ARTICLES nature neuroscience Epigenetic programming by maternal behavior 8 Ian CG Weaver, 2, Nadia Cervoni, Frances A Champagne, 2, Ana C D'Alessio Shakti Sharma! Jonathan R Seckl#, Sergiy Dymov Moshe Szyf2,3& Michael J Meaney,2 Here we report that increased pup licking and grooming(LG)and arched-back nursing(ABN) by rat mothers altered the offspring LG and abn were found to have differences in DNA methylation, as compared to offspring of low-LG-ABN'mothers. These g differences emerged over the first week of life, were reversed with cross-fostering, persisted into adulthood and were associated g with altered histone acetylation and transcription factor (NGFI-A)binding to the GR promoter. Central infusion of a histone a deacetylase inhibitor removed the group differences in histone acetylation, DNA methylation, NGFI-A binding GR expression and hypothalamic-pituitary-adrenal(HPA)responses to stress, suggesting a causal relation among epigenomic state, GR expression nd the maternal effect on stress responses in the offspring Thus we show that an epigenomic state of a gene can be established through behavioral programming, and it is potentially reversible 8 Through undefined epigenetic processes, maternal effects influence inhibits CRF synthesis and release, thus dampening HPA responses to s the development of defensive responses to threat in organisms rang- stress". The adult offspring of high- compared with low-LG-ABN o ing from plants to mammals,. In the rat, such effects are mediated by mothers show increased hippocampal GR expression and enhanced mission of individual differences in stress responses from mother to high-LG-ABN mothers show decreased hypothalamic CRF expre s offspring -. Mother-pup contact in the rat primarily occurs within sion and more modest HPA responses to stress. Eliminating the dif- e litter, licks and grooms her pups, and nurses while occasionally experience on HPA responses to stress in adulthood 2, suggesting that licking and grooming the pups. There are stable individual differ- the difference in hippocampal GR expression serves as a mechanism ences in two forms of maternal behavior-LG and ABN--over the for the effect of early experience on the development of individual first week of lactation6-10. Such naturally occurring variations in differences in HPA responses to stress o maternal behavior are associated with the development of individual In vivo and in vitro studies suggest that maternal LG and ABN differences in behavioral and HPA responses to stress in the offspring. increase GR gene expression in the offspring through increased sero As adults, the offspring of high-LG-ABN' mothers are less fearful and tonin(5-HT)activity at 5-HT7 receptors, and the subsequent activa show more modest HPA responses to stress than the offspring of low- tion of cAMP and CAMP-dependent protein kinase activity3-15Both LG-ABN mothers"-. Cross-fostering studies show that the biological the in vitro effect of 5-HT and the in vivo effect of maternal behavior offspring of low-LG-ABN mothers reared by high-LG-abn dams on gR gene expression are accompanied by an increased hippocam resemble the normal offspring of high-LG-ABN mothers(and vice pal expression of nerve growth factor-inducible protein A(NGFI-A,a versa"). These findings suggest that variations in maternal behavior transcription factor also known as egr-1, krox-24, zenk and zif-268) serve as a mechanism for the nongenomic transmission of individual The non-coding exon I region of the hippocampal GR includes a pre differences in stress reactivity across generations, 5. 9. The critical moter region, exon 17, containing a binding site for NGFI-A6 question concerns the mechanisms whereby these maternal effects, or(Fig. la). Splice variants of the GR mRNA containing the exon 17 other forms of environmental ' programming, are sustained over the sequence are found predominantly in the brain, and the expression of lifespan of the animal GR mRNAs containing the exon 1, sequence is increased in the off- Maternal behavior in the rat permanently alters the development of spring of high-LG-ABN mothers or following manipulations that HPA responses to stress through tissue-specific effects on gene increase maternal licking and grooming(Weaver, L.C. G. et al., Soc. expression. The magnitude of the HPA response to acute stress is a Neurosci. Abstr. 697. 15, 2001), suggesting that the use of this promoter function of hypothalamic corticotropin-releasing factor (CRF) is enhanced as a function of maternal care. Although these findings release, which activates the pituitary-adrenal system. There are also might explain the increased GR expression in the neonate, we are left adulatory influences, such as glucocorticoid negative feedback that with the question of how the effect of maternal care might persist into Hospital Research Center, 6875 LaSalle Blvd., Montreal, Quebec H4H 1R3, Canada. 