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Hippocampal gene expression changes underlying stress sensitization and recovery.

Gray JD, Rubin TG, Hunter RG, McEwen BS - Mol. Psychiatry (2013)

Bottom Line: Most genes increased by CRS were decreased after recovery but many remained altered and did not return to baseline.Quantitative reverse transcription-PCR (qRT-PCR) validated changes from the microarrays in known stress-induced genes and confirmed alterations in the NF-κB pathway genes, Nfkbia, RelA and Nfkb1.These findings establish a baseline profile of normal recovery and adaptation to stress.

View Article: PubMed Central - PubMed

Affiliation: Harold and Margaret Milliken Hatch Laboratory of Neuroendocrinology, The Rockefeller University, New York, NY, USA.

ABSTRACT
Chronic and acute stressors have been linked to changes in hippocampal function and anxiety-like behaviors. Both produce changes in gene expression, but the extent to which these changes endure beyond the end of stress remains poorly understood. As an essential first step to characterize abnormal patterns of gene expression after stress, this study demonstrates how chronic restraint stress (CRS) modulates gene expression in response to a novel stressor in the hippocampus of wild-type mice and the extent to which these changes last beyond the end of CRS. Male C57/bl6 mice were subjected to (1) a forced swim test (FST), (2) corticosterone (Cort) or vehicle injections, (3) CRS for 21 days and then a FST, or (4) allowed to recover 21 days after CRS and subjected to FST. Hippocampal mRNA was extracted and used to generate cDNA libraries for microarray hybridization. Naive acute stressors (FST and vehicle injection) altered similar sets of genes, but Cort treatment produced a profile that was distinct from both FST and vehicle. Exposure to a novel stress after CRS activated substantially more and different genes than naive exposure. Most genes increased by CRS were decreased after recovery but many remained altered and did not return to baseline. Pathway analysis identified significant clusters of differentially expressed genes across conditions, most notably the nuclear factor kappa-light-chain-enhancer of B cells (NF-κB) pathway. Quantitative reverse transcription-PCR (qRT-PCR) validated changes from the microarrays in known stress-induced genes and confirmed alterations in the NF-κB pathway genes, Nfkbia, RelA and Nfkb1. FST increased anxiety-like behavior in both the naive and recovery from CRS conditions, but not in mice 24h subsequent to their CRS exposure. These findings suggest that the effects of naive stress are distinct from Cort elevation, and that a history of stress exposure can permanently alter gene expression patterns in the hippocampus and the behavioral response to a novel stressor. These findings establish a baseline profile of normal recovery and adaptation to stress. Importantly, they will serve as a conceptual basis to facilitate the future study of the cellular and regional basis of gene expression changes that lead to impaired recovery from stress, such as those that occur in mood and anxiety disorders.

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qRT-PCR validates differences in genes identified by microarray known to be associated with the stress response and NfKb signalingBar graphs representing fold change from controls on genes measured by qRT-PCR. (A) Naïve FST significantly increased mRNA levels of cfos and nfkbia (**p<0.01 v. control). BDNF, GR, RelA and Nfkb1 all trended toward an increase but did not reach significance by qRT-PCR. (B) CRS (black bars) and CRS+FST (open bars) resulted in significant increases or decreases in several genes. Of note, BDNF is elevated by CRS, but decreased compared to control in CRS+FST (#p<0.05 between stress groups). The CRS levels of all Nfkb pathway members is elevated by CRS (Nfkbia **p<0.01, Rela ***p<0.001, Nfkb1 **p<0.01), but the addition of FST significantly decreased levels of nfkbia and nfkb1 (###p<0.001 and #p<0.05 respectively). (C) Significant genes in recovery from CRS (black bars) and Recovery from CRS + FST (open bars). BDNF showed a similar response pattern as the end of CRS (###p<0.001), however, Nfkbia and RelA were no longer significantly different from controls after recovery or novel FST. (*significantly different from control, #significantly different from heterotypic stress)
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Figure 2: qRT-PCR validates differences in genes identified by microarray known to be associated with the stress response and NfKb signalingBar graphs representing fold change from controls on genes measured by qRT-PCR. (A) Naïve FST significantly increased mRNA levels of cfos and nfkbia (**p<0.01 v. control). BDNF, GR, RelA and Nfkb1 all trended toward an increase but did not reach significance by qRT-PCR. (B) CRS (black bars) and CRS+FST (open bars) resulted in significant increases or decreases in several genes. Of note, BDNF is elevated by CRS, but decreased compared to control in CRS+FST (#p<0.05 between stress groups). The CRS levels of all Nfkb pathway members is elevated by CRS (Nfkbia **p<0.01, Rela ***p<0.001, Nfkb1 **p<0.01), but the addition of FST significantly decreased levels of nfkbia and nfkb1 (###p<0.001 and #p<0.05 respectively). (C) Significant genes in recovery from CRS (black bars) and Recovery from CRS + FST (open bars). BDNF showed a similar response pattern as the end of CRS (###p<0.001), however, Nfkbia and RelA were no longer significantly different from controls after recovery or novel FST. (*significantly different from control, #significantly different from heterotypic stress)

