Sharon A. Stevenson

Elevated stress sensitivity in corticotropin-releasing factor receptor 2 deficient mice decreases maternal, but not intermale aggression.

Maternal aggression is a form of aggression towards intruders by lactating females that is critical for defense of offspring. During lactation, fear and anxiety are reduced, the CNS is less responsive to the anxiogenic neuropeptide, corticotropin-releasing factor (CRF), and central injections of CRF inhibit maternal aggression. Together, these previous findings suggest that decreased CRF neurotransmission during lactation supports normal maternal aggression expression. Recent work indicates that mice deficient in CRF receptor 2 (CRFR2) display increased anxiety-like behaviors, have a hypersensitive stress response, and overproduce CRF. In this study, we examined both maternal and intermale aggression in wild-type (WT) and CRFR2-deficient mice. CRFR2-mutant mice exhibited significant deficits in maternal aggression on postpartum Day 4 relative to WT mice in terms of percentage displaying aggression, mean number of attacks, and mean time in aggressive encounters. However, time sniffing male intruder, pup retrieval, number of pups, and performance on the elevated plus maze were similar between genotypes. In contrast, intermale aggression did not differ between genotype in any measure on any of three consecutive test days. For neither form of aggression did sites of attacks on the intruder differ between genotype. Taken together, the results suggest that differences in stress sensitivity and the overproduction of CRF of the knockout (KO) mice specifically affects maternal, but not intermale aggression.

Urocortin 1 and 3 impair maternal defense behavior in mice.

Lactating female mice fiercely defend offspring while exhibiting decreased fear and anxiety. Recent work (J. S. Lonstein & S. C. Gammie, 2002) found that intracerebroventricular (icv) injections of corticotropin releasing factor (CRF), a putative anxiogenic peptide, inhibit maternal defense behavior. This study examines effects of CRF-related peptides, urocortin (Ucn) 1 and Ucn 3, on maternal aggression in mice. Intracerebroventricular injections of both Ucn 1 (0.2 microg) and Ucn 3 (0.5 microg) reduced aggression but not pup retrieval. c-Fos levels were elevated by intracerebroventricular injections of Ucn 1 (0.2 microg) and Ucn 3 (0.5 microg) in 2 and 6 brain regions, respectively; however, both triggered increases in bed nucleus of the stria terminalis dorsal (BNSTd) and lateral septum (LS). These findings suggest that CRF-related peptides similarly modulate maternal aggression and that BNSTd/LS may be critical sites for negative regulation of maternal aggression.

Deletion of corticotropin-releasing factor binding protein selectively impairs maternal, but not intermale aggression

Corticotropin-releasing factor (CRF) binding protein (CRF-BP) is a secreted protein that acts to bind and limit the activity of the neuropeptides, CRF and urocortin (Ucn) 1. We previously selected for high maternal defense (protection of offspring) in mice and found CRF-BP to be elevated in the CNS of selected mice. We also previously determined that both CRF and Ucn 1 are potent inhibitors of offspring protection when administered centrally. Thus, elevated CRF-BP could promote defense by limiting endogenous actions of CRF or Ucn 1. To test this hypothesis, we crossed the deletion for CRF-BP into the mice selected for high maternal defense and evaluated offspring protection and other maternal behaviors. CRF-BP knockout (KO) mice exhibited significant deficits in maternal aggression relative to wild-type (WT) mice in three different measures. Other maternal features were almost identical between groups, including dam and pup weight, litter size, nursing time, and pup retrieval. Both groups performed similarly in a forced swim stress test and aggression in both groups was reduced following the swim test. Virgin KO female mice exhibited higher levels of anxiety-like behavior in terms of decreased time in the light portion of the light/dark box test. For males, no differences in light/dark box or swim test were found. However, increased anxiety-like behavior in male KO mice was identified in terms of contact and approach to a novel object both with and without previous exposure to the swim test. No differences in isolation induced resident intruder male aggression were found between groups. Together, these results indicate that loss of CRF-BP selectively impairs maternal, but not intermale aggression and that loss of the gene induces anxiety-like behavior in males and females, but there are sex differences in terms of how that anxiety is revealed.

Altered gene expression in mice selected for high maternal aggression.

