As in previous years, a major focus of this project has been detailed longitudinal study of the behavioral and biological consequences of differential early social rearing, most notably comparing rhesus monkey infants reared by their biological mothers in pens containing adult males and other mothers with same-age infants for their first 6-7 months of life (MR), with monkeys separated from their mothers at birth, hand-reared in the labs neonatal nursery for their first month and then raised in small groups of same-age peers for their next 6 months (PR). In a third standard rearing environment, surrogate-peer rearing (SPR), infants are separated from their mothers and nursery reared just like PR infants, but then at 1 month are housed in individual cages containing an inanimate surrogate mother and additionally are placed in a play cage with 3 other like-reared peers for 2 hours daily for the next are 6 months. At 7 months of age, MR, PR, and SPR infants are all moved into one large pen, where they all live together until puberty. Thus, the differential social rearing occurs only for the first 6-7 months;thereafter MR, PR, and SPR all share the same physical and social environment. We previously demonstrated that PR monkeys cling more, play less, tend to be much more aggressive, and exhibit much greater behavioral and biological disruption during and immediately following short-term social separation at 6 months of age than MR monkeys, and they also exhibit deficits in serotonin metabolism (as indexed by chronically low values of CSF 5-HIAA), as do SPR monkeys, and they also have significantly lower levels of 5-HTT binding throughout many brain regions than do MR subjects. Many of these differences between MR and PR monkeys persist throughout the childhood years. Research in collaboration with colleagues from NIAAA has demonstrated that both PR and SPR monkeys also consume significantly more alcohol when placed in a happy hour situation as adolescents and young adults. This past year we published data extending these rearing condition differences to include developmental changes in plasma concentrations of BDNF and NGF (6), behavioral lateralization (4), acoustic startle response patterns following fluxotine treatment (11), and structural differences in various brain regions (15). Finally, we found that PR monkeys had chronically higher levels of cortisol obtained from hair samples than did MR and SPR monkeys throughout their first year of life, whereas during the second year SPR monkeys had higher levels than the other two rearing groups. During the past year we finished data collection and preliminary analyses for two projects comparing the results of genome-wide scans of blood and tissue samples collected from differentially reared monkeys. First, in collaboration with colleagues from McGill and Wake Forest Universities, we assessed methylation patterns in lymphocyte T cells and pfc obtained from 8 yr-old adult MR and SPR subjects who had been living in the same or comparable physical and social environments since 7 months of age. Over 4,000 genes, i.e. almost 1/5 of the entire genome, showed significant differences in methylation as a function of early experience in both T cells and pfc, with approximately half of the affected genes significantly more methylated in MR-derived samples and the remaining half significantly more methylated in SPR-derived samples. Additionally, there was considerable overlap among the specific genes in T cell and pfc that were differentially methylated: about 30% of the genes showed the same pattern of significant rearing condition differences in both T cells and pfc, whereas approximately 25% of the genes showed exactly the opposite pattern in T cells vs. pfc. Furthermore, there were a substantial number of genes in specific known pathways (e.g., various IL, monoamine transporter, and CRH pathways) that were differentially methylated as afunction of differential early experience again in both T cells and pfc. The second project utilizing genome-wide scans of samples obtained from differentially reared monkeys, a collaboration with colleagues from UCLA and the University of Chicago, involved microarray scanning of leukocyte samples obtained from MR, PR, and SPR infants at 7 months of age to determine possible differences in gene expression. As in the afore-mentioned case of differential methylation patters, significant differences in gene expression as a function of differential early experience were found throughout the entire genome. Relative to those of MR infants, genes upregulated in leukocytes from PR infants included the pro-inflammatory cytokine IL8, a diverse array of transcription factors, regulators of cell proliferation, and multiple T cell-associated transcripts. Genes notably suppressed in leukocytes from PR infants included those involved in Type 1 Interferon mediated antiviral responses, several immunoglobulin-encoding genes involved in B cell antibody production, several hematopoietic growth regulators, monocyte-associated gene products, and a variety of memory T cell-related markers. Analyses of gene expression patterns in SPR infants yielded results qualitatively similar to those of PR monkeys, but with differences with MR subject generally quantitatively less pronounced than those of PR subjects, albeit with a number of exceptions. Taken together, these two sets of genome-wide analyses demonstrate that the consequences of differential early experience for monkeys clearly extend to the level of gene methylation patterns and actual expression. Another major focus of recent research for this project has involved characterizing interactions between differential early social rearing and polymorphisms in several candidate genes (G X E interactions), most notably the 5-HTT-LPR gene and the MAO-A gene, for a variety of measures of behavioral and biological functioning throughout development in MR and PR rhesus monkeys. This past year we identified significant G x E interactions involving the 5-HTT-LPR polymorphism among MR infants whose mothers differed significantly in their care-giving patterns. Infants whose mothers exhibited low levels of ventral contact and grooming vocalized and explored less and were more passive in an open field test but only if they carried the "short" 5-HTT-LPR allele. In addition, in collaboration with colleagues from NIAAA and the University of Wurzburg, we identified additional functional polymorphisms in the corticotrophin releasing factor (CRH)2A gene (1), the mu opioid receptor gene (3) , the NPY gene (10), the DRD1 5UTR gene (12), the BDNF gene, and the NOS-1 gene. We were also able to characterized specific G x E interactions with respect to behavioral responses to social separation by juvenile rhesus monkeys for the 5-HTT-LPR and NPY polymorphisms, as well as in several measures of alcohol preference and consumption among young adult monkeys for the CRH, NPY, and DRD1 5UTR polymorphisms (1, 10, 12).