In the present study, positron emission tomography with the tracer alpha-[ 11 C]methyl-L-tryptophan ( 11 C-AMT) was used to compare 5-HT synthesis capacity in two groups of adult males from a 21-year longitudinal study (mean age ± SD: 27.1±0.7): individuals with a history of childhood-limited high physical aggression (C-LHPA; N = 8) and individuals with normal (low) patterns of physical aggression (LPA; N = 18). The C-LHPA males had significantly lower trapping of 11 C-AMT bilaterally in the orbitofrontal cortex and self-reported more impulsiveness. Despite this, in adulthood there were no group differences in plasma tryptophan levels, genotyping, aggression, emotional intelligence, working memory, computerized measures of impulsivity, psychosocial functioning/adjustment, and personal and family history of mood and substance abuse disorders.

Funding: This study was supported by an operating grant from the Canadian Institutes of Health Research (CIHR) to Drs. C. Benkelfat and R.E. Tremblay (MOP57838). Dr. L. Booij was supported by a postdoctoral fellowship from the Fonds de la recherche en santé Quebec (FRSQ) and by an award from the Netherlands Organization for Scientific research (NWO-VENI). The longitudinal study was supported by grants to Dr. R. E. Tremblay and collaborators from CIHR, FRSQ, FQRSC and SSHRC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The present study is part of a series of studies that applied a significant paradigm shift, from clinical studies of patient samples to a developmental epidemiological and longitudinal approach. Bio-psycho-social factors and outcomes were investigated with a population-based longitudinal cohort of young males from low socioeconomic area schools in Montreal (Canada) [19] . More specifically, the current study tested the hypothesis that adult males who were on a high physical aggression trajectory during elementary school and desisted during adolescence (childhood-limited high physical aggression, C-LHPA) still maintained lower regional brain 5-HT synthesis during adulthood compared to males who had a more normal pattern (low) of physical aggression (LPA) [3] . Based on results from previous neuro-imaging studies in animals and in patients with impulsive-aggressive behaviors [20] , [21] , the OBFC was chosen as primary volume of interest (VOI). All participants were assessed clinically, with a particular emphasis on current and recent impulsive aggressive behavior, frontal lobe function, emotional intelligence and psychosocial adjustment. Genotyping for the rate-limiting 5-HT synthesis enzyme, tryptophan-hydroxylase2 (TPH 2 ) and prospective measures of early and late life adversity were also collected in order to explore genetic and environmental influences on 5-HT metabolism. Brain 5-HT synthesis was estimated by means of positron emission tomography (PET) in combination with the injection of the synthetic analog of the 5-HT precursor tryptophan, alpha-[ 11 C]methyl-L-tryptophan ( 11 C-AMT) [22] , [23] , [24] .

Genetic epidemiology studies of aggressive behavior demonstrate that childhood physical aggression is highly heritable [14] , [15] , [16] . The neurobiological substrates underlying the individual variability in the development of physical aggression and violence are many, although there seems to be little agreement about their relative importance [17] . Generally, heuristic models support the view that gene by environment interactions manifest their effects through changes in brain structure and/or function, resulting into deficits in cortical top down control and/or facilitation of bottom up signalling triggered from limbic circuits [18] . One proposed mechanism contributing to reduced top down control and behavioral disinhibition is lower brain serotonin (5-HT) neurotransmission in the orbitofrontal cortex (OBFC) [18] .

In contrast to popular belief, longitudinal epidemiological research indicates that the sudden onset of physical aggression in adolescence is unusual [1] . Rather, physically aggressive behaviors can already be detected by 12 months of age, and their frequency peaks between the end of the second and fourth years [1] , [2] . In the majority of children, the frequency of physical aggression gradually decreases, starting before elementary school entry [1] , [3] . However, others start desisting only at the end of elementary school and others not until the end of adolescence [4] . A 60-year longitudinal study of juvenile delinquents concluded that very few show life-span high frequency of violent offending [5] . Among those who do express chronic physical aggression, impaired executive functioning is evident in adolescence and early adulthood, even after controlling for other cognitive-neuropsychological domains, intelligence quotient (IQ) and Attention Deficit Hyperactivity Disorder (ADHD) symptoms [4] , [6] , [7] , [8] . Longitudinal follow up of elementary school children with high levels of physical aggression demonstrates that they are at greater risk for substance abuse, anti-social personality, suicide, depression, spouse abuse and neglectful and abusive parenting [9] , [10] , [11] . However, the long-term outcomes for the physically aggressive children who desist with adolescence have not been studied [12] , [13] .

