Behavior In Adult Female Mice Research Articles

The Effect of Long-Term Moderate Static Magnetic Field Exposure on Adult Female Mice.

Because of the high cost and safety of ultra-high magnetic resonance imaging (MRI), its application has certain limitations. Whereas 0.5-3 T MRI has been widely applied in hospitals, static magnetic fields (SMFs) have been shown to improve mice mental health and have anti-tumor potentials. Here, we compared the effects of the upward and downward 150 mT SMF groups with the sham group on C57BL/6J adult female mice. Locomotor and exploratory activity were also measured by behavioral tests, including the open field and elevated plus test. Additionally, physiology, pathology indicators and gut microbiota were examined. We found that 150 mT SMFs long-term exposure enhanced locomotive and exploratory activity of mice, especially the downward 150 mT SMF. Compared with the downward 150 mT SMF group, the movement speed and distance in the center area of the sham group were increased by 65.99% (p < 0.0001) and 68.58% (p = 0.0038), respectively. Moreover, compared to the sham group, downward 150 mT SMF increased the number of entrances to the center area by 67.0% (p = 0.0082) and time in the center area by 77.12% (p = 0.0054). Additionally, we observed that upward 150 mT SMF improved the number of follicles (~2.5 times, p = 0.0325) and uterine glands through increasing the total antioxidant capacity and reducing lipid peroxidation level in mice. Gut microbiome analysis showed that 150 mT SMFs long-term exposure improved the microbiota abundance (Clostridium, Bifidobacterium, Ralstonia and Yaniella) in the genus level, which may affect metabolism, anxiety and behavior in adult female mice. Our results demonstrated that 150 mT SMFs long-term exposure not only had good biosafety, but also improved athletic performance, emotion and the function of ovarian, uterine and gut microbiota abundance in adult female mice, which unraveled the potential of moderate long-term SMF exposure in clinical applications.

Nicotine exposure through breastfeeding affects brain-derived neurotrophic factor and synaptic proteins levels in the brain of stressed adult female mice.

Nicotine has been used during pregnancy and lactation as a tobacco harm reduction strategy. However, it is unclear whether nicotine exposure during a critical development period negatively impacts stress responses in adulthood. This study investigated how nicotine, administered via breastfeeding, affects the brain-derived neurotrophic factor (BDNF), synaptic proteins levels, and anxiety-like behavior in adult female mice subjected to stress. Female Swiss mice were exposed to saline or nicotine (8mg/kg/day) through breastfeeding between their fourth and 17th postnatal days (P) via implanted osmotic mini pumps. The unpredictable chronic mild stress (UCMS) protocol was performed during their adulthood (P65) for 10 consecutive days, followed by the elevated plus maze (EPM) test 1 day after the protocol. Animals were euthanized and their blood, collected for plasma corticosterone measurements and their brain structures, dissected for BDNF and synaptic proteins analyses. We found no significant differences in corticosterone levels between groups (Saline/Non-stress, Nicotine/Non-stress, Saline/Stress, and Nicotine/Stress). The UCMS protocol hindered weight gain. Mice exposed to nicotine through breastfeeding with or without the UCMS protocol in adulthood showed higher grooming and head dipping frequency; decreased BDNF levels in cerebellum and striatum; increased postsynaptic density protein 95 (PSD-95), synapsin I, and synaptophysin levels in cerebellum; and decreased PSD-95 and synapsin I levels in brainstem. Our results indicate that nicotine exposure through breastfeeding leads to long-lasting behavioral effects and synaptic protein changes, most of which were independent of the UCMS protocol, even after a long nicotine-free period, highlighting the importance of further studies on nicotine exposure during development.

