The utility of the non-human primate model for studying gene by environment interactions in behavioral research


  • Supported by the NICHD and NIAAA Intramural Research Programs and the Deutsche Forschungsgemeinschaft (SFB 581, Le 629/4–2).

* C. S. Barr, NIAAA/NIH, NIHAC, PO Box 529, Bldg 112, Poolesville, MD, 20837, USA. E-mail:


Variation in the serotonin transporter gene-linked polymorphic region (5-HTTLPR) has been associated with anxiety and harm avoidance and is weakly associated with a number of neuropsychiatric disorders, including Type II alcoholism, which has a high rate of comorbidity with antisocial personality disorder. Studies have also demonstrated interactions between 5-HTTLPR variation and environmental stress on the incidence of depression. As in humans, there is a serotonin transporter gene promoter length polymorphism in rhesus macaques that produces similar decreases in transcriptional efficiency. Macaques with histories of early-life stress have been shown to exhibit impulsive aggression, incompetent social behavior and increased behavioral and endocrine responsivity to stress. In this paper, we review studies performed previously in our lab and present preliminary data examininng interactions between early rearing and serotonin transporter gene promoter variation on the incidences of play behavior and aggression in infant rhesus macaques. The data presented here highlight the importance of considering gene-environment interactions when studying childhood risk factors for aggression, anxiety and related neuropsychiatric disorders and support the use of the nonhuman primate for studing gene by environment interactions in behavioral research.

The ‘nature vs. nurture’ controversy over the development of personality is long-standing (for reviews, see Balaban 2002; Plomin et al. 1988). While, historically, many scientists held that personality was subject to evolutionary pressures and therefore was probably of genetic etiology, others maintained that personality was probably more strongly influenced by the environment. This debate extended further to the development of psychopathology and neuropsychiatric disease (Balaban 2002; Rutter et al. 1997). Today, it is more widely accepted that the development of personality is driven by both nature and nurture and that, moreover, there are potential interactions between genes and environment (G × E interactions) that may be at the root of the development of some personality traits and neuropsychiatric diseases (for review, see Gunzerath & Goldman 2003). With the advent of modern molecular and statistical tools, we are now capable of refining our approach so that specific G × E interactions in the development of personality traits can be revealed.

The non-human primate model is particularly useful for the study of G × E interactions in the development of psychopathology. Firstly, most psychopathology revolves around social functioning, and, compared to other laboratory animal species, non-human primates have complex behaviors and social structures that more closely approximate those present in humans. Secondly, because they are more closely related to humans than are the other laboratory animal species, they provide a useful model for the study of gene variation as it relates to certain temperament traits that can be readily characterized in the non-human. Finally, because the rearing environment of non-human primates, unlike that of humans, can be tightly controlled, they are particularly useful in the study of G × E interactions.

Of particular interest for the study of G × E interactions is variation within regulatory or coding regions of genes likely to be influenced by a stressful environment or, alternatively, of genes involved in the behavioral response to stress. One of the neurotransmitters implicated in behavioral aberrations observed in animals exposed to early life stress is serotonin, and serotonin plays a pivotal role in many forms of psychopathology, with the specific serotonin reuptake inhibitor (SSRIs) and other serotonin acting agents being some of the most widely prescribed psychotropic medications. During the early postnatal period, serotonin plays a pivotal role in the development of the central nervous system (CNS) (reviewed in Lauder 1983), and serotonin neurotransmission is involved in both activation and feedback control of the neuroendocrine stress axis (Weidenfeld et al. 2002). In turn, stress hormones are also involved in regulating the expression of certain serotonin system genes, including that for the serotonin transporter (Tafet et al. 2001).

The serotonin transporter is a protein critical to regulating serotonin function in brain, because serotonin's action in the synapse is terminated by reuptake. In mice, targeted disruption of the serotonin transporter gene results in increased ACTH and corticosterone responses to immobilization stress (Lanfumey et al. 2000; Li et al. 1999) as well as increased anxiety during the elevated plus maze and light/dark exploration tasks (Holmes et al. in press; Wichems et al. 2000). In humans, there is a common polymorphism involving a repeat length variation in the promoter region of the serotonin transporter gene (SCL6A4, or 5-HTTLPR). The short (s) serotonin transporter allele contains an attenuated promoter segment (5-HTTLPR) and, as such, shows diminished transcription relative to that of the long (l ) allele (Lesch et al. 1997). Variation in the serotonin transporter gene promoter is associated with certain anxiety-, depression- and aggression-related personality traits, such as neuroticism, harm avoidance and disagreeableness (Benjamin et al. 1998; Costa & McCrae 1997; Greenberg et al. 1998; Lesch et al. 1996; Mazzanti et al. 1998; Van Gestel et al. 2002). Moreover, variation of the 5-HTTLPR has been associated with certain neuropsychiatric diseases, including Type II alcoholism (Hallikainen et al. 1999; Saunder et al. 1998), which has a high rate of comorbidity with antisocial personality disorder (ASPD) and is characterized by its early onset and habitually impulsive violent behavior (Brown et al. 1994).