We previously applied selective breeding on outbred mice to increase maternal aggression (maternal defense). In this study, we compared gene expression within a continuous region of the central nervous system (CNS) involved in maternal aggression (hypothalamus and preoptic regions) between lactating selected (S) and nonselected control (C) mice (n= 6 per group). Using microarrays representing over 40 000 genes or expressed sequence tags, two statistical algorithms were used to identify significant differences in gene expression: robust multiarray and the probe logarithmic intensity error method. Approximately 200 genes were identified as significant using an intersection from both techniques. A subset of genes was examined for confirmation by real‐time polymerase chain reaction (PCR). Significant decreases were found in S mice for neurotensin and neuropeptide Y receptor Y2 (both confirmed by PCR). Significant increases were found in S mice for neuronal nitric oxide synthase (confirmed by PCR), the K+ channel subunit, Kcna1 (confirmed by PCR), corticotrophin releasing factor binding protein (just above significance using PCR; P= 0.051) and GABA A receptor subunit 1A (not confirmed by PCR, but similar direction). S mice also exhibited significantly higher levels of the neurotransmitter receptor, adenosine A1 receptor and the transcription factors, c‐Fos, and Egr‐1. Interestingly, for 24 genes related to metabolism, all were significantly elevated in S mice, suggesting altered metabolism in these mice. Together, this study provides a list of candidate genes (some previously implicated in maternal aggression and some novel) that may play an important role in the production of this behavior.

Altered maternal profiles in corticotropin-releasing factor receptor 1 deficient mice

BackgroundDuring lactation, the CNS is less responsive to the anxiogenic neuropeptide, corticotropin-releasing factor (CRF). Further, central injections of CRF inhibit maternal aggression and some maternal behaviors, suggesting decreased CRF neurotransmission during lactation supports maternal behaviors. In this study, we examined the maternal profile of mice missing the CRF receptor 1 (CRFR1). Offspring of knockout (CRFR1-/-) mice were heterozygote to offset possible deleterious effects of low maternal glucocorticoids on pup survival and all mice contained a mixed 50:50 inbred/outbred background to improve overall maternal profiles and fecundity.ResultsRelative to littermate wild-type (WT) controls, CRFR1-/- mice exhibited significant deficits in total time nursing, including high arched-back, on each test day. Consistent with decreased nursing, pups of CRFR1-deficient dams weighed significantly less than WT offspring. Licking and grooming of pups was significantly higher in WT mice on postpartum Day 2 and when both test days were averaged, but not on Day 3. Time off nest was higher for CRFR1-/- mice on Day 2, but not on Day 3 or when test days were averaged. Licking and grooming of pups did not differ on Day 2 when this measure was examined as a proportion of time on nest. CRFR1-/- mice showed significantly higher nest building on Day 3 and when tests were averaged. Mean pup number was almost identical between groups and no pup mortality occurred. Maternal aggression was consistently lower in CRFR1-/- mice and in some measures these differences approached, but did not reach significance. Because of high variance, general aggression results are viewed as preliminary. In terms of sites of attacks on intruders, CRFR1-/- mice exhibited significantly fewer attacks to the belly of the intruder on Day 5 and when tests were averaged. Performance on the elevated plus maze was similar between genotypes. Egr-1 expression differences in medial preoptic nucleus and c-Fos expression differences in bed nucleus of stria terminalis between genotype suggest possible sites where loss of gene alters behavioral output.ConclusionTaken together, the results suggest that the presence of an intact CRFR1 receptor supports some aspects of nurturing behavior.

Effects of Daily and Acute Restraint Stress During Lactation on Maternal Aggression and Behavior in Mice

A decreased reactivity to stressors during lactation might heighten the expression of maternal care (including defense of offspring) by minimizing the extent to which stress can impact maternal care. Although stressors applied during pregnancy have variable effects on maternal aggression (or defense of offspring), to date no study has examined the effects of stress applied during the postpartum period on maternal aggression. In this study, we examined the effects of both daily and acute restraint stress (30 min) applied postpartum on maternal aggression and other maternal behaviors. Daily restraint (ending 2 h before testing) did not alter any measure of maternal behavior, including nursing, licking and grooming of pups and pup retrieval, or any measure of maternal aggression. In contrast, acute stress significantly impaired total time aggressive and number of attacks, but pup retrieval was normal. c-Fos levels were significantly elevated in a number of brain regions in association with acute stress, including lateral septum (LS), caudal periaqueductal gray and medial amygdala (MeA), suggesting possible sites where stress reactivity could alter aggression. Together, the results indicate that acute restraint stress impairs maternal aggression and provide a starting point for future studies examining how stress reactivity pathways may intersect with maternal aggression pathways.