Given the finding in the C-LHPA group of higher self-reported impulsiveness relative to the LPA group, and studies showing that low 5-HT neurotransmission is a biological risk factor for both aggressive behaviors and behavioral disinhibition, (e.g. [18] , [28] ), the primary VOI analysis was rerun, including the BIS score as a covariate. The results of aggression trajectory on normalized K* in the OBFC were only slightly attenuated, indicating a multivariate effect of aggression trajectory (F(8,16) = 3.82, P = 0.01; partial eta squared: 0.66), and univariate statistics showing lower normalized K* in the left lateral OBFC (F(1,23) = 15.68, P = 0.001; partial eta squared: 0.41) and in the right lateral OBFC (F(1,23) = 6.29, P = 0.02; partial eta squared: 0.22) in the C-LHPA group, relative to the LPA group.

A significant difference in the expected direction was observed between the C-LHPA and LPA groups on a retrospective measure of aggression in childhood and adolescence, but no significant difference was observed for current aggression ( Table 1 ). However, the C-LHPA group exhibited significantly higher self-reported scores for impulsivity (Barratt Impulsivity Scale; BIS). Nonetheless, the two groups did not differ for the total number of commission errors (CE), nor for the number of CE in the Reward-Punishment component of the Go/NoGo task, a measure previously reported to be increased in clinical samples endowed with high impulsivity and/or aggression [25] , [26] , [27] .

Discussion

The present study investigated brain 5-HT synthesis in a community sample of healthy adult males characterized by high levels of physical aggression during the primary school years and a desisting pattern during adolescence (C-LHPA). We compared them to males recruited from the same cohort with a normal (low) physical aggression trajectory during childhood and adolescence (LPA). We found no differences in aggressive behavior, mood state, social status and neuropsychological measures of working memory, emotional intelligence and impulsivity between the C-LHPA and LPA groups, yet the C-LHPA group demonstrated markedly lower 5-HT synthesis in the bilateral OBFC in adulthood relative to those with a normal aggression trajectory. In fact, the magnitude of the reduction of 11C-AMT uptake and trapping in the OBFC was about twice the one previously reported in patient samples with impulse control disorders [27], [33]. This observation forces a re-examination of the low 5-HT hypothesis as a central tenet of the biology of violence.

5-HT plays a significant role in neurodevelopment. In humans, 5-HT neurons are constituted approximately 5 weeks after gestation [34], 5-HT fibers grow in the cortex prenatally, and within a short period of time, between 12–14 weeks of gestation, the thalamocortical axons transiently express the 5-HT transporter, crucial for the fine-tuning of cortical development [35]. 5-HT levels continue to increase throughout the first two to five years of life and then gradually decrease until adult levels are reached (approximately age 14) to a level approximating 50% of the peak values of early childhood [36]. Research in mice has shown that altered 5-HT availability during a critical window of early development, through genetic or pharmacological manipulations and/or environmental events, can result in structural and/or functional alterations in brain neurotransmission with deleterious consequences on emotion regulation in adulthood [37], [38]. We presume that relatively low 5-HT synthesis capacity partially accounts for high levels of physical aggression in childhood. However, the substantial decrease in frequency of physical aggression during adolescence, as observed in our C-LHPA group, cannot be directly attributed to a relative increase in 5-HT synthesis capacity, at least not from the level observed at 27 years of age. We suggest that low brain 5-HT in the C-LHPA group may operate as a vulnerability trait, but other protective environmental factors, as well as brain maturation and new learning, may have come into play with adolescence and early adulthood to support impulse control despite low 5-HT. Indeed, a 20 year longitudinal study of pre-pubertal adolescents examining sensation-seeking and impulsivity found that by age 15, both measures demonstrated a steady linear decline, fitting well with the developmental pattern of other self-regulatory tasks that tap self-regulation (Go/NoGo, Stroop, Antisaccades), as well as the maturational time course of the brain circuits that subserve impulse control [39]. Consistent with this study, many of the differences in the behavioral phenotype noted between our two groups in childhood had disappeared at the 21 year follow-up; and both groups were indistinguishable on the basis of psychosocial status in adulthood. We suggest that, while 5-HT neurotransmission tends to remain stable over time, from childhood to adulthood [40], experience, brain maturation and/or environmental support play a major role in the near universal reduction in frequency of physical aggression with age, even in the most delinquent males [5].