Prenatal Androgen Excess Impairs Sexual Behavior in Adult Female Mice: Perspective on Sexual Dysfunction in PCOS

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders worldwide, affecting 5-20% of reproductive aged women [1]. PCOS is characterised by androgen excess, oligo- or anovulation, and polycystic ovarian morphology [1]. PCOS patients also experience sexual dysfunction, including decreased sexual desire, increased sexual dissatisfaction and gender dysphoria [2-4]. The origins of PCOS-related sexual difficulties remain unidentified, but may be related to impaired central mechanisms regulating sexual behaviours. Prenatally androgenized (PNA) mice recapitulate the PCOS phenotype and exhibit alterations in the neuronal network regulating reproductive function [5], providing a powerful, pathology-based model to unravel the biological origins of sexual dysfunction in PCOS. Here, we aimed to determine whether female sexual behaviours are impaired in the PNA mouse model of PCOS. To model PCOS, female dams received injections of dihydrotestosterone (PNA) or oil vehicle (VEH) daily from gestational day 16-18. Adult female offspring were ovariectomized and implanted with a silastic capsule of estradiol to examine the female-typical sexual behaviour: lordosis as well as partner preference. We also examined a potential masculinisation of the brain by replacing the estradiol implant by a testosterone implant then testing the female for male-like sexual behaviours. PNA females exhibited significantly reduced lordosis behaviour compared to VEH females (p<0.01). In contrast, partner preference and male-like sexual behaviour were not different between PNA and VEH females. In addition, using Open-field test and elevated-plus maze, we observed no effect of prenatal androgen exposure on locomotion and anxiety. These results highlight, for the first time, that prenatal exposure to the non-aromatisable androgen, DHT, impairs female receptivity only without masculinisation. These findings support the use of the PNA mouse model of PCOS to identify the neuronal targets of prenatal androgen action and to determine the mechanisms by which prenatal androgen excess impairs lordosis. Taken together, this study introduce a novel perspective on the origins of sexual dysfunction in women with PCOS and indicate the need for further investigation into the mechanisms of androgen excess on the female brain and sexual function. [1] Lizneva D et al, Fertil Steril. 2016;106:6-15. [2] Fliegner M et al, Geburtshilfe Frauenheilkd. 2019;79:498-509.[3] Kowalczyk R et al, Acta Obstet Gynecol Scand. 2012;91:710-4.[4] Mansson M et al, Eur J Obstet Gynecol Reprod Biol. 2011;155:161-5. [5] Ruddenklau A, Campbell RE. Endocrinology. 2019 Oct 1;160(10):2230-2242.

Estradiol Increases Microglial Response to Lipopolysaccharide in the Ventromedial Hypothalamus during the Peripubertal Sensitive Period in Female Mice.

Sensitive periods are times of development during which the effects of experience are unusually strong and long lasting. The peripubertal period has emerged as one such sensitive period, and a single administration of lipopolysaccharide (LPS) during this time reduces hormone-induced sexual behavior in adult female mice. During periods of high synaptic turnover, maturation, and elimination, as occurs during this sensitive period, microglia are particularly active. Estradiol also regulates microglial numbers, morphology, and activation. In addition, a good deal of evidence suggests that estradiol may confer this vulnerability to the effects of a stressor during the peripubertal period. Therefore, we investigated the effects of estradiol on microglial morphology, cytokine levels, and the sickness response to LPS. Estradiol levels were manipulated by implanting an estradiol-filled SILASTIC capsule (or oil-filled control) in ovariectomized mice or by administering the aromatase inhibitor, formestane (or oil control), to ovary-intact mice. We found that (1) estradiol elevates basal microglial Iba1 immunoreactivity in the ventromedial nucleus of the hypothalamus (VMH), (2) LPS induces higher levels of proinflammatory cytokines in the presence of estradiol, and (3) LPS causes hypothermia in the presence of estradiol. Taken together, these data suggest that estradiol enhances the effect of LPS during the pubertal sensitive period.