Mentions: Genes predicted by the literature to be changed by stress that were identified as significant on the microarrays were validated using qRT-PCR. cFos is an early response gene that has been shown to be rapidly activated in response to stress and is a marker of recent neural activity (17) that was significantly elevated in all the FST conditions by microarray (Table 1). cFos was significantly increased in naïve FST and CRS+FST by qRT-PCR (t(6)=5.04, p<0.01, t(6)=3.10, p<0.05), and was increased but did not reach significance (p=0.11) in the CRS+Rec+FST condition (Fig. 2A). Brain derived neurotrophic factor (BDNF) has been shown by numerous groups to be highly dynamic in the hippocampus in response to stress (18). Microarrays identified BDNF as significantly increased in the FST animals that had a stress history and trended toward an increase in naïve FST animals (Table 1). qRT-PCR showed a trend toward an increase of BDNF in the naïve stress and a significant increase after CRS (t(6)=3.32, p<0.05), but no change in the CRS+FST condition (Fig. 2B). After recovery from CRS, basal levels of BDNF remained significantly elevated compared to non-stress controls (t(6)=3.37, p<0.05), and exposure to a novel stress suppressed the elevated BDNF levels back below baseline (t(6)=2.79, p<0.05). Finally, the glucocorticoid receptor (GR) gene (Nr3c1), an essential transcription factor mediating stress-induced changes in the hippocampus (19), was not altered by any of the acute stress conditions, but trended toward a decrease after CRS on the microarrays. qRT-PCR confirmed the decrease after CRS as significant (t(14)=3.516, p<0.01), but also found it to be further reduced by heterotypic stress (CRS+FST)(Fig. 2C, t(10)=6.59, p<0.0001). Nr3c1 levels were found to be significantly elevated in CRS+Recovery (t(6)=3.02, p<0.05), but were not altered by exposure to an acute heterotypic stressor. Together, these data suggest that the microarrays were accurate in identifying genes altered by stress and, moreover, they revealed genes of interest, some of which respond differently in naïve, CRS and recovered CRS conditions.


Hippocampal gene expression changes underlying stress sensitization and recovery.

Gray JD, Rubin TG, Hunter RG, McEwen BS - Mol. Psychiatry (2013)

qRT-PCR validates differences in genes identified by microarray known to be associated with the stress response and NfKb signalingBar graphs representing fold change from controls on genes measured by qRT-PCR. (A) Naïve FST significantly increased mRNA levels of cfos and nfkbia (**p<0.01 v. control). BDNF, GR, RelA and Nfkb1 all trended toward an increase but did not reach significance by qRT-PCR. (B) CRS (black bars) and CRS+FST (open bars) resulted in significant increases or decreases in several genes. Of note, BDNF is elevated by CRS, but decreased compared to control in CRS+FST (#p<0.05 between stress groups). The CRS levels of all Nfkb pathway members is elevated by CRS (Nfkbia **p<0.01, Rela ***p<0.001, Nfkb1 **p<0.01), but the addition of FST significantly decreased levels of nfkbia and nfkb1 (###p<0.001 and #p<0.05 respectively). (C) Significant genes in recovery from CRS (black bars) and Recovery from CRS + FST (open bars). BDNF showed a similar response pattern as the end of CRS (###p<0.001), however, Nfkbia and RelA were no longer significantly different from controls after recovery or novel FST. (*significantly different from control, #significantly different from heterotypic stress)
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4061278&req=5

