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From the Office of Dr. David Crews
Focus of Research
Note: (numbers in parentheses indicate publication number)

6. Epigenetic Modifications and the Development of Behavior.(NIMH R21 MH068273 and NIEHS R21-ES017538)

Epigenetic modifications and the development of behavior involve two distinct mechanisms. “Transgenerational” effects can be observed if the environmental factors that bring about the epigenetic modification simply continue to persist. For example, if the diet, behavior, or environmental toxicant such as lead continues to be present in the environment, then epigenetic modification will be manifest each generation. This environmentally-induced epigenetic state can be reversed by removal of the factor or addition of a different environmental factor. This mitotic transgenerational effect is termed ‘Context Dependent’ epigenetic change. Reviews of this work can be found n articles 301, 316, 324, 334, 342, 347, and 351. ‘Germline Dependent Imprinted’ epigenetic modification is fundamentally different than Context Dependent epigenetic modification. Here the imprint is mediated through the germline and tends to be sex-linked. That is, a genomically imprinted epigenetic modification is transferred to subsequent generations because the change in the epigenome is incorporated into the germline. Thus, the effect is manifest each generation in the absence of the causative agent. For example, in such instances the DNA methylation of heritable epialleles are passed through to subsequent generations rather than being erased as occurs normally during gametogenesis and shortly after fertilization. There are two reasonably large bodies of research indicating that in rodents, the prenatal environment (who your fetal neighbors are) and the postnatal period (the nature and quantity of maternal care) influence the adult behavioral phenotype and underlying brain neurochemistry. Normally, litter composition reflects the sex ratio produced at birth, but this continuity does not mean that the pre- and postnatal periods are without their own specific influence, or that the cumulative process has unique emergent properties that are unrecognized. The research demonstrating that prenatal (intra-uterine) sex ratio influences adult behavior has not continued to control for sex ratio of the litter postnatally, leaving open the possibility that the behavior of the mother to the newborn pups may contribute to the phenomena. Research on the importance of maternal behavior in shaping adult behavior is typically associated with the work by Meaney and colleagues who find that the quality and frequency of maternal behavior results in differences in stress reactivity and brain neurochemistry in the offspring. It is important to keep in mind though that this work focuses on extremes in mothering types, that is, 1 standard deviation above and below the mean in mothering ability, not the average mothers. In addition, this work has not taken into account the prenatal sex ratio of the pregnant mother, which has been shown to affect mothering quality. In our paper to be published in Psychological Science, we disassociated these two life periods, controlling for each and then reassembling litters. The results were startling. That is, the sexuality of the adult male is due to the sex ratio of the litter and not the prenatal sex ratio (intra-uterine position) or the quality of maternal behavior a pup receives. After controlling for prenatal sex ratio we find that males raised in female-biased litters exhibit less mounting compared to males raised in litters of equal sex ratio or in male-biased litters. Further, males from female-biased litters are less attractive to sexually receptive females. These differences are not erased by sexual experience, suggesting that the effects of the sibling environment are permanent. Surprisingly these males compensate for their lower attractiveness by being more efficient copulators. The paper in Frontiers in Behavioral Neuroscience details a similar approach with genetically modified mice. Typically, the investigator will take individuals without consideration of the litter in which they were born. This we show is a mistake as the litter has the inherent confound of varying sex ratio and, in knockout mice created by breeding heterozygotes, the additional confound of genotype (i.e., the ratio of wildtype, heterozygote, or knockout male and female offspring). Deconstructing these two confounds demonstrates that the both the sex and genotype of the sibling shapes the individual's adult behaviors and the brain mechanisms that underlie them. Specifically, working with a common mouse model (estrogen receptor ? knockout), litters were reconstituted shortly after to birth to control for Sex ratio and Genotype ratio. We found that when adult, both males and females showed significantly altered (and in different directions) according to the Sex and Genotype of their littermates. Even more importantly, the patterns of metabolic activity in brain (limbic nuclei) were organized differently depending upon this early family environment. Thus, complex behavioral traits, as well as their functional neural networks, are altered fundamentally by the litter environment in which the individual is raised. This work is also important because most studies today focus at the level of the control and consequences of gene action and hence miss the larger picture. The animal is viewed almost as a 'host' carrying the gene of interest. In this genocentric age the ever-increasing use of genetically modified animals in behavioral neuroscience research makes in imperative that practitioners be aware of this important formative element. The finding that functional neural systems can be re-organized depending upon the composition of the litter in which the individual develops is startling yet yields a deeper understanding of how neural systems are organized early in life. When taken together, this work illuminates the importance of experience in behavioral development. We can never lose sight of the continuity of life processes, and that it can be profitably viewed as the cumulation of discrete segments. Each period emerges from what goes before and, at the same time, sets the stage for what follows. Thus, each period has its own characteristic ethologies and particular contribution to the behavioral phenotype. Our task is to determine how these contributions can be deconstructed so that each period can be studied both in its own right and how it interacts with the other periods. Our current work involves the interaction of germline-dependent and context-dependent epigenetic modifications. Here the specific questions is whether the transgenerational epigenetic imprint modifies the effects of stress?