Maternal Separation Early in Life Alters the Expression of Genes Npas4 and Nr1d1 in Adult Female Mice: Correlation with Social Behavior

Early-life stress affects neuronal plasticity of the brain regions participating in the implementation of social behavior. Our previous studies have shown that brief and prolonged separation of pups from their mothers leads to enhanced social behavior in adult female mice. The goal of the present study was to characterize the expression of genes (which are engaged in synaptic plasticity) Egr1, Npas4, Arc, and Homer1 in the prefrontal cortex and dorsal hippocampus of adult female mice with a history of early-life stress. In addition, we evaluated the expression of stress-related genes: glucocorticoid and mineralocorticoid receptors (Nr3c1 and Nr3c2) and Nr1d1, which encodes a transcription factor (also known as REVERBα) modulating sociability and anxiety-related behavior. C57Bl/6 mice were exposed to either maternal separation (MS, 3 h once a day) or handling (HD, 15 min once a day) on postnatal days 2 through 14. In adulthood, the behavior of female mice was analyzed by some behavioral tests, and on the day after the testing of social behavior, we measured the gene expression. We found increased Npas4 expression only in the prefrontal cortex and higher Nr1d1 expression in both the prefrontal cortex and dorsal hippocampus of adult female mice with a history of MS. The expression of the studied genes did not change in HD female mice. The expression of stress-related genes Nr3c1 and Nr3c2 was unaltered in both groups. We propose that the upregulation of Npas4 and Nr1d1 in females with a history of early-life stress and the corresponding enhancement of social behavior may be regarded as an adaptation mechanism reversing possible aberrations caused by early-life stress.

Stress early in life leads to cognitive impairments, reduced numbers of CA3 neurons and altered maternal behavior in adult female mice.

The hippocampus is a crucial part of the limbic system involved both in cognitive processing and in the regulation of responses to stress. Adverse experiences early in life can disrupt hippocampal development and lead to impairment of the hypothalamic-pituitary-adrenal axis response to subsequent stressors. In our study, two types of early-life stress were used: prolonged separation of pups from their mothers (for 3 hours/day, maternal separation, MS) and brief separation (for 15 minutes/day, handling, HD). In the first part of our study, we found that adult female mice (F0) who had experienced MS showed reduced locomotor activity and impairment of long-term spatial and recognition memory. Analysis of various hippocampal regions showed that MS reduced the number of mature neurons in CA3 of females, which is perhaps a crucial hippocampal region for learning and memory; however, neurogenesis remained unchanged. In the second part, we measured maternal care in female mice with a history of early-life stress (F0) as well as the behavior of their adult offspring (F1). Our results indicated that MS reduced the level of maternal care in adult females (F0) toward their own progeny and caused sex-specific changes in the social behavior of adult offspring (F1). In contrast to MS, HD had no influence on female behavior or hippocampal plasticity. Overall, our results suggest that prolonged MS early in life affects the adult behavior of F0 female mice and hippocampal neuronal plasticity, whereas the mothers' previous experience has effects on the behavior of their F1 offspring through disturbances of mother-infant interactions.

Early-life stress links 5-hydroxymethylcytosine to anxiety-related behaviors

ABSTRACTEnvironmental stress contributes to the development of psychiatric disorders, including posttraumatic stress disorder and anxiety. While even acute stress alters gene expression, the molecular mechanisms underlying these changes remain largely unknown. 5-hydroxymethylcytosine (5hmC) is a novel environmentally sensitive DNA modification that is highly enriched in the brain and is associated with active transcription of neuronal genes. Here we examined behavioral and molecular alterations in adult mice that experienced an early-life stress before weaning (postnatal day 12 to 18) and found anxiety-like behaviors in adult female mice that were accompanied by correlated disruptions of hypothalamic 5hmC and gene expression in 118 genes, revealing potentially functional 5hmC (i.e., gene regulation). These genes are known and potentially novel stress-related targets, including Nr3c2, Nrxn1, Nfia, and Clip1, that have a significant enrichment for neuronal ontological functions, such as neuronal development and differentiation. Sequence motif predictions indicated that 5hmC may regulate gene expression by mediating transcription factor binding and alternative splicing of many of these transcripts. Together, these findings represent a critical step toward understanding the effects of early environment on the neuromolecular mechanisms that underlie the risk to develop anxiety disorders.

Pubertal exposure to di-(2-ethylhexyl) phthalate influences social behavior and dopamine receptor D2 of adult female mice.