2McGill Program for the Study of Behaviour, Genes and Environment ology and Therapeutics, McGill University, 3655 Sir William Osler Promenade, Montreal, Quebec H3G 1Y6, Canada. Molecular Centre, Edinburgh University, Western General Hospital, Edinburgh EH4 2XU, UK. Correspondence should be addressed to M.J. M meaney @mcgill. ca)or M.S. ( moshe szyf@mcgill. ca). d online 27 June 2004; corrected 27 July 2004(details online); doi: 10.1038/nn1276 NATURE NEUROSCIENCE VOLUME 7 NUMBER 8 AUGUST 2004 847ARTICLES Through undefined epigenetic processes, maternal effects influence the development of defensive responses to threat in organisms rang￾ing from plants to mammals1,2. In the rat, such effects are mediated by variations in maternal behavior, which serve as the basis for the trans￾mission of individual differences in stress responses from mother to offspring3–5. Mother-pup contact in the rat primarily occurs within the context of a nest-bout, which begins when the mother approaches the litter, licks and grooms her pups, and nurses while occasionally licking and grooming the pups6. There are stable individual differ￾ences in two forms of maternal behavior—LG and ABN—over the first week of lactation6–10. Such naturally occurring variations in maternal behavior are associated with the development of individual differences in behavioral and HPA responses to stress in the offspring. As adults, the offspring of ‘high-LG-ABN’ mothers are less fearful and show more modest HPA responses to stress than the offspring of ‘low￾LG-ABN’ mothers6–9. Cross-fostering studies show that the biological offspring of low-LG-ABN mothers reared by high-LG-ABN dams resemble the normal offspring of high-LG-ABN mothers (and vice versa9). These findings suggest that variations in maternal behavior serve as a mechanism for the nongenomic transmission of individual differences in stress reactivity across generations4,5,9. The critical question concerns the mechanisms whereby these maternal effects, or other forms of environmental ‘programming’, are sustained over the lifespan of the animal. Maternal behavior in the rat permanently alters the development of HPA responses to stress through tissue-specific effects on gene expression. The magnitude of the HPA response to acute stress is a function of hypothalamic corticotropin-releasing factor (CRF) release, which activates the pituitary-adrenal system. There are also modulatory influences, such as glucocorticoid negative feedback that inhibits CRF synthesis and release, thus dampening HPA responses to stress11. The adult offspring of high- compared with low-LG-ABN mothers show increased hippocampal GR expression and enhanced glucocorticoid feedback sensitivity7,9. Predictably, adult offspring of high-LG-ABN mothers show decreased hypothalamic CRF expres￾sion and more modest HPA responses to stress7. Eliminating the dif￾ference in hippocampal GR levels abolishes the effects of early experience on HPA responses to stress in adulthood12, suggesting that the difference in hippocampal GR expression serves as a mechanism for the effect of early experience on the development of individual differences in HPA responses to stress5. In vivo and in vitro studies suggest that maternal LG and ABN increase GR gene expression in the offspring through increased sero￾tonin (5-HT) activity at 5-HT7 receptors, and the subsequent activa￾tion of cAMP and cAMP-dependent protein kinase activity13–15. Both the in vitro effect of 5-HT and the in vivo effect of maternal behavior on GR gene expression are accompanied by an increased hippocam￾pal expression of nerve growth factor-inducible protein A (NGFI-A, a transcription factor also known as egr-1, krox-24, zenk and zif-268). The non-coding exon 1 region of the hippocampal GR includes a pro￾moter region, exon 17, containing a binding site for NGFI-A16 (Fig. 1a). Splice variants of the GR mRNA containing the exon 17 sequence are found predominantly in the brain, and the expression of GR mRNAs containing the exon 17 sequence is increased in the off￾spring of high-LG-ABN mothers or following manipulations that increase maternal licking and grooming16 (Weaver, I.C.G. et al., Soc. Neurosci. Abstr. 697.15, 2001), suggesting that the use of this promoter is enhanced as a function of maternal care. Although these findings might explain the increased GR expression in the neonate, we are left with the question of how the effect of maternal care might persist into 1Douglas Hospital Research Center, 6875 LaSalle Blvd., Montréal, Québec H4H 1R3, Canada. 2McGill Program for the Study of Behaviour, Genes and Environment and 3Department of Pharmacology and Therapeutics, McGill University, 3655 Sir William Osler Promenade, Montréal, Québec H3G 1Y6, Canada. 4Molecular Medicine Centre, Edinburgh University, Western General Hospital, Edinburgh EH4 2XU, UK. Correspondence should be addressed to M.J.M. (michael.meaney@mcgill.ca) or M.S. (moshe.szyf@mcgill.ca). Published online 27 June 2004; corrected 27 July 2004 (details online); doi:10.1038/nn1276 Epigenetic programming by maternal behavior Ian C G Weaver1,2, Nadia Cervoni3, Frances A Champagne1,2, Ana C D’Alessio3, Shakti Sharma1, Jonathan R Seckl4, Sergiy Dymov3, Moshe Szyf2,3 & Michael J Meaney1,2 Here we report that increased pup licking and grooming (LG) and arched-back nursing (ABN) by rat mothers altered the offspring epigenome at a glucocorticoid receptor (GR) gene promoter in the hippocampus. Offspring of mothers that showed high levels of LG and ABN were found to have differences in DNA methylation, as compared to offspring of ‘low-LG-ABN’ mothers. These differences emerged over the first week of life, were reversed with cross-fostering, persisted into adulthood and were associated with altered histone acetylation and transcription factor (NGFI-A) binding to the GR promoter. Central infusion of a histone deacetylase inhibitor removed the group differences in histone acetylation, DNA methylation, NGFI-A binding, GR expression and hypothalamic-pituitary-adrenal (HPA) responses to stress, suggesting a causal relation among epigenomic state, GR expression and the maternal effect on stress responses in the offspring. Thus we show that an epigenomic state of a gene can be established through behavioral programming, and it is potentially reversible. NATURE NEUROSCIENCE VOLUME 7 | NUMBER 8 | AUGUST 2004 847 © 2004 Nature Publishing Group http://www.nature.com/natureneuroscience