Genotype and Rearing Over the past 4 years, we have investigated a common polymorphism involving an insertion/deletion in the promoter region for the serotonin transporter gene (5-HTTLPR). In a study performed in collaboration with Mary Schneider and Gary Kraemer at the University of Wisconsin, Dr. Newman showed that genotype affects the outcome of fetal alcohol exposure. Fetal alcohol exposed (FA-exposed) carriers of the s rh5-HTTLPR allele exhibited increased neonatal irritability and increased limbic-hypothalamic-pituitary-adrenal (HPA) axis reactivity compared to FA-exposed monkeys homozygous for the l allele. Consistent with other studies performed by Dr. Barr and her team, genetic predisposition and FA-exposure combine to substantially alter HPA axis responses to stressors, indicating that this may be a risk factor for the expression of behavioral and mental disorders in some environments, and a protective factor in others. This study is important in showing that the effect of FA-exposure on behavioral and neuroendocrine regulation is dependent not only on alcohol exposure but that genotype must also be considered when assessing risk and outcome. Most of the studies performed here within the Poolesville laboratory have investigated the effect of long-term or permanent maternal deprivation, but studies in rodents suggest that more subtle, short-term maternal separations may also result in HPA deficits. In a collaboration with M. Sanchez, P. Plotsky, and J. Winslow at Yerkes, Dr. Newman investigated the short- and long-term consequences of repeated maternal separation during sensitive periods of social and emotional development (3-6 months of age). We examined immediate effects of the separation protocol on hypothalamic-pituitary adrenal (HPA) axis function (diurnal cortisol rhythm and reactivity) of infants and mothers, as well as on the mother-infant relationship. We also studied long-term effects on emotionality and HPA axis function. Repeated short-term maternal separations produced significant alterations in mother-infant contact, as well as a sex-dependent increase cortisol reactivity to the separations. In addition, when compared to their half-sibling controls these separations resulted in long-term alterations in HPA axis activity and emotionality, including a flattened diurnal secretory rhythm of cortisol and elevated acoustic startle reactivity that remained for several months after the end of the separation protocol. Moreover, this effect appeared to be lasting, with the cortisol reactivity to the separations predicting alterations in cortisol diurnal rhythm (flattening) and increased emotionality (increased acoustic startle response) at later ages. The infants? cortisol reactivity to maternal separation and daily diurnal rhythm, however, varied showing a 5-HTT genotype x separation experience interaction. Consistent with other studies by Dr. Barr, the effects were different in males and females. Our results are consistent with the hypothesis that early adverse parental experiences may alter normal physiological and emotional development, posing a risk factor for the development of anxiety/mood disorders during childhood and adolescence. But they also demonstrate that sex and genetic factors play an important role modulating this vulnerability. NPY, MOR, and COMT Other rhesus genes that have been studied in our laboratory over the last year include those for Neuropeptide Y (NPY) and the mu opiod receptor (MOR). In humans, a functional variant in the NPY gene (T1128C, or Leu7Pro) influences propeptide processing, leading to decreased release of mature NPY from neuron terminals. Because NPY has anxiolytic properties, this polymorphism would be expected to be associated with anxiety and anxiety-related disorders, and, in fact, the Leu7Pro has been associated with Type I alcoholism. Dr. Barr has sequenced the coding regions, the exon/intron boundaries and a portion of the 5?flanking region of the rhNPY gene, and found there to be 96% sequence similarity between rhesus and humans for the coding regions and 87% similarity in noncoding regions. Although she has not identified any orthologues to the functional Leu7Pro polymorphism, she identified 6 SNPs in the 5?flanking region of the rhesus NPY gene. One of these is adjacent to a GRE 1/2 site, which could potentially disrupt glucocorticoid receptor-mediated regulation of NPY transcription. Consistent with the demonstrated role for corticosteroids in induction of NPY, Dr. Barr found that animals carrying this gene variant exhibit decreased cerebrospinal fluid levels of NPY. Although they have not yet begun this phase of the NPY project, Dr. Barr and Mr. Stephen Lindell will be working together to generate pDsRed NPY constructs for the purpose of performing functional assays. Whether any of the NPY variants influence behavioral phenotypes (for example, stress reactivity or alcohol consumption) or whether they interact with early infant rearing, are also yet to be tested. In humans, a variant in the Mu opiod receptor gene (MORA118G) produces a 3-fold increase in affinity and has been associated with increased subjective euphoria following alcohol administration in human subjects. Some studies have also demonstrated this polymorphism to be associated with naltrexone response and with low basal levels of corticosteroids. Dr. Barr has found that an orthologous polymorphism in rhesus macaques (MORC77G) is associated with low levels of cortisol and, moreover, that there is an association between the G allele and increased alcohol preference, particularly among males. This is of interest, given that male alcoholics and drug abusers are more likely to respond to mu opiod receptor blockade than are females. Whether the MORC77G polymorphism and an individual?s sex also affect naltrexone treatment response in macaques is yet to be studied. COMT is a catabolic enzyme that metabolizes monoamines and is an important candidate gene in human neuropsychiatric disease. During the past year, Dr. Newman found two single nucleotide polymorphisms within coding regions, and there are early indications that others are present as well. ARRAYS We have studied the 5-HTTLPR polymorphism extensively in our lab and have demonstrated interactions between rearing experience and rh5-HTTLPR on many behavioral phenotypes of interest, including affective responding, behavioral withdrawal, and HPA axis response to stress, but only among animals exposed to early adversity. Studies identifying the molecular players underlying these GxE interactions are yet to be examined. We have harvested whole brains from 18 male rhesus macaques (5 mother-reared l/l, 4 mother-reared l/s, 5 peer-reared l/l, and 4 peer-reared l/s) for performing a study utilizing microarray technology to screen for rearing and genotype effects on mRNA levels in the brain. Using gene filters with approximately 6000 human cDNAs, Dr. Barr has performed pilot studies in collaboration with Stan Watson, Juan Lopez and Robert Thompson at the University of Michigan, using cerebrocortical tissue (Area 46) from 8 of the archived rhesus brains. They have identified more than 500 genes that are differentially expressed in peer- and mother-reared rhesus. These preliminary data also suggest the expression of a subset of these genes may be modified by rh5-HTTLPR genotype. Currently, we are working on verifying our results using TaqMan and are testing other platforms to determine which will work optimally with macaque brain tissue.
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