Terri M. Driessen

Genes showing altered expression in the medial preoptic area in the highly social maternal phenotype are related to autism and other disorders with social deficits

BackgroundThe mother-child relationship is the most fundamental social bond in mammals, and previous studies indicate that the medial preoptic area (MPOA) contributes to this increase in sociability. It is possible that the same genes that lead to elevated sociability in one condition (the maternal state) might also be dysregulated in some disorders with social deficits (e.g. autism). In this study, we examined whether there was enrichment (greater than chance overlap) for social deficit disorder related genes in MPOA microarray results between virgin and postpartum female mice. We utilized microarrays to assess large scale gene expression changes in the MPOA of virgin and postpartum mice. The Modular Single Set Enrichment Test (MSET) was used to determine if mental health disorder related genes were enriched in significant microarray results. Additional resources, such as ToppCluster, NIH DAVID, and weighted co-expression network analysis (WGCNA) were used to analyze enrichment for specific gene clusters or indirect relationships between significant genes of interest. Finally, a subset of microarray results was validated using quantitative PCR.ResultsSignificant postpartum MPOA microarray results were enriched for multiple disorders that include social deficits, including autism, bipolar disorder, depression, and schizophrenia. Together, 98 autism-related genes were identified from the significant microarray results. Further, ToppCluser and NIH DAVID identified a large number of postpartum genes related to ion channel activity and CNS development, and also suggested a role for microRNAs in regulating maternal gene expression. WGCNA identified a module of genes associated with the postpartum phenotype, and identified indirect links between transcription factors and other genes of interest.ConclusionThe transition to the maternal state involves great CNS plasticity and increased sociability. We identified multiple novel genes that overlap between the postpartum MPOA (high sociability) and mental health disorders with low sociability. Thus, the activity or interactions of the same genes may be altering social behaviors in different directions in different conditions. Maternity also involves elevated risks for disorders, including depression, psychosis, and BPD, so identification of maternal genes common to these disorders may provide insights into the elevated vulnerability of the maternal brain.

Large Scale Expression Changes of Genes Related to Neuronal Signaling and Developmental Processes Found in Lateral Septum of Postpartum Outbred Mice

Coordinated gene expression changes across the CNS are required to produce the mammalian maternal phenotype. Lateral septum (LS) is a brain region critically involved with aspects of maternal care, and we recently examined gene expression of whole septum (LS and medial septum) in selectively bred maternal mice. Here, we expand on the prior study by 1) conducting microarray analysis solely on LS in virgin and postpartum mice, 2) using outbred mice, and 3) evaluating the role of sensory input on gene expression changes. Large scale changes in genes related to neuronal signaling were identified, including four GABAA receptor subunits. Subunits α4 and δ were downregulated in maternal LS, likely reflecting a reduction in the extrasynaptic, neurosteroid-sensitive α4/δ containing receptor subtype. Conversely, subunits ε and θ were increased in maternal LS. Fifteen K+ channel related genes showed altered expression, as did dopamine receptors Drd1a and Drd2 (both downregulated), hypocretin receptor 1 (Hcrtr1), kappa opioid receptor 1 (Oprk1), and transient receptor potential channel 4 (Trpc4). Expression of a large number of genes linked to developmental processes or cell differentiation were also altered in postpartum LS, including chemokine (C-X-C) motif ligand 12 (Cxcl12), fatty acid binding protein 7 (Fabp7), plasma membrane proteolipid (Pllp), and suppressor of cytokine signaling 2 (Socs2). Additional genes that are linked to anxiety, such as glutathione reductase (Gsr), exhibited altered expression. Pathway analysis also identified changes in genes related to cyclic nucleotide metabolism, chromatin structure, and the Ras gene family. The sensory presence of pups was found to contribute to the altered expression of a subset of genes across all categories. This study suggests that both large changes in neuronal signaling and the possible terminal differentiation of neuronal and/or glial cells play important roles in producing the maternal state.

Development of a versatile enrichment analysis tool reveals associations between the maternal brain and mental health disorders, including autism

BackgroundA recent study of lateral septum (LS) suggested a large number of autism-related genes with altered expression in the postpartum state. However, formally testing the findings for enrichment of autism-associated genes proved to be problematic with existing software. Many gene-disease association databases have been curated which are not currently incorporated in popular, full-featured enrichment tools, and the use of custom gene lists in these programs can be difficult to perform and interpret. As a simple alternative, we have developed the Modular Single-set Enrichment Test (MSET), a minimal tool that enables one to easily evaluate expression data for enrichment of any conceivable gene list of interest.ResultsThe MSET approach was validated by testing several publicly available expression data sets for expected enrichment in areas of autism, attention deficit hyperactivity disorder (ADHD), and arthritis. Using nine independent, unique autism gene lists extracted from association databases and two recent publications, a striking consensus of enrichment was detected within gene expression changes in LS of postpartum mice. A network of 160 autism-related genes was identified, representing developmental processes such as synaptic plasticity, neuronal morphogenesis, and differentiation. Additionally, maternal LS displayed enrichment for genes associated with bipolar disorder, schizophrenia, ADHD, and depression.ConclusionsThe transition to motherhood includes the most fundamental social bonding event in mammals and features naturally occurring changes in sociability. Some individuals with autism, schizophrenia, or other mental health disorders exhibit impaired social traits. Genes involved in these deficits may also contribute to elevated sociability in the maternal brain. To date, this is the first study to show a significant, quantitative link between the maternal brain and mental health disorders using large scale gene expression data. Thus, the postpartum brain may provide a novel and promising platform for understanding the complex genetics of improved sociability that may have direct relevance for multiple psychiatric illnesses. This study also provides an important new tool that fills a critical analysis gap and makes evaluation of enrichment using any database of interest possible with an emphasis on ease of use and methodological transparency.