Among macaques, animals reared without adults in peer-only groups show CNS serotonin impairments and have increased frequencies of serious aggression (Higley et al. 1996a; Higley, in press). Peer-reared (PR) animals also exhibit less complex social behaviors, spend more time in withdrawal, achieve lower social rank and consume more alcohol than do animals reared with their mothers (MR) (Higley et al. 1991; Higley et al. 1996a; Higley et al. 1996b; Suomi et al. 1992). Nevertheless, even within homogeneous peer-reared groups, certain peer-reared animals are not as severely affected by early deleterious rearing experiences, suggesting a role for genetic influence in response to adversity. In the rhesus macaque, there is an analogous 21 bp length variant, rh5-HTTLPR, located in the same region as the serotonin transporter gene promoter polymorphism identified in humans (Lesch et al. 1997). Although different in sequence and in its precise location, the short (s) length variant results in decreased transcriptional efficiency of the serotonin transporter and, as such, shares the nomenclature of the 5-HTTLPR variant in humans (Bennett et al. 2002).

Using the rhesus macaque model, we have been able to demonstrate interactions between rearing experience and rh5-HTTLPR on many phenotypes of interest (Table 1). The first report of a G × E interaction in non-human primates demonstrated that rh-5-HTTLPR genotype and rearing condition interact to influence the cerebrospinal fluid concentrations of 5-hydroxy-3-indoleacetic acid (5-HIAA), a marker for susceptibility to aggressive behavior and alcohol consumption in both macaques and humans. In three recent studies, we demonstrated that the s allele adversely affects neonatal responding, alcohol sensitivity and hypothalamic-pituitary-adrenal axis activity during social separation stress, but only among PR animals (Barr et al. 2003a; Barr et al. 2003b; Champoux et al. 2002). Conversely, monkeys reared by their mothers were not differentiated by genotype.

Table 1. : Effect of rh5-HTTLPR in rhesus macaques
ReferencePhenotypeMain effect of rh5-HTTLPR?G × E?
Bennett et al. (2002)CSF 5-HIAANY
Champoux et al. (2002)Neonatal assessmentY (affective responding)Y (orientation)
Barr et al. (2003)Alcohol sensitivityYY

Neonatal assessment is designed to test an individual's orienting ability, motor maturity, reflex functioning and temperament across the first few months month of life. Studies in human infants have found that carriers of the 5-HTTLPRs allele have impaired orientation abilities (Auerbach et al. 1999; Ebstein et al. 1998). Using a similar method of assessment among rhesus infants, we have shown that PR, but not MR, l/s animals have significantly lower orientation and motor maturity scores, sensitive measures of CNS integrity and future affective difficulties (Champoux et al. 2002). Consistent with an association of the s allele with anxiety in people, there is also an effect of the s allele on measures of anxiety and affective responding during testing of infant macaques, independent of rearing environment. In another study we demonstrated that, at 6 months of age, neuroendocrine stress axis responses to chronic social separation are higher among heterozygote monkeys (Barr et al. submitted). However, during the acute phase of this stressor, the effect of rh5-HTTLPR gene variation on hypothalamic-pituitary-adrenal output is observed only among PR animals.

Interactions between gene variation and rearing condition remain into adolescence and adulthood. Certain intermediate phenotypes, such as a decreased level of response to alcohol, are thought to be markers for susceptibility to alcoholism and alcohol abuse (Schuckit 1994). The level of response to alcohol has been associated with 5-HTTLPR variation in human populations (Schuckit et al. 1999). For the purpose of determining whether differences in intoxication scores among alcohol-naïve macaques correlated with prospective alcohol consumption, animals were given an intravenous dose of alcohol at 5 years of age, immediately prior to entering an alcohol consumption study. Alcohol sensitivity was affected by rh5-HTTLPR genotype. However, when animals were divided according to rearing condition, this effect was observed only among animals reared in PR groups (Barr et al. 2003b).

To illustrate how behavioral data can be collected longitudinally to understand developmental outcomes, we show preliminary data from a two-year longitudinal study of 18 rhesus macaques (9 males, 9 females), collected during 10 separate five-minute scoring sessions at ages corresponding to infancy and early childhood. Using these data, we found an interaction between rearing condition and rh5–HTTLPR interaction on both social play and aggression. Infants reared in mother-infant dyads tend to engage in play with age-mates less frequently than peer-reared infants because, having been raised with their mothers, who are far less likely to engage in playful behavior than are infants, they are less socially experienced at this early age (Suomi 1979). Among PR infants, however, those that carry the s allele do not differ from MR infants in their frequency of play, suggesting that, among these more playful animals, serotonin transporter gene promoter variation is associated with a reticence to engage in rough play with age-mates (Fig. 1). This is consistent with findings in humans demonstrating relationships between the l/s genotype and anxiety, harm avoidance and amygdala response to stressful stimuli (Greenberg et al. 1998; Hariri et al. 2002; Lesch et al. 1996).

