ARTICLES panied by an increase in hippocampal GR expression. This hypoth- thylated exon 1, GR promoter contributes to the attenuation of Gr esis is supported by the results(Fig 5a)showing that hippocampal expression in low-LG-ABN adult offspring, whereas increased NGFI GR expression was significantly increased in TSA-treated offspring A binding to the hyperacetylated and hypomethylated response ele- of low-LG-ABN mothers to levels that were comparable to those of ment on the exon 1, Gr promoter in the offspring of the either the vehicle- or TSA-treated offspring of high-LG-abn high-LG-ABN mothers would serve to maintain the differences in mothers. ANOVA revealed highly significant main effects of Group gene expression. DNA methylation represents a stable epigenetic (F=7.4, P=0.01)and Dose(F=24.8, P<00001), as well as a sig- mark; therefore, our findings provide an explanation for the enduring nificant Group x Dose interaction effect(F=3. 1, P=0.048). Post- effect on mother-infant interactions over the first week of postnat hoc analysis indicated that 100 ng/ml TSA treatment significantly life on HPA responses to stress in the offspring. increases hippocampal GR expression in the offspring of low-LG ABN mothers (vehicle-treated low-LG-ABN vS. 100 ng/ml TSA- DISCUSSION 8 treated low-LG-ABN,"P < 0.001), such that there is no longer a Further studies are required to determine how maternal behavior significant difference in hippocampal GR expression between the alters the epigenetic status of the exon 1, GR promoter. In addition, ffspring of low-or high-LG-ABN mothers(100 ng/mI TSA treated the exact causal relationship between DNA methylation and altered low-LG-ABN vS 100 ng/ml TSA treated high-LG-ABN, P>0.90). histone acetylation and NGFI-A binding remains to be defined. Ithough TSA treatment significantly induced GR expression in Nevertheless, our findings provide the first evidence that maternal low-LG-ABN adult offspring, global abundance of protein in the behavior produces stable alterations of DNA methylation and chro- s hippocampus was not apparently increased, as indicated by the matin structure, providing a mechanism for the long-term effects of equal a-tubulin immunoreactivity(Fig. 5a). In comparing the maternal care on gene expression in the offspring. These studies offer a vehicle-treated groups, note that the effect of maternal care on GR an opportunity to clearly define the nature of gene-environment gene expression.9 is subtler than the more pronounced effect on interactions during development and how such effects result in the the methylation status of the 5 CpG dinucleotide(site 16) within sustained environmental programming of gene expression and the exon 1, promoter(Fig 4b).However, in previous studies 6 we function over the lifespan. It is important to note that maternal E found evidence for multiple promoters reg ulating ects on expression of defensive responses, such as increased g Pession suggesting that exon 1, is but one of the regulatory HPA activity, are a common theme in biology. such that the magni- 8 sequences determining GR expression within the hippocamp ude of the maternal influence on the development of HPA and behavioral responses to stress in the rat should not be surprising o Reversal of maternal effect on HPA responses to stress Maternal effects on defensive responses to threat are apparent in As adults, the offspring of high-LG-ABN mothers show more mod- plants, insects and reptiles. Such effects commonly follow from the est HPA responses to stress than the offspring of low-LG-ABn exposure of the mother to the same or similar forms of threat and mothers. The effect of maternal care on HPA responses to stress may represent examples whereby the experience of the mother is seems to be, in part, associated with differences in hippocampal Gr translated through an epigenetic mechanism of inheritance into levels and glucocorticoid negative feedback sensitivity?. Given that phenotypic variation in the offspring. Thus, maternal effects could TSA treatment reversed the group difference in hippocampal gr result in the transmission of adaptive responses across genera- expression, we examined the adrenocortical responses to stress in a tions,. Indeed, among mammals, natural selection may have separate cohort of vehicle- and TSA-treated animals. Central infu- shaped offspring to respond to subtle variations in parental behavior sion of TSA completely eliminated the maternal effect on HPA as a forecast of the environmental conditions they will ultimately responses to acute stress( Fig 5b). Statistical analysis of the plasma face once they become independent of the parent. Epigenetic modi- corticosterone data revealed significant effects of Group(F=4.3, fications of targeted regulatory sequences in response to even rea- P=0.048), Treatment(F=4.3, P=0.046)and Time (F=27.3, sonably subtle variations in environmental conditions might then P<00001), as well as a significant Group X Treatment interaction serve as a major source of epigenetic