We found significant heritability for behavioral inhibition, i.e. the duration of freezing in response to the NEC challenge, in this population of macaques. We also found a significant genetic effect on a related behavior, vigilance or orienting to the intruder. Threat-induced freezing in rhesus monkeys and the analogous behavioral inhibition in humans are adaptive responses reflecting underlying anxiety, and in certain situations are protective (Kalin & Shelton 1989
). Increased vigilance associated with freezing is adaptive because it facilitates ongoing evaluation of potential risk. However, extreme levels of behavioral inhibition and hypervigilance reflect excessive anxiety. In human children, this is associated with increased risk for developing anxiety and depressive disorders as well as comorbid substance abuse (Caspi et al. 1996
; Kagan et al. 1988
; Kalin & Shelton 2003
; Rosenbaum et al. 1993
Using young rhesus monkeys, we previously described behavioral and physiological components of the anxious endophenotype revealed by the human intruder challenge. In addition to excessive freezing and vigilance behavior, this endophenotype includes increased pituitary–adrenal activity (Kalin et al. 1998a
), increased cerebrospinal fluid concentrations of the anxiogenic neuropeptide corticotrophin-releasing factor (Kalin et al. 2000
) and asymmetric right frontal brain electrical activity (Kalin et al. 1998b
). Increased cortisol and asymmetric right frontal brain activity also occur in extremely inhibited children (Kagan et al. 1988
; Schmidt et al. 1999
). Furthermore, individual differences in monkey freezing behavior are positively correlated with differences in metabolic activity of the amygdala and bed nucleus of the stria terminalis as assessed with high-resolution positron emission tomography imaging (Kalin et al. 2005
). These brain structures are components of the neural circuitry associated with the processing of emotion, fear and anxiety. Human adults with a childhood history of extreme behavioral inhibition also exhibit increased amygdala reactivity (Schwartz et al. 2003
The present study demonstrates that behavioral expression of this anxious endophenotype is significantly influenced by genetic differences among animals. We also note that much of the variation in behavioral response to this test is not attributable to genetic variation but to age, sex and unidentified environmental factors. The variance components approach applied to animals from multigeneration pedigrees efficiently exploits kinship information to test the hypothesis that individual behaviors are influenced by genetic differences among animals and simultaneously quantifies the relative significance of genetic and environmental factors.
Williamson et al. (2003)
also used variance components methods to examine the heritability of anxiety and fearfulness in a different pedigree of young rhesus monkeys. They found that the latency to leave the mother and explore a novel play room, latency to inspect a novel food item and duration of exploratory behavior while separated from their mother were all significantly heritable. While Williamson et al. (2003)
did not directly assess freezing behavior, they did measure ‘movement’ during a modified human intruder test. Neither the amount of movement nor vocalizations during the NEC test were significantly heritable (Williamson et al 2003
), which is concordant with the results in our study.
Fairbanks et al. (2004)
demonstrated the heritability of approach behaviors expressed by adolescent and adult vervet monkeys (Chlorocebus aethiops
) exposed to an unfamiliar conspecific. Although exposure to unfamiliar conspecifics may not be testing the same elements of temperament as response to a human intruder, this result is also consistent with our finding of significant heritability of responses to potentially dangerous stimuli. Overall, the results of Williamson et al. (2003)
and Fairbanks et al. (2004)
are consistent with our conclusion that individual variation in specific elements of primate behavior related to anxiety and fear is influenced by genetic differences among animals. Furthermore, these genetic effects are attributable to additive genetic variance, without the involvement of genotype × environment interaction effects.
