Stephen G. Lindell

Variation at the mu-opioid receptor gene (OPRM1) influences attachment behavior in infant primates

In a variety of species, development of attachment to a caregiver is crucial for infant survival and partly mediated by the endogenous opioids. Functional mu-opioid receptor gene polymorphisms are present in humans (OPRM1 A118G) and rhesus macaques (OPRM1 C77G). We hypothesized that rhesus infants carrying a gain-of-function OPRM1 77G allele would experience increased reward during maternal contact and would, therefore, display increased measures of attachment. We collected behavioral data from rhesus macaques (n = 97) during early infancy and at 6 months of age, across four cycles of maternal separation (4 days) and reunion (3 days). Animals were genotyped for the OPRM1 C77G polymorphism, and the effects of this allele on attachment-related behaviors were analyzed. Infants carrying the G allele exhibited higher levels of attachment behavior during early infancy. During prolonged periods of maternal separation, although infant macaques homozygous for the C allele exhibited decreases in their levels of distress vocalization with repeated separation, this response persisted in G allele carriers. The OPRM1 77G allele also affected social preference during reunion. C/G infants spent increasing amounts of time in social contact with their mothers as a function of repeated separation and were less likely to interact with other individuals in the social group, a pattern not observed among infants with the C/C genotype. These findings suggest a role for OPRM1 variation in the expression of attachment behavior in human subjects, especially as a function of separation from the caregiver.

Early maternal rejection affects the development of monoaminergic systems and adult abusive parenting in rhesus macaques (Macaca mulatta)

This study investigated the effects of early exposure to variable parenting style and infant abuse on cerebrospinal fluid (CSF) concentrations of monoamine metabolites and examined the role of monoaminergic function in the intergenerational transmission of infant abuse in rhesus monkeys (Macaca mulatta). Forty-three infants reared by their biological mothers and 15 infants that were cross-fostered at birth and reared by unrelated mothers were followed longitudinally through their first 3 years of life or longer. Approximately half of the infants were reared by abusive mothers and half by nonabusive controls. Abused infants did not differ from controls in CSF concentrations of 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA), or 3-methoxy-4-hydroxyphenylgycol (MHPG). Abused infants, however, were exposed to higher rates of maternal rejection, and highly rejected infants had lower CSF 5-HIAA and HVA than low-rejection infants. The abused females who became abusive mothers in adulthood had lower CSF 5-HIAA than the abused females who did not. A similar trend was also observed among the cross-fostered females, suggesting that low serotonergic function resulting from early exposure to high rates of maternal rejection plays a role in the intergenerational transmission of infant abuse.

Neurobiological characteristics of rhesus macaque abusive mothers and their relation to social and maternal behavior

Previous studies have reported hyperactivation of catecholaminergic systems and elevated concentrations of corticotropin-releasing-hormone (CRH) in the cerebrospinal fluid (CSF) of child maltreatment victims or combat veterans with post-traumatic stress disorder (PTSD). This study investigated the CSF concentrations of CRH and monoamine metabolites in rhesus macaque mothers that physically abused their infants and had themselves been abused as infants. Ten abusive mothers and 10 controls served as study subjects. All animals were sampled for CSF during pregnancy and the postpartum period. Focal observations of social and maternal behavior were also made. Abusive mothers had significantly higher CSF concentrations of CRH and 5-HIAA than controls. Across both subjects and controls, higher CRH, 5-HIAA and MHPG concentrations were associated with anti-social behavior patterns including a high frequency of maternal aggression, infant rejection, and a low frequency of contacts received from other individuals. These findings are consistent with those of previous primate and human studies and suggest that the neurobiological alterations associated with infant abuse may play an important role in the occurrence of maladaptive behavior in adulthood, including the perpetuation of infant abuse across generations.

Association between the recombinant human serotonin transporter linked promoter region polymorphism and behavior in rhesus macaques during a separation paradigm

Human studies have suggested an association between a variable length polymorphism in the serotonin transporter gene promoter region and vulnerability to anxiety and depression. Relative to the long (l) allele, the short (s) allele increases the risk of developing depression in individuals exposed to stressful life events. An orthologue of the human variant is present in rhesus macaques and allows for studies in animals exposed to stress. Here, we used an established model of early life stress exposure, in which rhesus macaques are raised without adults in a group of peers (peer-only reared [PR]), or with their mothers. At 6 months of age, animals were subjected to 4-day long social separations for 4 consecutive weeks, with 3 days of reunion in between. Data were collected during both the acute (Day 1) and chronic phases (Days 2-4) of separation. Behavioral factors were separately extracted for each phase of separation. For acute separation, the behavioral factors generated were despair and behavioral pathology and, for the chronic phase despair, agitation, and behavioral pathology. During both phases of social separation, PR l/s animals were more likely to exhibit pathological behaviors, whereas PR l/l monkeys show higher levels of despair compared to the other three groups. These findings indicate that early stress affects the behavioral response to separation differently as a function of recombinant human serotonin transporter linked polymorphic repeat genotype and suggest that carriers of the s allele are not only more anxious but may also be more vulnerable to developing behavioral pathology in the face of chronic adversity.