variation in gene expression and effect(F=7.7, P=0.009). Post-hoc analysis revealed that TSA treat- function, and ultimately as a process mediating such maternal ment significantly decreased basal plasma corticosterone in the off- effects. We propose that effects on chromatin structure such as those pring of low-LG-aBn mothers to levels comparable to those of described here serve as an intermediate process that imprints high-LG-ABN animals. Thus, plasma corticosterone responses to dynamic environmental experiences on the fixed genome, resulting restraint stress in the vehicle-treated adult offspring of low-LG- in stable alterations in phenotype ABN mothers were significantly(P 0.01) greater than those of TSA- and vehicle-treated adult offspring of high-LG-ABN mothers METHODS response to stress in the offspring of high-LG-ABN mothers was from Long-Evans hooded rats born in our colony from animals originally unaffected by TSA treatment obtained from Charles River Canada(St Constant, Quebec). All procedures In summary, central infusion of the HDAC inhibitor TSA enhanced were performed according to guidelines developed by the Canadian Council histone H3-K9 acetylation of the exon 1, GR promoter in the off- on Animal Care and protocol approved by the McGill University Animal Care spring of the low-LG-ABN mothers, increased NGFI-A binding to its Committee. Maternal behavior was scored and the adoption study was per- cognate sequence, induced hypomethylation of CpG dinucleotide formed as previously described. For further methodological details, see sequences in the promoter and eliminated the maternal effect on hi Supplementary Methods online. pocampal GR expression and the HPA response to stress. Sodium bisulfite mapping. Sodium bisulfite mapping was performed a These findings are consistent with idea that the maternal effect on previously described21, 22. The rat GR exon 1, genomic region(GenBank GR expression and HPA responses to stress is mediated by alterations accession number AJ271870)of the sodium bisulfite-treated hippocampal in chromatin structure. We propose that the reduced binding of DNA (50 ng/ml) was subjected to PCR amplification using outside primers NGFl-A to its response element on the hypoacetylated and hyperme-(forward: 1646-TTTTTTAGGTTTTTTTAGAGGG-1667: reverse: 1930- 852 VOLUME 7 NUMBER 8 AUGUST 2004 NATURE NEUROSCIENCEARTICLES panied by an increase in hippocampal GR expression. This hypoth￾esis is supported by the results (Fig. 5a) showing that hippocampal GR expression was significantly increased in TSA-treated offspring of low-LG-ABN mothers to levels that were comparable to those of either the vehicle- or TSA-treated offspring of high-LG-ABN mothers. ANOVA revealed highly significant main effects of Group (F = 7.4, P = 0.01) and Dose (F = 24.8, P < 0.0001), as well as a sig￾nificant Group × Dose interaction effect (F = 3.1, P = 0.048). Post￾hoc analysis indicated that 100 ng/ml TSA treatment significantly increases hippocampal GR expression in the offspring of low-LG￾ABN mothers (vehicle-treated low-LG-ABN vs. 100 ng/ml TSA￾treated low-LG-ABN, *P < 0.001), such that there is no longer a significant difference in hippocampal GR expression between the offspring of low- or high-LG-ABN mothers (100 ng/ml TSA treated low-LG-ABN vs. 100 ng/ml TSA treated high-LG-ABN, P > 0.90). Although TSA treatment significantly induced GR expression in low-LG-ABN adult offspring, global abundance of protein in the hippocampus was not apparently increased, as indicated by the equal α-tubulin immunoreactivity (Fig. 5a). In comparing the vehicle-treated groups, note that the effect of maternal care on GR gene expression7,9 is subtler than the more pronounced effect on the methylation status of the 5′ CpG dinucleotide (site 16) within the exon 17 promoter (Fig. 4b). However, in previous studies16 we found evidence for multiple promoters regulating hippocampal GR expression suggesting that exon 17 is but one of the regulatory sequences determining GR expression within the hippocampus. Reversal of maternal effect on HPA responses to stress As adults, the offspring of high-LG-ABN mothers show more mod￾est HPA responses to stress than the offspring of low-LG-ABN mothers7. The effect of maternal care on HPA responses to stress seems to be, in part, associated with differences in hippocampal GR levels and glucocorticoid negative feedback sensitivity7. Given that TSA treatment reversed the group difference in hippocampal GR expression, we examined the adrenocortical responses to stress in a separate cohort of vehicle- and TSA-treated animals. Central infu￾sion of TSA completely eliminated the maternal effect on HPA responses to acute stress (Fig. 5b). Statistical analysis of the plasma corticosterone data revealed significant effects of Group (F = 4.3, P = 0.048), Treatment (F = 4.3, P = 0.046) and Time (F = 27.3, P < 0.0001), as well as a significant Group × Treatment interaction effect (F = 7.7, P = 0.009). Post-hoc analysis revealed that TSA treat￾ment