α1-Noradrenegic receptor antagonism disrupts female songbird responses to male song

Female songbirds respond behaviorally to differences in male song structure. Past data suggest a complex role for norepinephrine in female responses to song. Here, we examined the effects of central infusions of the α(1)-noradrenergic receptor antagonist prazosin on female European starling (Sturnus vulgaris) responses to nest boxes broadcasting male song. Prazosin disrupted female preferential responses to male starling song over the less biologically relevant purple martin (Progne subis) song. Prazosin decreased female responses to male starling song in a linear dose-response fashion; whereas, it affected responses to purple martin song in a U-shaped dose-response fashion. Results suggest that the role of norepinephrine in female responses to male song differs depending upon drug dose and the biological relevance of the song stimulus.

Intermale Aggression in Corticotropin-releasing Factor Receptor 1 Deficient Mice

The anxiogenic neuropeptide, corticotropin-releasing factor (CRF), has a complex effect on intermale aggression. CRF receptor 1 (CRFR1) is the primary receptor for CRF and in this study, we examined in detail isolation-induced intermale aggression in CRFR1 deficient mice. All mice contained a mixed 50:50 inbred/outbred background to improve aggressive performance. Mice were isolated for 4 weeks prior to 2 consecutive days of aggression testing using the resident-intruder paradigm. Mice were also tested for anxiety on the elevated plus maze. Relative to littermate wild-type (WT) controls, CRFR1-mutant mice exhibited normal levels of intermale aggression over the 2 test days in terms of percentage showing aggression, number of attacks, time aggressive, and latency to first attack. In terms of sites of attacks on intruders, CRFR1-deficient mice attacked the ventral portion of the mid-section (including belly) significantly less frequently than WT males on test day 1, but these differences did not reach significance on test day 2. No other differences in sites of attacks were observed. Tail rattling also did not differ between groups. Importantly, KO males showed decreased anxiety relative to WT mice (consistent with previous reports) as evidenced by spending significantly more time on the open arms and significantly less time on the closed arms of the elevated plus maze. Plus maze performance did not correlate with any measure of levels of aggression, suggesting a dissociation between altered levels of anxiety and aggressive performance. Taken together, the results suggest that the activation CRFR1 is not necessary for the normal production of isolation-induced intermale aggression.

Behavioral and pharmacological assessment of a potential new mouse model for mania

Bipolar disorder (BPD) is a devastating long-term disease for which a significant symptom is mania. Rodent models for mania include psychostimulant-induced hyperactivity and single gene alterations, such as in the Clock or DAT genes, but there is still a pressing need for additional models. Recently, our lab isolated a line of mice, termed Madison (MSN), that exhibit behavioral characteristics that may be analogous to symptoms of mania. In this study we quantified possible traits for mania and tested the response to common anti-BPD drugs in altering the behavioral profiles observed in this strain. Relative to other mouse lines, MSN mice showed significant elevations of in-cage hyperactivity levels, significant decreases in daytime sleep, and significant increases in time swimming in the forced swim test. In terms of sexual behavior, the MSN mice showed significantly higher number of mounts and a trend toward higher time mounting. In separate studies, olanzapine and lithium (or respective controls) were administered to MSN mice for at least 2weeks and response to treatments was evaluated. Olanzapine (1mg/kg/day) significantly decreased in-cage hyperactivity and significantly increased time sleeping. Lithium (0.2-0.4% in food) significantly decreased in-cage hyperactivity. Given the behavioral phenotypes and the response to anti-BPD treatments, we propose that MSN mice may provide a possible new model for understanding the neural and genetic basis of phenotypes related to mania and for developing pharmaceutical treatments.

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.