Influence of parenting style on the offspring's behaviour and CSF monoamine metabolite levels in crossfostered and noncrossfostered female rhesus macaques

We investigated the association between variation in parenting style and the offsprings behaviour and CSF monoamine metabolite (5-HIAA, HVA, and MHPG) levels in rhesus monkeys. Study subjects were 25 two-year-old females reared by their biological mothers and 15 same-aged females that were crossfostered at birth and reared by unrelated mothers. Subjects that were rejected more by their mothers in the first 6 months of life engaged in more solitary play and had lower CSF concentrations of 5-HIAA than subjects that were rejected less. The relation between these variables was generally similar in crossfostered and noncrossfostered females. CSF levels of 5-HIAA were negatively correlated with rates of scratching, a behavioural indicator of anxiety. These results suggest that that early exposure to high rates of maternal rejection can result in higher anxiety later in life, and that this effect may be mediated by serotonergic mechanisms. Variation in maternal protectiveness did not affect offspring behaviour and neither protectiveness nor rejection affected CSF levels of HVA and MHPG. CSF levels of MHPG, however, were negatively correlated with solitary play behaviour and avoidance of other individuals, suggesting that individuals with lower CSF MHPG were more fearful and socially phobic than those with higher CSF MHPG. Taken together, these findings suggest that individual differences in anxiety and fearfulness in young rhesus monkeys are accounted for, at least in part, by variation in CSF levels of monoamine metabolites, and that the development of brain monoamine systems, particularly serotonin, can be affected by early exposure to variable maternal behaviour.

Functional CRH variation increases stress-induced alcohol consumption in primates

Corticotropin-releasing factor (CRF), encoded by the CRH gene, is a key integrator of stress responses, and, as such, CRH gene variation may contribute to individual differences in susceptibility to stress-related pathology. In rhesus macaques, a single nucleotide polymorphism (SNP) is found within the CRH promoter (−248C→ T). Here, we assessed whether this variant influenced stress responding and, because increased CRF system activity drives alcohol drinking in rodents, we examined whether it predicted voluntary alcohol consumption as a function of prior stress exposure. Using a hypothalamic nuclear extract, we showed that the −248 T allele resulted in increased DNA protein interactions relative to the C allele. In vitro, the T allele resulted in CRH promoter activity that was higher following both stimulation with forskolin and treatment with dexamethasone. Endocrine and behavioral responses to social separation stress (release of ACTH and cortisol, and suppression of environmental exploration, respectively) were higher among carriers of the T allele, particularly among those exposed to early adversity in the form of peer rearing. We also found that T allele carriers with a history of early life adversity consumed more alcohol in a limited-access paradigm. Our data suggest that CRH promoter variation that confers increased stress reactivity increases the risk for alcohol use disorders in stress-exposed individuals.

CRH Haplotype as a Factor Influencing Cerebrospinal Fluid Levels of Corticotropin-Releasing Hormone, Hypothalamic-Pituitary-Adrenal Axis Activity, Temperament, and Alcohol Consumption in Rhesus Macaques

CONTEXT Both highly stress-reactive and novelty-seeking individuals are susceptible to alcohol use disorders. Variation in stress reactivity, exploration, and response to novelty have been attributed to differences in corticotropin-releasing hormone (CRH) system function. As such, CRH gene variation may influence risk for alcohol use and dependence. OBJECTIVE To determine whether CRH variation influences relevant intermediate phenotypes, behavior, and alcohol consumption in rhesus macaques. DESIGN We sequenced the rhesus macaque CRH locus (rhCRH) and performed cladistic clustering of haplotypes. In silico analysis, gel shift, and in vitro reporter assays were performed to identify functional variants. Cerebrospinal fluid (CSF) and blood samples were obtained, and levels of CRH and corticotropin (ACTH) were measured by radioimmunoassay. Behavioral data were collected from macaques during infancy. Among adolescent/adult animals, we recorded responses to an unfamiliar conspecific and measured levels of ethanol consumption. SETTING National Institutes of Health Animal Center. PARTICIPANTS Rhesus macaques. MAIN OUTCOME MEASURES Animals were genotyped for a single-nucleotide polymorphism disrupting a glucocorticoid response element, rhCRH -2232 C>G, and the effects of this allele on CSF levels of CRH, plasma levels of ACTH, behavior, and ethanol consumption were assessed by analysis of variance. RESULTS We show that -2232C>G alters DNA x protein interactions and confers decreased sensitivity of the CRH promoter to glucocorticoids in vitro. Consistent with the known effects of glucocorticoids on CRH expression in the brain, carriers of the G allele had lower CSF levels of CRH but higher levels of ACTH. Infants carrying the G allele were more exploratory and bold, and among adolescent and adult male macaques, the G allele was associated with exploratory/bold responding to an unfamiliar male. Adults with the C/G genotype also exhibited increased alcohol consumption in the social group, a model for high-risk alcohol-seeking behavior. CONCLUSION Haplotypes that differ in terms of corticosteroid sensitivity have been identified in humans. Our data may suggest that functionally similar CRH variants could influence risk for externalizing disorders in human subjects.