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

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.

Glutamic acid decarboxylase 65 and 67 expression in the lateral septum is up-regulated in association with the postpartum period in mice.

The postpartum period in mammals undergoes a variety of physiological adaptations, including metabolic, behavioral and neuroendocrine alterations. GABA signaling has been strongly linked to various emotional states, stress responses and offspring protection. However, whether GABA signaling may change in the lateral septum (LS), a core brain region for regulating behavioral, emotional and stress responses in postpartum mice has not previously been examined. In this study, we tested whether the expression of two isoforms of glutamic acid decarboxylase (GAD), GAD65 (GAD2) and GAD67 (GAD1), the rate-limiting enzyme for GABA synthesis, exhibits altered expression in postpartum mice relative to nonmaternal, virgin mice. Using microdissected septal tissue from virgin and age-matched postpartum females, quantitative real-time PCR and Western blotting were carried out to assess GAD mRNA and protein expression, respectively. We found both protein and mRNA expression of GAD67 in the whole septum was up-regulated in postpartum mice. By contrast, no significant difference in the whole septum was observed in GAD65 expression. We then conducted a finer level of analysis using smaller microdissections and found GAD67 to be significantly increased in rostral LS, but not in caudal LS or medial septum (MS). Further, GAD65 mRNA expression in rostral LS, but not in caudal LS or MS was also significantly elevated in postpartum mice. These findings suggest that an increased GABA production in rostral LS of the postpartum mice via elevated GAD65 and GAD67 expression may contribute to multiple alterations in behavioral and emotional states, and responses to stress that occur during the postpartum period. Given that rostral LS contains GABA neurons that are projection neurons or local interneurons, it still needs to be determined whether the function of elevated GABA is for local or distant action or both.

Sexually-motivated song is predicted by androgen-and opioid-related gene expression in the medial preoptic nucleus of male European starlings (Sturnus vulgaris)

Across vertebrates, communication conveys information about an individuals motivational state, yet little is known about the neuroendocrine regulation of motivational aspects of communication. For seasonally breeding songbirds, increases in testosterone in spring stimulate high rates of sexually-motivated courtship song, though not all birds sing at high rates. It is generally assumed that testosterone or its metabolites act within the medial preoptic nucleus (POM) to stimulate the motivation to sing. In addition to androgen receptors (ARs) and testosterone, opioid neuropeptides in the POM influence sexually-motivated song production, and it has been proposed that testosterone may in part regulate song by modifying opioid systems. To gain insight into a possible role for androgen-opioid interactions in the regulation of communication we examined associations between sexually-motivated song and relative expression of ARs, mu opioid receptors (muORs), and preproenkephalin (PENK) in the POM (and other regions) of male European starlings using qPCR. Both AR and PENK expression in POM correlated positively with singing behavior, whereas muOR in POM correlated negatively with song. Furthermore, the ratio of PENK/muOR expression correlated negatively with AR expression in POM. Finally, in the ventral tegmental area (VTA), PENK expression correlated negatively with singing behavior. Results support the hypothesis that ARs may alter opioid gene expression in POM to fine-tune singing to reflect a males motivational state. Data also suggest that bidirectional relationships may exist between opioids and ARs in POM and song, and additionally support a role for opioids in the VTA, independent of AR activity in this region.

Addiction and reward-related genes show altered expression in the postpartum nucleus accumbens

Motherhood involves a switch in natural rewards, whereby offspring become highly rewarding. Nucleus accumbens (NAC) is a key CNS region for natural rewards and addictions, but to date no study has evaluated on a large scale the events in NAC that underlie the maternal change in natural rewards. In this study we utilized microarray and bioinformatics approaches to evaluate postpartum NAC gene expression changes in mice. Modular Single-set Enrichment Test (MSET) indicated that postpartum (relative to virgin) NAC gene expression profile was significantly enriched for genes related to addiction and reward in five of five independently curated databases (e.g., Malacards, Phenopedia). Over 100 addiction/reward related genes were identified and these included: Per1, Per2, Arc, Homer2, Creb1, Grm3, Fosb, Gabrb3, Adra2a, Ntrk2, Cry1, Penk, Cartpt, Adcy1, Npy1r, Htr1a, Drd1a, Gria1, and Pdyn. ToppCluster analysis found maternal NAC expression profile to be significantly enriched for genes related to the drug action of nicotine, ketamine, and dronabinol. Pathway analysis indicated postpartum NAC as enriched for RNA processing, CNS development/differentiation, and transcriptional regulation. Weighted Gene Coexpression Network Analysis (WGCNA) identified possible networks for transcription factors, including Nr1d1, Per2, Fosb, Egr1, and Nr4a1. The postpartum state involves increased risk for mental health disorders and MSET analysis indicated postpartum NAC to be enriched for genes related to depression, bipolar disorder (BPD), and schizophrenia. Mental health related genes included: Fabp7, Grm3, Penk, and Nr1d1. We confirmed via quantitative PCR Nr1d1, Per2, Grm3, Penk, Drd1a, and Pdyn. This study indicates for the first time that postpartum NAC involves large scale gene expression alterations linked to addiction and reward. Because the postpartum state also involves decreased response to drugs, the findings could provide insights into how to mitigate addictions.