DEHP, one of the most commonly phthalates used in plastics and many other products, is an environmental endocrine disruptor (EED). Puberty is another critical period for the brain development besides the neonatal period and is sensitive to EEDs. Social behavior is organized during puberty, so the present study is to investigate whether pubertal exposure to DEHP influenced social behavior of adult female mice. The results showed that pubertal exposure to DEHP for 2 weeks did not change the serum level of 17β-estradiol and the weight of uterus of adult females, but decreased the number of grid crossings and the frequency of rearing, and increased grooming in open field. DEHP reduced the open arm entries and the time spent in open arms in the elevated plus maze. DEHP reduced mutual sniffing and grooming between unfamiliar conspecifics in social play task and reduced the right chamber (containing unfamiliar female mouse) entries and the frequency of sniffing unfamiliar female mouse. DEHP at 1 mg kg(-1) d(-1) reduced the time spent in right chamber. Furthermore, Western blot analyses showed that DEHP decreased the levels of estrogen receptor β (ERβ), dopamine receptor D2, and the phosphorylation of ERKs in striatum. These results suggest that pubertal exposure to DEHP impaired social investigation and sociability and influenced anxiety-like state of adult female mice. The decreased activity of ERK1/2, and the down-regulated D2 and ERβ in striatum may be associated with the DEHP-induced changes of emotional and social behavior in mice.

Airborne molecules released from male mouse urine affect female exploratory behavior

Male mouse urine delivers a wide range of molecules, which may be involved in intraspecific chemical communication. These include the Major Urinary Proteins (MUP), which bind volatile odorant molecules and slowly release them from urine marks. The aim of this work is to evaluate the role of volatile molecules in eliciting exploratory behavior, in comparison to MUP. Female mice were exposed to male mouse urine, either diluted or not, or to MUP stripped of ligands. Gas chromatography and mass spectrometry of the stimuli were performed to verify the presence and identify the odorant molecules in urine and to assess the absence of MUP ligands. The exploratory behavior of adult female mice was analyzed in a cage, in the presence of two stimuli on opposite sides, but preventing direct contact with them. Four stimuli were presented in pairs: adult male mouse urine, MUP stripped of ligands, urine diluted 100 times and water as control. The results show that adult female mice explore urine, as little as 150 nl, but do not explore MUP stripped of ligands. These data show that male urine airborne molecules, effective at very low doses, mediate initial stimulus exploration by female mice.

Prenatal nicotine exposure increases anxiety and modifies sensorimotor integration behaviors in adult female mice

Prenatal exposure to nicotine (PNE) has been associated with a myriad of physiological, cognitive, and behavioral effects in the developing offspring. In this study, CD-1 dams were given injections of nicotine or control vehicle throughout gestation and their offspring were raised to 6 months of age. Adult mice were administered a battery of behavioral tests (the Suok test, the elevated platform test, and the elevated plus maze test) to assess anxiety and sensorimotor integration. PNE resulted in a decreased likelihood of jumping during the elevated platform test and decreased directed exploration in the Suok test, both indicative of increased anxiety. Also, PNE mice showed increased numbers of missteps while traversing an elevated rod in the Suok test, demonstrating altered sensorimotor integration. No significant differences were found in falls, segments traveled, latency to leave the central zone, vegetative responses, risk assessment behaviors, or autogroom behaviors. The elevated plus maze test revealed no significant differences between groups. No significant differences in body and brain weights, or cortical thickness within motor, somatosensory, and visual cortices were observed between PNE and control mice. Although neuroanatomical effects of PNE may be rescued as development progresses, defects in sensorimotor integration and increased anxiety persist into adulthood.