Figure 2: qRT-PCR validates differences in genes identified by microarray known to be associated with the stress response and NfKb signalingBar graphs representing fold change from controls on genes measured by qRT-PCR. (A) Naïve FST significantly increased mRNA levels of cfos and nfkbia (**p<0.01 v. control). BDNF, GR, RelA and Nfkb1 all trended toward an increase but did not reach significance by qRT-PCR. (B) CRS (black bars) and CRS+FST (open bars) resulted in significant increases or decreases in several genes. Of note, BDNF is elevated by CRS, but decreased compared to control in CRS+FST (#p<0.05 between stress groups). The CRS levels of all Nfkb pathway members is elevated by CRS (Nfkbia **p<0.01, Rela ***p<0.001, Nfkb1 **p<0.01), but the addition of FST significantly decreased levels of nfkbia and nfkb1 (###p<0.001 and #p<0.05 respectively). (C) Significant genes in recovery from CRS (black bars) and Recovery from CRS + FST (open bars). BDNF showed a similar response pattern as the end of CRS (###p<0.001), however, Nfkbia and RelA were no longer significantly different from controls after recovery or novel FST. (*significantly different from control, #significantly different from heterotypic stress)
Mentions: Genes predicted by the literature to be changed by stress that were identified as significant on the microarrays were validated using qRT-PCR. cFos is an early response gene that has been shown to be rapidly activated in response to stress and is a marker of recent neural activity (17) that was significantly elevated in all the FST conditions by microarray (Table 1). cFos was significantly increased in naïve FST and CRS+FST by qRT-PCR (t(6)=5.04, p<0.01, t(6)=3.10, p<0.05), and was increased but did not reach significance (p=0.11) in the CRS+Rec+FST condition (Fig. 2A). Brain derived neurotrophic factor (BDNF) has been shown by numerous groups to be highly dynamic in the hippocampus in response to stress (18). Microarrays identified BDNF as significantly increased in the FST animals that had a stress history and trended toward an increase in naïve FST animals (Table 1). qRT-PCR showed a trend toward an increase of BDNF in the naïve stress and a significant increase after CRS (t(6)=3.32, p<0.05), but no change in the CRS+FST condition (Fig. 2B). After recovery from CRS, basal levels of BDNF remained significantly elevated compared to non-stress controls (t(6)=3.37, p<0.05), and exposure to a novel stress suppressed the elevated BDNF levels back below baseline (t(6)=2.79, p<0.05). Finally, the glucocorticoid receptor (GR) gene (Nr3c1), an essential transcription factor mediating stress-induced changes in the hippocampus (19), was not altered by any of the acute stress conditions, but trended toward a decrease after CRS on the microarrays. qRT-PCR confirmed the decrease after CRS as significant (t(14)=3.516, p<0.01), but also found it to be further reduced by heterotypic stress (CRS+FST)(Fig. 2C, t(10)=6.59, p<0.0001). Nr3c1 levels were found to be significantly elevated in CRS+Recovery (t(6)=3.02, p<0.05), but were not altered by exposure to an acute heterotypic stressor. Together, these data suggest that the microarrays were accurate in identifying genes altered by stress and, moreover, they revealed genes of interest, some of which respond differently in naïve, CRS and recovered CRS conditions.

Bottom Line: Most genes increased by CRS were decreased after recovery but many remained altered and did not return to baseline.Quantitative reverse transcription-PCR (qRT-PCR) validated changes from the microarrays in known stress-induced genes and confirmed alterations in the NF-κB pathway genes, Nfkbia, RelA and Nfkb1.These findings establish a baseline profile of normal recovery and adaptation to stress.

View Article: PubMed Central - PubMed

Affiliation: Harold and Margaret Milliken Hatch Laboratory of Neuroendocrinology, The Rockefeller University, New York, NY, USA.

ABSTRACT
Chronic and acute stressors have been linked to changes in hippocampal function and anxiety-like behaviors. Both produce changes in gene expression, but the extent to which these changes endure beyond the end of stress remains poorly understood. As an essential first step to characterize abnormal patterns of gene expression after stress, this study demonstrates how chronic restraint stress (CRS) modulates gene expression in response to a novel stressor in the hippocampus of wild-type mice and the extent to which these changes last beyond the end of CRS. Male C57/bl6 mice were subjected to (1) a forced swim test (FST), (2) corticosterone (Cort) or vehicle injections, (3) CRS for 21 days and then a FST, or (4) allowed to recover 21 days after CRS and subjected to FST. Hippocampal mRNA was extracted and used to generate cDNA libraries for microarray hybridization. Naive acute stressors (FST and vehicle injection) altered similar sets of genes, but Cort treatment produced a profile that was distinct from both FST and vehicle. Exposure to a novel stress after CRS activated substantially more and different genes than naive exposure. Most genes increased by CRS were decreased after recovery but many remained altered and did not return to baseline. Pathway analysis identified significant clusters of differentially expressed genes across conditions, most notably the nuclear factor kappa-light-chain-enhancer of B cells (NF-κB) pathway. Quantitative reverse transcription-PCR (qRT-PCR) validated changes from the microarrays in known stress-induced genes and confirmed alterations in the NF-κB pathway genes, Nfkbia, RelA and Nfkb1. FST increased anxiety-like behavior in both the naive and recovery from CRS conditions, but not in mice 24h subsequent to their CRS exposure. These findings suggest that the effects of naive stress are distinct from Cort elevation, and that a history of stress exposure can permanently alter gene expression patterns in the hippocampus and the behavioral response to a novel stressor. These findings establish a baseline profile of normal recovery and adaptation to stress. Importantly, they will serve as a conceptual basis to facilitate the future study of the cellular and regional basis of gene expression changes that lead to impaired recovery from stress, such as those that occur in mood and anxiety disorders.

Show MeSH
Related in: MedlinePlus