Figure 1.

Interaction between rh5-HTTLPR and rearing environment on social play in infant macaques.‘Social play’ was defined as initiating play by ‘play face’, wrestling (rough and tumble), chasing, tagging, swatting, bobbing, biting, pulling, lunging, mouthing and receiving play from the other animal. Data is presented as mean duration of social play during a 5-minute scoring session. Since there were no main effects based on sex, sex was not considered in the analysis. There were main effects of both rearing condition (F1,17 = 8.7, P= 0.01) and rh5-HTTLPR genotype (F1,17 = 6.7, P = 0.02) on social play during infancy. There was also an interaction between these variables (F1,17 = 6.1, P= 0.03), such that PR l/l animals played more frequently than the other groups studied (Fisher's PLSD, P < 0.05). Although two animals shared a grandparent (1 MR l/s female and 1 MR l/s male), exclusion of these animals from the analysis produced similar results.

Previous studies from our laboratory have demonstrated that, as adults, animals reared in peer-only groups can be particularly aggressive. Although more animals need to be studied, our data suggest that there is an interaction between rearing condition and the serotonin transporter gene to influence the prevalence of aggression in macaques at an age corresponding to childhood in humans (Fig. 2). Typically, aggressive behavior among macaques begins to develop during the second year of life. In our observations of this small group of animals we noted that, without provocation, PR juveniles with the l/s genotype exhibited relatively high rates of aggressive behavior, suggesting that the low activity variant of the serotonin transporter gene promoter may augment the influence of early maternal absence, resulting in increased frequency of aggressive behavior at a time early in psychosocial development. Although they are meant to be illustrative and, with a small sample size, are preliminary, these findings are supportive of those from Greenberg et al. (1998), which demonstrate an association between the l/s genotype and disagreeableness in humans. This is also of interest given the literature supporting both genetic and environmental roles in the etiology of aggressive disorders, such as antisocial personality disorder and conduct disorder, both of which are associated with childhood aggression (Bohman et al. 1982; Brennan & Mednick 1993; Carey 1996; Caspi et al. 2002; Holmes et al. 2001; Johnson et al. 1996).

Figure 2.

Interaction between rh5-HTTLPR and rearing environment on aggression in juvenile macaques.‘Aggression’ was defined as: bites, hairpulls, aggressive chases, hitting or slapping. Data is presented as mean rates of aggression within a 5-minute scoring session. Since there were no main effects based on sex, sex was not considered in the analysis. There was an interaction between rearing and rh5-HTTLPR genotype on the incidence of aggression (F1,17 = 4.7, P= 0.05). Among juveniles with the l/s genotype, those that were reared with peers were more likely to be aggressive than were mother-reared animals (Fisher's PLSD, P < 0.04), but among mother-reared animals, this effect was not present. Exclusion of two related animals from the analysis yielded similar results.

Risk for psychiatric disease may not be identical between individuals with similar genetic backgrounds, but instead may vary according to life-history experiences. A recent study by Caspi et al. (2003) demonstrated that 5-HTTLPR gene variation increased the likelihood of developing depressive symptoms, especially among individuals with a stressful life history (Caspi et al. 2003). The failure of studies to replicate associations between the serotonin transporter gene promoter variant and certain disease states in humans may be partially explained by differences between subjects' developmental backgrounds and histories. In humans, both social experience and serotonin neurotransmission are likely to be important in the etiology of antisocial behavior as well as in anxiety-related personality traits. From the preliminary results presented here, we infer that both social experience and serotonin neurotransmission may be important in early willingness to participate in social play, for learning behavioral inhibition and in preventing the development of aggressive behavior in the rhesus macaque. Although further studies are warranted, these data suggest that rh5-HTTLPR genotype may affect social behavior and more importantly, that serotonin transporter gene promoter variation and environment interact to influence these behaviors.

Our work supports the notion that it is essential to consider environmental influence when determining the effect of genetic variation on behavioral phenotype (Table 1). It may be that certain gene systems are particularly sensitive to environmental influence and therefore to G × E interactions. Since variables such as early life stress cannot be tightly controlled in human subjects, non-human primate models for the study of gene effects, environmental influence and G × E interactions will be important to our understanding of the development of temperament and of the pathogenesis of psychopathology and neuropsychiatric disease.