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Gene expression changes in the septum: possible implications for microRNAs in sculpting the maternal brain.

Zhao C, Saul MC, Driessen T, Gammie SC - PLoS ONE (2012)

Bottom Line: These genes were previously found to be differentially regulated in other brain regions during lactation.Unexpectedly, enrichment analysis revealed a high number of microRNAs, transcription factors, or conserved binding sites (177 with corrected P-value <0.05) that were significantly linked to maternal upregulated genes, while none were linked to downregulated genes.Together, this study provides new insights into genes (along with possible mechanisms for their regulation) that are involved in septum-mediated adaptations during the postpartum period.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, University of Wisconsin-Madison, Madison, Wisconsin, United States of America. czhao23@wisc.edu

ABSTRACT
The transition from the non-maternal to the maternal state is characterized by a variety of CNS alterations that support the care of offspring. The septum (including lateral and medial portions) is a brain region previously linked to various emotional and motivational processes, including maternal care. In this study, we used microarrays (PLIER algorithm) to examine gene expression changes in the septum of postpartum mice and employed gene set enrichment analysis (GSEA) to identify possible regulators of altered gene expression. Genes of interest identified as differentially regulated with microarray analysis were validated with quantitative real-time PCR. We found that fatty acid binding protein 7 (Fabp7) and galanin (Gal) were downregulated, whereas insulin-like growth factor binding protein 3 (Igfbp3) was upregulated in postpartum mice compared to virgin females. These genes were previously found to be differentially regulated in other brain regions during lactation. We also identified altered expression of novel genes not previously linked to maternal behavior, but that could play a role in postpartum processes, including glutamate-ammonia ligase (Glul) and somatostatin receptor 1 (Sstr1) (both upregulated in postpartum). Genes implicated in metabolism, cell differentiation, or proliferation also exhibited altered expression. Unexpectedly, enrichment analysis revealed a high number of microRNAs, transcription factors, or conserved binding sites (177 with corrected P-value <0.05) that were significantly linked to maternal upregulated genes, while none were linked to downregulated genes. MicroRNAs have been linked to placenta and mammary gland development, but this is the first indication they may also play a key role in sculpting the maternal brain. Together, this study provides new insights into genes (along with possible mechanisms for their regulation) that are involved in septum-mediated adaptations during the postpartum period.

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Schematic representation of the brain regions (boxed areas) dissected for gene array analysis.Distance from Bregma in the rostrocaudal planes is indicated. Reprinted and modified from The Allen Mouse Brain Atlas (reference atlas version 1, 2008). Abbreviations: LSc, caudal part of lateral septal nucleus; LSr, rostral part of lateral septal nucleus; MS, medial septal nucleus; VL, lateral ventricle; aco, anterior commissure, olfactory limb.
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pone-0038602-g002: Schematic representation of the brain regions (boxed areas) dissected for gene array analysis.Distance from Bregma in the rostrocaudal planes is indicated. Reprinted and modified from The Allen Mouse Brain Atlas (reference atlas version 1, 2008). Abbreviations: LSc, caudal part of lateral septal nucleus; LSr, rostral part of lateral septal nucleus; MS, medial septal nucleus; VL, lateral ventricle; aco, anterior commissure, olfactory limb.

Mentions: On postpartum Days 6–8, brains were removed from lactating females between 10∶00 and 12∶00 h. Brains from age-matched, virgin females were also removed on the same day and dissections were alternated between the two groups so that an equal number of dissections from each group were obtained. The animals were lightly anaesthetized with isoflurane, killed by cervical dislocation and then decapitated. Following decapitation, virgin females were examined for stage of estrous cycle using a vaginal lavage [65], [66]. Female mice in vaginal proestrus were not used because this stage is associated with mating in mice. For the microarray study, the stages of the six virgin mice were estrous, diestrous I, and diestrous II (N = 2 each). Although estrous stage can influence gene expression [57], the use of mice from different estrous states (excluding proestrus) provides a generic view of the virgin female and should provide insights into reliable differences between maternal and virgin mice. The whole brain was removed, snap frozen in isopentane on dry ice, and then stored at −80°C until sliced. Brain sections were sliced in a cryostat (Leica, CM1850, Bannockburn, IL, USA) with the first two sections at 300 microns and the last section at 200 microns and then mounted on glass slides. Target tissue was captured using a micropunch technique [67]. Microdissection of frozen brain sections was made with Brain Punch Set (Stoelting, Wood Dale, IL, USA) under a dissecting microscope. The whole septum including lateral and medial parts (Fig. 2) was collected bilaterally from Bregma 1.10 to 0.14 and pooled, so that each mouse provided one sample of the septum. Microdissections from ten animals in each group were flash frozen on dry ice and stored at −80°C until being processed for either gene array analysis or real-time PCR. Total RNA was extracted and prepared in matched pairs (virgin versus lactating) using an Aurum Total RNA Fatty and Fibrous Tissue Kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Following isolation, the integrity of RNA was assessed using Agilent RNA 6000 Nano Chips with Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). The purity of RNA was tested, and the yield of RNA was determined using NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Purified total RNA was stored at −80°C until processed.


