One of the most powerful advantages of the primate model that our laboratory has developed is the capacity to closely control and investigate the role of genes and environment using a developmental paradigm. 1. During the past year, our research included studies of genetic and environmental mechanisms that produce low cerebrospinal fluid concentrations of 5-hydroxyindoleacetic acid (CSF 5-HIAA) concentrations and alcohol consumption. We continued our pedigree analyses investigating genetic and environmental influences on alcohol consumption. In an analysis of factors affecting alcohol consumption in nonhuman primates that for the first time utilized a large population sample, we were able to show that age, sex, drinking conditions, rearing background, and strain were important variables influencing alcohol consumption. When these where controlled for, heritable influences contributions accounted for about 30% of the variance in alcohol consumption in macaques. In that same study, we replicated earlier findings showing that monkeys reared in peer-only groups, without adults present, were more likely to consume alcohol in excess.2. Molecular genetic studies were also pursued. In collaboration with Drs. K. Peter Lesch and Armin Heils at the University of Wurzberg, Germany, we found an association between length variation in the serotonin transporter gene regulatory region (5-HTTLPR) CSF 5-HIAA concentrations and competitive aggression. In humans the 5-HTTLPR is associated with anxiety, affective disorders, and substance abuse, but inconsistently so. The principle finding from our manuscript, a genotype/environment interaction effect on CSF 5-HIAA, suggests a potential source to explain serotonin variability in the human literature. Data showed a similar effect on aggression, with monkeys having the short allele exhibiting more aggression. Unlike the effect on CSF 5-HIAA concentrations, this augmenting effect on aggression appeared to be independent of rearing condition. This developmental model was also used to assess the developmental pathways for differences in behavior and CSF 5-HIAA concentrations. In a collaboration with Drs. Maribeth Champoux and Stephen Suomi in NICHD, we assessed the contribution of genetic and environmental variables on development during a standardized neonatal examination of macaque infants. Assessments were conducted on postnatal days 14 and 30 on 322 laboratory-born infants. Test items were condensed into Orientation, Motor Maturity, Activity, and State Control clusters, measures of motoric development, cognition, and temperament. Heritability values significantly greater than zero (p < .05) were obtained for State Control and Orientation clusters on Days 14 and 30, and for the Activity cluster on Day 14 only, but not for Motor Maturity. Heritability values changed with age, with the pattern of change differing across clusters. In second study with this group, we found that neonatal monkeys with the short allele were deficient in these neonatal measures designed to measure CNS maturity and development. Other data show that more aggressive strains, such as the Chinese rhesus are more likely to possess the less efficient serotonin transporter genotype (5HTTLPR-short allele) first characterized by Lesch. These findings suggest that low CSF 5-HIAA concentrations result, at least in part, from an altered gene expression in the transporter region, but that early rearing experiences affect its phenotypic expression. This may result in behavior differences that can be tracked developmentally.3. Three other projects involving analysis of other candidate serotonergic genes are underway. In a collaboration with Drs. David Goldman and Andrew Bergen he monkeys have been typed for an MAO-A polymorphism similar to one associated with panic disorders in humans. By analyzing monkeys acute response to a social stressor we found preliminary evidence for an association the MAO-A genotype and stress reactivity. In a second project, the serotonin 1A receptor was assessed as a target for candidate gene analysis, and a large set of monkeys genotyped for a 5HT1A polymorphism. Analyses are underway to assess the phenotypic expression of the discovered polymorphisms. THROMBOCYTE-MAO (trbcMAO) is in part genetically determined and is stable throughout life. There are a number of studies which have shown a correlation between trbcMAO and personality traits such as sensation seeking and impulsiveness, which in turn underlies connections between low trbcMAO and vulnerability for substance abuse and violent behavior. A third project involved a collaboration with Marie Asberg and Lars Oreland. We found a correlation between trbcMAO and CSF 5-HIAA in nonhuman primates. Moreover, we found a positive correlation between trbcMAO, impaired social dominance and excessive alcohol intake. These findings suggest that trbcMAO may act as a biological marker for the functioning of the central serotonin system which is related to social functioning and excessive alcohol consumption.4. A computerized apparatus was developed that enabled us to identify individual monkeys living in large social groups, dispense alcohol to them, and measure individual consumption patterns, thus allowing us to characterize large numbers of monkeys as high and low alcohol consumers. At the recommendation of our Scientific Counselors, during the past year, the apparatus was modified to provide a clearer choice between our sweetened alcohol solution and the sweetened vehicle that the alcohol solution is made from. This methodology will allow us for the first time to study chronic alcohol consumption for 24-hours a day and provide a technical solution to allow other researchers interested in modeling alcohol consumption in primates to perform such studies using limited personnel resources.5. A growing body of evidence indicates that corticotrophin releasing hormone (CRH) plays an important role in many of the classic behavioral and physiological responses to stress, including anxiety, activation of the hypothalamic-pituitary adrenal (HPA) axis, and stimulation of the sympathetic and adrenomedullary systems. This is important for