents assessed for risky drinking behavior.
Main Outcome Measures: Genotype profiles for GLAST; N-methyl-d-aspartate-receptor subunits NR1, NR2A, and NR2B; MGLUR5; NNOS; PRKG2; CAMK4; the regulatory subunit of PI3K; and CREB were analyzed for association with alcohol dependence using multivariate statistical analysis. Risky adolescent drinking was tested using the transmission disequilibrium test.
Resutls: Analysis of study 1 revealed that NR2A and MGLUR5 have the greatest relevance for human alcohol dependence among the genes selected with odds ratios of 2.35 and 1.69, respectively. Replication analysis in study 2 confirmed an association of alcohol dependence with NR2A (odds ratio, 2.01) but showed no association with MGLUR5. Combined analysis of study 1 and study 2 exhibited a more significant association on the Cochran-Mantel-Haenszel test (P < .001) for NR2A; NR2A was associated with positive family history, early onset of alcoholism, and maximum number of drinks in adults as well as risky drinking patterns in adolescents.
Conclusion: Genetic variations in NR2A have the greatest relevance for human alcohol dependence among the glutamatergic genes selected for their known alteration of alcohol effects in animal models. [less]

MATERIALS AND METHODS: Mice were trained in the 5-CSRTT and then were injected intraperitoneally (i.p.) with 0, 0.5, 1 and 2 g/kg ethanol before testing under standard parameters and in a long inter-trial interval (ITI) session, which promotes the emergence of premature responses, a measure of poor inhibitory control. To examine if the effects of ethanol in the 5-CSRTT were due to its actions at GABA(A) receptors or at NMDA receptors, the GABA(A) receptor agonist diazepam (1 and 2 mg/kg, i.p.) and the non-competitive NMDA antagonist ketamine (10 and 20 mg/kg, i.p.) were tested in long ITI sessions. RESULTS: Ethanol did not affect attention or impulsivity in the standard procedure, but increased premature responding in long ITI sessions. The effects of ethanol were mimicked by diazepam in both strains of mice, whereas ketamine increased premature responding only in the CD1 strain. CONCLUSIONS: Ethanol's ability to increase impulsivity in the 5-CSRTT is mediated by both common and different neurotransmitter systems in the two strains of mice and is dependent on the task's parameters. Furthermore, ethanol did not decrease response accuracy, suggesting that attentional mechanisms are preserved after acute ethanol in mice and that the increases in impulsive behaviour are independent of attentional performance. [less]

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We demonstrate repeatable, reproducible assessment of ongoing pain that is independent of patient self-report. In a cross-over trial design, 16 participants requiring bilateral removal of lower-jaw third molars underwent pain-free pre-surgical pCASL scans. Following extraction of either left or right tooth, repeat scans were acquired during post-operative ongoing pain. When pain-free following surgical recovery, the pre/post-surgical scanning procedure was repeated for the remaining tooth. Voxelwise statistical comparison of pre and post-surgical scans was performed to reveal rCBF changes representing ongoing pain. In addition, rCBF values in predefined pain and control brain regions were obtained. rCBF increases (5–10%) representing post-surgical ongoing pain were identified bilaterally in a network including primary and secondary somatosensory, insula and cingulate cortices, thalamus, amygdala, hippocampus, midbrain and brainstem (including trigeminal ganglion and principal-sensory nucleus), but not in a control region in visual cortex. rCBF changes were reproducible, with no rCBF differences identified across scans within-session or between post-surgical pain sessions. This is the first report of the cerebral representation of ongoing post-surgical pain without the need for exogenous tracers. Regions of rCBF increases are plausibly associated with pain and the technique is reproducible, providing an attractive proposition for testing interventions for on-going pain that do not rely solely on patient self-report. Our findings have the potential to improve our understanding of the cerebral representation of persistent painful conditions, leading to improved identification of specific patient sub-types and implementation of mechanism-based treatments.
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Comprehensive behavioural and neuropsychological characterization, functional and structural neuroimaging and genome-wide association analyses of 2000 14-year-old adolescents are combined with functional genetics in animal and human models. Results will be validated in 1000 adolescents from the Canadian Saguenay Youth Study. The sample will be followed up longitudinally at the age of 16 years to investigate the predictive value of genetics and intermediate phenotypes for the development of frequent psychiatric disorders. This review describes the strategies the IMAGEN consortium used to meet the challenges posed by large-scale multicentre imaging-genomics investigations. We provide detailed methods and Standard Operating Procedures that we hope will be helpful for the design of future studies. These include standardization of the clinical, psychometric and neuroimaging-acquisition protocols, development of a central database for efficient analyses of large multimodal data sets and new analytic approaches to large-scale genetic neuroimaging analyses.

 

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