Suppression of Alcohol Preference by Naltrexone in the Rhesus Macaque: A Critical Role of Genetic Variation at the μ-Opioid Receptor Gene Locus

BACKGROUND The role of a nonsynonymous A118G polymorphism of the human micro-opioid receptor gene (OPRM1) for alcohol reward and therapeutic efficacy of naltrexone remains controversial. A functionally equivalent OPRM1 C77G polymorphism in rhesus macaques allows this to be addressed under controlled experimental conditions. METHODS Twenty-one rhesus macaques (13 female rhesus macaques, 8 male rhesus macaques) were genotyped for OPRM1 C77G and studied during 1-hour sessions for preference between an aspartame-sweetened alcohol solution (8.4% vol/vol) and a nonalcoholic control fluid in a baseline session followed by naltrexone (1 mg/kg) and vehicle treatment in a counterbalanced within-subject design. RESULTS Mixed-model analysis of variance controlling for baseline and sex showed a highly significant (p = .003) interaction between genotype and treatment. Post hoc analysis showed that vehicle-treated 77G carriers had markedly higher alcohol preference than 77C homozygous subjects (p = .001). Following naltrexone administration, 77G carriers decreased their preference (p = .002) and no longer differed from 77C homozygous subjects. In contrast, the latter group was unaffected by treatment and, in fact, showed a trend-level increase of preference following naltrexone. CONCLUSIONS These results support a critical pharmacogenetic role of OPRM1 variation for therapeutic efficacy of naltrexone.

Gene–Environment Interactions and Response to Social Intrusion in Male and Female Rhesus Macaques

BACKGROUND Genetic factors interact with environmental stressors to moderate risk for human psychopathology, but sex may also be an important mediating factor. Different strategies for coping with environmental stressors have evolved in males and females, and these differences may underlie the differential prevalence of certain types of psychopathology in the two sexes. In this study, we investigated the possibility of sex-specific gene-environment interactions in a nonhuman primate model of response to social threat. METHODS Rhesus macaques (77 males and 106 females) were exposed to an unfamiliar conspecific. Using factor analysis, we identified three behavioral factors characterizing the response to social threat. Monkeys were genotyped for the serotonin transporter-linked polymorphism (5-HTTLPR), and the effects of genotype, early life stress, and sex on behavioral responses were evaluated. RESULTS Factor analysis produced five factors: High-Risk Aggression, Impulsivity/Novelty-Seeking, Gregariousness/Boldness, Harm Avoidance, and Redirected Aggression. Overall, males displayed higher levels of High-Risk Aggression and Gregariousness/Boldness than females. Levels of High-Risk Aggression in males carrying the s allele were significantly higher if they were also exposed to early adversity in the form of peer rearing. CONCLUSIONS Our findings support those from studies in humans suggesting that males are more vulnerable to externalizing or aggression-related disorders. The results highlight the importance of interactions that exist among behavior, genes, and the environment and suggest that sex differences in vulnerability to psychopathology may be grounded in our evolutionary history.

Functional NPY Variation as a Factor in Stress Resilience and Alcohol Consumption in Rhesus Macaques

CONTEXT Neuropeptide Y (NPY) counters stress and is involved in neuroadaptations that drive escalated alcohol drinking in rodents. In humans, low NPY expression predicts amygdala response and emotional reactivity. Genetic variation that affects the NPY system could moderate stress resilience and susceptibility to alcohol dependence. OBJECTIVE To determine whether functional NPY variation influences behavioral adaptation to stress and alcohol consumption in a nonhuman primate model of early adversity (peer rearing). DESIGN We sequenced the rhesus macaque NPY locus (rhNPY) and performed in silico analysis to identify functional variants. We performed gel shift assays using nuclear extract from testes, brain, and hypothalamus. Levels of NPY in cerebrospinal fluid were measured by radioimmunoassay, and messenger RNA levels were assessed in the amygdala using real-time polymerase chain reaction. Animals were exposed to repeated social separation stress and tested for individual differences in alcohol consumption. Animals were genotyped for -1002 T > G, and the data were analyzed using analysis of variance. SETTING National Institutes of Health Animal Center. Subjects Ninety-six rhesus macaques. Main Outcome Measure Behavior arousal during social separation stress and ethanol consumption. RESULTS The G allele altered binding of regulatory proteins in all nuclear extracts tested, and -1002 T > G resulted in lower levels of NPY expression in the amygdala. Macaques exposed to adversity had lower cerebrospinal fluid NPY levels and exhibited higher levels of arousal during stress, but only as a function of the G allele. We also found that stress-exposed G allele carriers consumed more alcohol and exhibited an escalation in intake over cycles of alcohol availability and deprivation. CONCLUSIONS Our results suggest a role for NPY promoter variation in the susceptibility to alcohol use disorders and point to NPY as a candidate for examining gene x environment interactions in humans.