The validity and significance of the observations presented in this study, however, rest upon the following considerations: (1) When the technique was originally implemented, it was argued that the brain regional uptake of 11C-AMT might reflect blood-brain transport rather than brain 5-HT trapping and thereby, 5-HT synthesis [41]. Experimental evidence accumulated over more than 10 years [23], [42], [43], [44], [45], [46] now provides a firm basis for a consensus that brain regional 11C-AMT trapping represents an acceptable index of 5-HT synthesis. Of particular interest is the report of Patlak plots of 11C-AMT brain uptake estimated in primates, with a slope significantly different from zero, and providing unequivocal evidence for brain trapping of the tracer [24]. (2) The study was conducted between 2002 and 2007. An older version of the SPM software (SPM99) was used for exploratory image analysis. However, reanalysis of the image data with a newer version of SPM (SPM2) yielded similar results. Newer versions of SPM have now been released (SPM5 and SPM8), although much of the add-on functionality(ies) are for functional Magnetic Resonance Imaging (fMRI) analysis. The use of pre-processing steps (co-registration/realignment) relying on tools developed and validated in-house (MINC tools) at the Montreal Neurological Institute (MNI) has made the use of newer versions of SPM less critical for PET investigations. (3) The interpretation of the results rests upon the assumption that normalized K* is a stable measure over time [47]. Cerebrospinal fluid (CSF) concentrations of 5-Hydroxyindoleacetic acid (5-HIAA) in humans and monkeys are highly correlated over time [40]. In addition, in humans, low levels of CSF 5-HIAA have already been found in infants (age between birth and 3 months) from parents with anti-social personally disorders [48]. These studies support the idea that lower 5-HT synthesis in young adults as observed in the present study would most likely indicate a neurochemical vulnerability present since childhood. (4) Our study was limited to males. The developmental pattern of aggressive behavior [49], as well as many aspects of brain 5-HTneurotransmission, including 11C-AMT uptake and trapping, are influenced by gender [22], it would be of much interest to investigate the extent to which the present results generalize to females. (5) 5-HT neurotransmission exercises an inhibitory tonic control on behavior; low central 5-HT facilitates behavioral disinhibition and its most extreme clinical manifestation, impulsive aggression [50], [51]. Whether aggressive behavior at school reflected increased impulsivity was not specifically tested at that age. This was however, unlikely, since measures of impulsivity collected in the laboratory in adolescence and during early adulthood, by and large, did not discriminate between trajectories. Furthermore, co-varying out the PET data for BIS scores measured in adulthood in both trajectories, did not affect the results.

In summary, this study compared brain regional 11C-AMT uptake and trapping, a proxy measure of 5-HT synthesis, in a group of young adult males on a childhood-limited high physical aggression trajectory and in a group of young males from the same population who were on a normal (low) trajectory of physical aggression during childhood and adolescence. As predicted, the C-LHPA group exhibited lower 11C-AMT in the OBFC, with an effect size about twice of what was previously reported in patient samples endowed with an impulsive aggression phenotype. Surprisingly however, in the presence of relatively low 5-HT synthesis in the C-LHPA group, neither groups differed markedly in adulthood in their levels of aggressive behavior, nor in their behavioral, neurocognitive and psychosocial outcomes. This observation is interpreted as reflecting the mitigating effects of brain maturation, learning and supportive environment in the case of participants from the high aggression “desisters” trajectory. Altogether, these data emphasize a less deterministic model with a role for familial and other environmental factors operating at different developmental time periods. More generally, these observations may lead to a re-examination of the low 5-HT diathesis theory, as a risk factor in the pathogenesis of impulsive aggression.