Gene expression changes in the septum: possible implications for microRNAs in sculpting the maternal brain.

Zhao C, Saul MC, Driessen T, Gammie SC - PLoS ONE (2012)

Schematic representation of the brain regions (boxed areas) dissected for gene array analysis.Distance from Bregma in the rostrocaudal planes is indicated. Reprinted and modified from The Allen Mouse Brain Atlas (reference atlas version 1, 2008). Abbreviations: LSc, caudal part of lateral septal nucleus; LSr, rostral part of lateral septal nucleus; MS, medial septal nucleus; VL, lateral ventricle; aco, anterior commissure, olfactory limb.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368935&req=5

pone-0038602-g002: Schematic representation of the brain regions (boxed areas) dissected for gene array analysis.Distance from Bregma in the rostrocaudal planes is indicated. Reprinted and modified from The Allen Mouse Brain Atlas (reference atlas version 1, 2008). Abbreviations: LSc, caudal part of lateral septal nucleus; LSr, rostral part of lateral septal nucleus; MS, medial septal nucleus; VL, lateral ventricle; aco, anterior commissure, olfactory limb.
Mentions: On postpartum Days 6–8, brains were removed from lactating females between 10∶00 and 12∶00 h. Brains from age-matched, virgin females were also removed on the same day and dissections were alternated between the two groups so that an equal number of dissections from each group were obtained. The animals were lightly anaesthetized with isoflurane, killed by cervical dislocation and then decapitated. Following decapitation, virgin females were examined for stage of estrous cycle using a vaginal lavage [65], [66]. Female mice in vaginal proestrus were not used because this stage is associated with mating in mice. For the microarray study, the stages of the six virgin mice were estrous, diestrous I, and diestrous II (N = 2 each). Although estrous stage can influence gene expression [57], the use of mice from different estrous states (excluding proestrus) provides a generic view of the virgin female and should provide insights into reliable differences between maternal and virgin mice. The whole brain was removed, snap frozen in isopentane on dry ice, and then stored at −80°C until sliced. Brain sections were sliced in a cryostat (Leica, CM1850, Bannockburn, IL, USA) with the first two sections at 300 microns and the last section at 200 microns and then mounted on glass slides. Target tissue was captured using a micropunch technique [67]. Microdissection of frozen brain sections was made with Brain Punch Set (Stoelting, Wood Dale, IL, USA) under a dissecting microscope. The whole septum including lateral and medial parts (Fig. 2) was collected bilaterally from Bregma 1.10 to 0.14 and pooled, so that each mouse provided one sample of the septum. Microdissections from ten animals in each group were flash frozen on dry ice and stored at −80°C until being processed for either gene array analysis or real-time PCR. Total RNA was extracted and prepared in matched pairs (virgin versus lactating) using an Aurum Total RNA Fatty and Fibrous Tissue Kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Following isolation, the integrity of RNA was assessed using Agilent RNA 6000 Nano Chips with Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). The purity of RNA was tested, and the yield of RNA was determined using NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Purified total RNA was stored at −80°C until processed.

Bottom Line: These genes were previously found to be differentially regulated in other brain regions during lactation.Unexpectedly, enrichment analysis revealed a high number of microRNAs, transcription factors, or conserved binding sites (177 with corrected P-value <0.05) that were significantly linked to maternal upregulated genes, while none were linked to downregulated genes.Together, this study provides new insights into genes (along with possible mechanisms for their regulation) that are involved in septum-mediated adaptations during the postpartum period.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, University of Wisconsin-Madison, Madison, Wisconsin, United States of America. czhao23@wisc.edu

ABSTRACT
The transition from the non-maternal to the maternal state is characterized by a variety of CNS alterations that support the care of offspring. The septum (including lateral and medial portions) is a brain region previously linked to various emotional and motivational processes, including maternal care. In this study, we used microarrays (PLIER algorithm) to examine gene expression changes in the septum of postpartum mice and employed gene set enrichment analysis (GSEA) to identify possible regulators of altered gene expression. Genes of interest identified as differentially regulated with microarray analysis were validated with quantitative real-time PCR. We found that fatty acid binding protein 7 (Fabp7) and galanin (Gal) were downregulated, whereas insulin-like growth factor binding protein 3 (Igfbp3) was upregulated in postpartum mice compared to virgin females. These genes were previously found to be differentially regulated in other brain regions during lactation. We also identified altered expression of novel genes not previously linked to maternal behavior, but that could play a role in postpartum processes, including glutamate-ammonia ligase (Glul) and somatostatin receptor 1 (Sstr1) (both upregulated in postpartum). Genes implicated in metabolism, cell differentiation, or proliferation also exhibited altered expression. Unexpectedly, enrichment analysis revealed a high number of microRNAs, transcription factors, or conserved binding sites (177 with corrected P-value <0.05) that were significantly linked to maternal upregulated genes, while none were linked to downregulated genes. MicroRNAs have been linked to placenta and mammary gland development, but this is the first indication they may also play a key role in sculpting the maternal brain. Together, this study provides new insights into genes (along with possible mechanisms for their regulation) that are involved in septum-mediated adaptations during the postpartum period.

Show MeSH
Related in: MedlinePlus