LAUSR

Background Determining whether stress will lead to future stress-resilience or vulnerability depends on a delicate balance of a probably adjustable stress response set-point. The adjustment of this set-point is most likely effective during sensory postnatal development and involves the hypothalamus-pituitary-adrenal (HPA) axis. Here we demonstrate that heat stress during the critical period of thermal control establishment in 3-day-old chicks, renders habituated or sensitized response, a week later, depending on the ambient temperature i.e. moderate heat lead to future heat resistance while harsh temperatures lead to heat vulnerability. Furthermore, these changes are governed by epigenetic modifications, specifically DNA methylation (5-methycytosine - 5mc) and DNA hydroximethylation (5-hydroxymethylcytosine - 5hmc). Methods In this study we made use of several innovative methods, primarily: RNA and DNA purification Reverse transcription to cDNA qRT-PCR Global hydroxymethylated DNA (5-hmC) Quantification, ELISA Global DNA methylation (5mC) Quantification, ELISA Bisulfit treatment followed by ligation of DNA to plasimd and transformation to E.coli DNA sequencing of specific targets of interest from the CRH gene, from the cloned DNA Western blotting Chromatin immunoprecipitation (ChIP) Immunohistology Intracranial injection of different treatments to live 3 day old chicks. Results After concluding that CRH plays a significant role in determining either heat resilience or vulnerability response later in life, we examine epigenetic changes as a potential mechanism. Interestingly, global 5mc% and global 5hmc% changed significantly between the two different heat conditioned groups, a week after their conditioning. Resilient chicks displayed low 5mc% alongside high 5hmc%, while vulnerable chicks displayed an opposite pattern of high 5mc% and low 5hmc%. This pattern was repeated when specific CpG methylation sites along the CRH gene were evaluated, indicating these dynamic changes contribute to the differences in the expression levels of CRH, and therefore might take part in the formation of the habituation and resilience phenomena. By injecting Parp (poly ADP-ribose polymerase) inhibitor - which is required for activation of Tet (ten-eleven translocation demethylation intermediate family) activity, the 5hmc% declined, along with a decline in 5mc%. This in turn resulted in obliteration of the ambient temperature resilience showed previously by non-injected chicks, when compared to their control counterparts. Discussion Long term changes in body temperature, mRNA expression and Cort levels of chicks conditioned at different intensities of stress, define the basis of resilience versus vulnerability response later in life. Here we show that these changes are directly affected by CRH, demonstrated by injecting CRH or CRH-antisense which inverted the "normal" response. Furthermore, we propose that DNA methylation and de-methylation take part in this long term phenomena as indicated both by global and gene specific 5mc% and 5hmc% one week after heat conditioning. Moreover, injection of PAPR inhibitor, which is required for activation of the Tet demethylation intermediate family activity, resulted in obliteration of the ambient temperature resilience showed previously by non-injected chicks. It is highly plausible to assume that the mechanism underlying the balance between heat stress resilience and vulnerability is regulated by epigenetic adaptations.