Human studies have reported and replicated associations of neuroticism, major depression and other anxiety-related traits with the s
allele of the serotonin transporter locus (Caspi et al. 2003
; Kendler et al. 2005
; Lesch et al. 1996
; Schinka et al. 2004
; Sen et al. 2004
). Furthermore, previous studies in rhesus monkeys have detected an effect of a similar polymorphism in the same gene on adrenocorticotropic hormone levels (Barr et al. 2004b
) and alcohol preference (Barr et al. 2004a
). However, we found no relationship between serotonin transporter promoter repeat genotype and either duration of freezing or duration of orienting to the intruder. Most of the published effects of the rhesus serotonin transporter genotype depend on an interaction between serotonin transporter genotype and the rearing environment experienced by the animal (Barr et al. 2004a
; Bennett et al. 2002
), suggesting that like some human studies, the rhesus serotonin transporter promoter polymorphism may exhibit its strongest influence in combination with adverse environmental experience. However, one study has reported an effect on the age at which male macaques disperse from their natal social groups under normal mother-rearing conditions (Trefilov et al. 2000
We note that not all studies of the human serotonin transporter polymorphism are concordant (Lasky-Su et al. 2005
; Munafo et al. 2005
; Surtees et al. 2006
; Willis-Owen et al. 2005
). Most relevant to our findings are studies examining the relationship between the SLC6A4 short (s
) allele and childhood behavioral inhibition or shyness. In a large sample of preschool children, no relationship was found between observed shyness and the s
allele (Schmidt et al. 2002
), whereas among second graders, the s
allele was unexpectedly associated with decreased levels of shyness (Arbelle et al. 2003
). In contrast, Battaglia et al. (2005)
concluded that the s
allele was associated with increased shyness among third and fourth graders. Finally, Fox et al. (2005)
reported a gene × environment interaction such that 7-year olds with the s
allele and low levels of social support showed increased inhibition when exposed to unfamiliar peers.
Bethea et al. (2004)
reported a genetic association study investigating the effect of the serotonin transporter promoter polymorphism on anxiety-related behaviors in young rhesus monkeys. In the same population studied by Williamson et al. (2003)
, Bethea and colleagues analyzed a series of behavioral tests, including the modified human intruder challenge. They report that SLC6A4 promoter repeat genotypes (also called 5HTTLPR genotypes) are associated with one behavior during the human intruder test: the number of threat displays given by monkeys while the human stared directly at them. No association was found with any behaviors expressed during the NEC test, although SLC6A4 genotypes were reported to be associated with activity during a Free Play test and fear grimaces displayed to a threatening mechanical toy.
It is not clear how to interpret these genetic associations reported by Bethea et al. (2004)
. The same behavioral methods were used to study the same population of monkeys by Williamson et al. (2003)
, and the latter study did not find significant heritability for any of the behaviors reported by Bethea et al.
to be associated with serotonin transporter genotypes. Bethea et al.
did not provide an explanation for why they might find a genetic association with 5HTTLPR genotypes, while Williamson et al.
could not detect any genetic effect across the entire genome. Bethea et al.
also reported that they did not make use of the pedigree relationships among their study subjects in their analysis, although they acknowledge that some of the animals used were likely to be genealogically related. The analysis of heritability using variance components methods as we report here depends on using kinship relationships among individuals to estimate quantitative genetic effects, but the genetic association approach used by Bethea et al. (2004)
depends on the assumption that animals are genealogically unrelated. Bethea et al.
acknowledged that this is a concern regarding their analyses and suggested that it is possible that individuals exhibiting high levels of anxiety in their study may be influenced by other genetic polymorphisms but not by SLC6A4 (5HTTLPR) specifically.
In general, the available data from humans and rhesus monkeys suggest that the serotonin transporter polymorphism does influence specific behavioral and neurobiological phenotypes, but its effects are not detected on all anxiety-related traits. It may be that among children and young rhesus monkeys, the major effects of serotonin transporter repeat unit polymorphism depend on interaction with adverse environmental experience. Like the situation for SLC6A4 and human psychopathology, it is not yet clear how much effect the serotonin transporter polymorphism has among monkeys raised in essentially normal circumstances. Because all our study subjects were raised with their mothers, as opposed to the peer-rearing condition employed in other studies (Barr et al. 2004a
), we did not investigate genotype × environment interaction. We note, however, that several studies of human temperament and nonhuman primate behavior (Fairbanks et al. 2004
; Williamson et al. 2003
; this study) document additive genetic effects on anxiety-related traits that do not depend on abnormal developmental or environmental circumstances to be manifest.
In summary, we examined individual variation in behavioral responses to a standardized behavioral challenge, the human intruder test (Kalin & Shelton 1989 2003
) among a large number of young rhesus monkeys from a multigeneration pedigree. Knowledge of the kinship relationships among animals allowed us to estimate the proportion of phenotypic variation attributable to additive genetic variation and to simultaneously estimate the influence of age and sex. We found significant heritability for two out of five behaviors. Both freezing and orienting to intruder are part of an anxious endophenotype we have demonstrated to be physiologically similar to extreme behavioral inhibition in children (Kalin & Shelton 2003
). In addition to testing for overall genetic heritability, we tested for, but could not detect, specific effects of the serotonin transporter promoter polymorphism.