significantly decreased basal plasma corticosterone in the off￾spring of low-LG-ABN mothers to levels comparable to those of high-LG-ABN animals. Thus, plasma corticosterone responses to restraint stress in the vehicle-treated adult offspring of low-LG￾ABN mothers were significantly (P < 0.01) greater than those of TSA- and vehicle-treated adult offspring of high-LG-ABN mothers or TSA-treated offspring of Low-LG-ABN mothers. The HPA response to stress in the offspring of high-LG-ABN mothers was unaffected by TSA treatment. In summary, central infusion of the HDAC inhibitor TSA enhanced histone H3-K9 acetylation of the exon 17 GR promoter in the off￾spring of the low-LG-ABN mothers, increased NGFI-A binding to its cognate sequence, induced hypomethylation of CpG dinucleotide sequences in the promoter and eliminated the maternal effect on hip￾pocampal GR expression and the HPA response to stress. These findings are consistent with idea that the maternal effect on GR expression and HPA responses to stress is mediated by alterations in chromatin structure. We propose that the reduced binding of NGFI-A to its response element on the hypoacetylated and hyperme￾thylated exon 17 GR promoter contributes to the attenuation of GR expression in low-LG-ABN adult offspring, whereas increased NGFI￾A binding to the hyperacetylated and hypomethylated response ele￾ment on the exon 17 GR promoter in the offspring of the high-LG-ABN mothers would serve to maintain the differences in gene expression. DNA methylation represents a stable epigenetic mark; therefore, our findings provide an explanation for the enduring effect on mother-infant interactions over the first week of postnatal life on HPA responses to stress in the offspring. DISCUSSION Further studies are required to determine how maternal behavior alters the epigenetic status of the exon 17 GR promoter. In addition, the exact causal relationship between DNA methylation and altered histone acetylation and NGFI-A binding remains to be defined. Nevertheless, our findings provide the first evidence that maternal behavior produces stable alterations of DNA methylation and chro￾matin structure, providing a mechanism for the long-term effects of maternal care on gene expression in the offspring. These studies offer an opportunity to clearly define the nature of gene-environment interactions during development and how such effects result in the sustained ‘environmental programming’ of gene expression and function over the lifespan. It is important to note that maternal effects on the expression of defensive responses, such as increased HPA activity, are a common theme in biology1,2 such that the magni￾tude of the maternal influence on the development of HPA and behavioral responses to stress in the rat should not be surprising. Maternal effects on defensive responses to threat are apparent in plants, insects and reptiles. Such effects commonly follow from the exposure of the mother to the same or similar forms of threat and may represent examples whereby the experience of the mother is translated through an epigenetic mechanism of inheritance into phenotypic variation in the offspring. Thus, maternal effects could result in the transmission of adaptive responses across genera￾tions1,2,5. Indeed, among mammals, natural selection may have shaped offspring to respond to subtle variations in parental behavior as a forecast of the environmental conditions they will ultimately face once they become independent of the parent5. Epigenetic modi￾fications of targeted regulatory sequences in response to even rea￾sonably subtle variations in environmental conditions might then serve as a major source of epigenetic variation in gene expression and function, and ultimately as a process mediating such maternal effects. We propose that effects on chromatin structure such as those described here serve as an intermediate process that imprints dynamic environmental experiences on the fixed genome, resulting in stable alterations in phenotype. METHODS Animals and maternal behavior. The animals used in all studies were derived from Long-Evans hooded rats born in our colony from animals originally obtained from Charles River Canada (St. Constant, Québec). All procedures were performed according to guidelines developed by the Canadian Council on Animal Care and protocol approved by the McGill University Animal Care Committee. Maternal behavior was scored32 and the adoption study was per￾formed9 as previously described. For further methodological details, see Supplementary Methods online. Sodium bisulfite mapping. Sodium bisulfite mapping was performed as previously described21,22. The rat GR exon 17 genomic region (GenBank accession number AJ271870) of the sodium bisulfite-treated hippocampal DNA (50 ng/ml) was subjected to PCR amplification using outside primers (forward: 1646-TTTTTTAGGTTTTTTTAGAGGG-1667; reverse: 1930- 852 VOLUME 7 | NUMBER 8 | AUGUST 2004 NATURE NEUROSCIENCE © 2004 Nature Publishing Group http://www.nature.com/natureneuroscience