Endogenous CNS Expression of Neurotensin and Neurotensin Receptors Is Altered during the Postpartum Period in Outbred Mice

Neurotensin (NT) is a neuropeptide identical in mice and humans that is produced and released in many CNS regions associated with maternal behavior. NT has been linked to aspects of maternal care and previous studies have indirectly suggested that endogenous NT signaling is altered in the postpartum period. In the present study, we directly examine whether NT and its receptors exhibit altered gene expression in maternal relative to virgin outbred mice using real time quantitative PCR (qPCR) across multiple brain regions. We also examine NT protein levels using anti-NT antibodies and immunohistochemistry in specific brain regions. In the medial preoptic area (MPOA), which is critical for maternal behaviors, mRNA of NT and NT receptor 3 (Sort1) were significantly up-regulated in postpartum mice compared to virgins. NT mRNA was also elevated in postpartum females in the bed nucleus of the stria terminalis dorsal. However, in the lateral septum, NT mRNA was down-regulated in postpartum females. In the paraventricular nucleus of the hypothalamus (PVN), Ntsr1 expression was down-regulated in postpartum females. Neurotensin receptor 2 (Ntsr2) expression was not altered in any brain region tested. In terms of protein expression, NT immunohistochemistry results indicated that NT labeling was elevated in the postpartum brain in the MPOA, lateral hypothalamus, and two subregions of PVN. Together, these findings indicate that endogenous changes occur in NT and its receptors across multiple brain regions, and these likely support the emergence of some maternal behaviors.

Medial prefrontal cortex: genes linked to bipolar disorder and schizophrenia have altered expression in the highly social maternal phenotype

The transition to motherhood involves CNS changes that modify sociability and affective state. However, these changes also put females at risk for post-partum depression and psychosis, which impairs parenting abilities and adversely affects children. Thus, changes in expression and interactions in a core subset of genes may be critical for emergence of a healthy maternal phenotype, but inappropriate changes of the same genes could put women at risk for post-partum disorders. This study evaluated microarray gene expression changes in medial prefrontal cortex (mPFC), a region implicated in both maternal behavior and psychiatric disorders. Post-partum mice were compared to virgin controls housed with females and isolated for identical durations. Using the Modular Single-set Enrichment Test (MSET), we found that the genetic landscape of maternal mPFC bears statistical similarity to gene databases associated with schizophrenia (5 of 5 sets) and bipolar disorder (BPD, 3 of 3 sets). In contrast to previous studies of maternal lateral septum (LS) and medial preoptic area (MPOA), enrichment of autism and depression-linked genes was not significant (2 of 9 sets, 0 of 4 sets). Among genes linked to multiple disorders were fatty acid binding protein 7 (Fabp7), glutamate metabotropic receptor 3 (Grm3), platelet derived growth factor, beta polypeptide (Pdgfrb), and nuclear receptor subfamily 1, group D, member 1 (Nr1d1). RT-qPCR confirmed these gene changes as well as FMS-like tyrosine kinase 1 (Flt1) and proenkephalin (Penk). Systems-level methods revealed involvement of developmental gene networks in establishing the maternal phenotype and indirectly suggested a role for numerous microRNAs and transcription factors in mediating expression changes. Together, this study suggests that a subset of genes involved in shaping the healthy maternal brain may also be dysregulated in mental health disorders and put females at risk for post-partum psychosis with aspects of schizophrenia and BPD.

Genetic and neuroendocrine regulation of the postpartum brain.

Changes in expression of hundreds of genes occur during the production and function of the maternal brain that support a wide range of processes. In this review, we synthesize findings from four microarray studies of different maternal brain regions and identify a core group of 700 maternal genes that show significant expression changes across multiple regions. With those maternal genes, we provide new insights into reward-related pathways (maternal bonding), postpartum depression, social behaviors, mental health disorders, and nervous system plasticity/developmental events. We also integrate the new genes into well-studied maternal signaling pathways, including those for prolactin, oxytocin/vasopressin, endogenous opioids, and steroid receptors (estradiol, progesterone, cortisol). A newer transcriptional regulation model for the maternal brain is provided that incorporates recent work on maternal microRNAs. We also compare the top 700 genes with other maternal gene expression studies. Together, we highlight new genes and new directions for studies on the postpartum brain.