alcohol researchers because stress and anxiety are thought to play a primary role in excessive alcohol consumption. In new initiative, we administered a non-peptide CRH type 1 receptor antagonist, antalarmin, to monkeys under stress. This collaboration with Drs. Phil Gold and Kamal Habib in NIMH showed that antalarmin significantly reduced indices of anxiety while enhancing environmental exploration, a behavior that typically occurs in the context of a non-threatening environment. In addition, antalarmin significantly inhibited stress-induced activation of the HPA axis, sympathomedullary system, and stress-induced increases in CSF CRH. These data demonstrate a significant role for CRH in the regulation of stress system in primates and suggest that CRH antagonists may be helpful in treating human disorders associated with stress system dysregulation such as excessive alcohol consumption, as well as anxiety disorders, and depression. Early life parental deprivation has been shown to predispose monkeys to major depression, excessive alcohol consumption, and anxiety disorders later in adulthood. In a second series of studies, we separated infant rhesus monkeys from their mother for a period of four weeks at the age of six months. CSF samples from those seventeen monkeys were obtained before and weekly after separating them from their mothers. In this study we found a significant correlation between the time after separation and CSF levels of CRH (Pearson r = 0.4814, p < 0.0001), suggesting that social stress induces a vigorous CRH response in infant monkeys.6. Our new program of study designed to investigate cognitive functioning and impulsivity in monkeys prone to high alcohol consumption and/or low CSF 5-HIAA concentrations continued this year. Under the direction of Dr. Allyson Bennett, new methodologies were initiated to test monkey cognitive skills using a computerized joystick system. This methodology allows experimenters to vary experimental demands to test specific parameters of cognition in primates. Studies compared subjects reared in one of three social conditions: In social groups with adults present (A.- Parental-reared) and in one of two social groups without adults present (B.- chronic peer-access i.e., Peer-reared, or (C.- daily exposure but limited to only a few hours, i.e., Surrogate-peer- reared). Results showed that subjects reared with their parents learned new cognitive tasks rapidly. Peer-reared subjects learned the same cognitive tasks, but at a slower rate than the mother- reared subjects. On the other hand, even after a large number of sessions the Surrogate-peer-reared subjects failed to learn the cognitive tasks. In a subsequent study, Dr. Bennett found that monkeys with low CSF 5-HIAA concentrations were more likely to engage in potentially risky behaviors when a reinforcer was present. When presented with an opaque black box that was baited with reinforcing food, monkeys with low CSF 5-HIAA concentrations were more likely to quickly touch the box, and spent more time in close proximity. When the box was constructed out of material that allowed the subjects to see inside the box, there was no difference between the monkeys with high and low CSF 5-HIAA concentrations in the time to touch or sit in close proximity to the black box. CSF HVA on the other hand, was not related to time to approach the boxes, but after the subjects approached the stimulus, it was related to the time spent manipulating both the clear and opaque boxes.7. Measurements of heart rate variability are widely used to assess both central as well as peripheral nervous system functioning. An important neuromodulator affecting reflex control of heart rate is serotonin. In a series of studies with Dr. Paolo DePetrillo, we found that monkeys with low CSF 5-HIAA concentrations exhibited high heart rate variability, suggesting reduced temporal regulation of the autonomic system in subjects with low CSF 5-HIAA concentrations. In a second series of studies conducted with Drs. Salem and Hibbeln (LMBB), we continued our investigation of the role of essential fatty acids in CNS serotonin functioning by assessing heart rate variability in subjects fed diets devoid of or rich in essential fatty acids. Subjects fed a diet high in essential fatty acids as infants were shown as adolescents to exhibit higher heart rate variability when compared to subjects fed a diet low in essential fatty acids, suggesting the importance of essential fatty acids in normative development.8. In most of our studies to date, the principle measure of CNS serotonin functioning is the removal of CSF to assay for serotonin metabolites. In humans, the invasiveness of this procedure clearly precludes frequent sampling and makes it especially difficult to perform such studies in children. As an alternative, the prolactin response to fenfluramine has been widely used to assess CNS serotonin functioning. However, obtaining a prolactin response after the fenfluramine challenge is also invasive. Not only must fenfluramine be injected, typically, blood is sampled every 30 minutes for up to 5 hours following fenfluramine administration. Prolactin is also found in the saliva. We hypothesized that salivary prolactin concentrations would correlate positively with cerebrospinal fluid (CSF) 5-hydroxyindoleacetic acid (5-HIAA). Salivary prolactin concentrations were positively correlated with CSF 5-HIAA concentrations. These findings support the possibility of using salivary prolactin concentrations as an index of CNS serotonin turnover in humans.9. A growing body of research indicates that the 5-HT system shows circannual seasonal variation in humans. Seasonal changes of CSF 5-HIAA concentrations indicated that CSF 5-HIAA concentrations were significantly increased in the fall (October and November), the height of the breeding season, with no evidence of differences between winter and spring. This finding suggests that like humans, nonhuman primates also show seasonal variation in CNS serotonin functioning, and suggest an evolutionary basis for seasonal variation in CNS serotonin functioning.
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