Intraspecific Communication Through Chemical Signals in Female Mice: Reinforcing Properties of Involatile Male Sexual Pheromones

In rodents, social and reproductive behaviors critically depend on chemical signals, including sexual pheromones that have been suggested (but not demonstrated) to be rewarding. In this work, we analyze this issue by studying the chemoinvestigatory behavior of adult female mice (without experience with male-derived chemicals) toward 1) the synthetic odorant citralva, 2) bedding soiled by different conspecifics (females, males, and castrated males), and 3) volatiles derived from bedding soiled by males and castrated males (confronted in 2-choice tests). We also study whether these chemical signals are able to induce conditioned place preference, a reliable test for rewarding properties of stimuli. The results show that involatile, male-derived chemicals elicit an intense and sustained chemoinvestigation and, more importantly, are the only tested chemical signals that induce conditioned place preference. In contrast, volatile, male-derived chemicals are not significantly chemoinvestigated. Bedding soiled by castrated males induces a transient chemoinvestigation, likely directed to steroid-independent, biologically relevant chemical signals, whereas the intense chemoinvestigation of female-soiled bedding shows a slow habituation. Finally, females did not explore significantly citralva-odorized bedding. The present work constitutes the first demonstration of the unconditioned reinforcing properties of involatile (likely detected by the vomeronasal organ) steroid-dependent chemical signals in mammals.

Neonatal testosterone administration, but not in utero contiguity to males, augments the display of male sexual behavior by testosterone-treated adult female mice

Male copulatory behavior of adult female mice given slow-release capsules of testosterone was examined in animals that developed in utero contiguous to two males (mFm) or to two females (fFf). Other females of unspecified uterine position which were injected with testosterone propionate on the day of birth as well as intact males also were examined. mFm and fFf females did not differ on any measure; latency to the first mount, number of mount bouts, number of mount bouts with genital thrusting. The perinatally androgenized females exhibited more mount bouts and more bouts accompanied by genital thrusting than did mFm and fFf subjects. The former also displayed more mount bouts with thrusting on the second pair of tests than males. Lastly, a greater proportion of perinatally androgenized females than mFm or fFf animals displayed male sexual behavior two weeks following removal of the testosterone-containing capsule.

Infanticide and parental behavior in wild female house mice: Effects of ovariectomy, adrenalectomy and administration of oxytocin and prostaglandin F 2α

We have previously observed that about 60% of virgin, wild female house mice (Mus musculus), that were the offspring of wild mice trapped in Missouri, exhibited infanticide when a single 2-day-old pup was placed into each female's home cage. But, by the end of pregnancy, there was a significant increase (to 90%) in the proportion of wild female mice that exhibited infanticide. At parturition, infanticide was inhibited and parental behavior was induced in all females. In the present study we examined the influence of ovarian and adrenal hormones, as well as two hormones associated with pregnancy and parturition: oxytocin and prostaglandin F 2α (PGF), in regulating infanticide and parental behavior in wild female house mice. The presence or absence of gonadal and adrenal hormones did not influence the frequency of infanticidal behavior in adult female mice. Subcutaneous injections of either oxytocin or PGF inhibited infanticide in previously infanticidal pregnant females 1 hr after injection, but only oxytocin served to also facilitate parental behavior (most PGF-treated females left the pup untouched). Oxytocin, but not PGF, was effective in inhibiting infanticide in previously infanticidal virgin females. These findings suggest that, by themselves, ovarian and adrenal hormones do not mediate the exhibition of infanticide by wild female mice, but the lack of response to PGF in virgin females suggests that endocrine changes during pregnancy (possibly changes in steroids) may modulate the inhibitory effects of PGF on infanticide.

Studies on opponent status and steroid mediation of aggression in female mice

The first experiment explored the effects of testosterone or estradiol-17 β on the aggressive behavior of adult female mice paired against olfactory bulbectomized male opponents. Ovariectomized females implanted with Silastic capsules containing testosterone repeatedly attacked their opponents, but females implanted with estradiol remained nonaggressive. In a second experiment, females implanted with testosterone were highly aggressive toward intact male opponents. These females were less aggressive toward intact females, but they did not distinguish behaviorally between male and female opponents as distinctly as intact males did. This suggests that androgen-treated females are less responsive than are males to “aggression-inhibiting” stimuli from female opponents.

Testosterone-induced aggression in adult female mice

Silastic implants of testosterone (T) and injections of testosterone propionate (TP) were used to study the effects of ovariectomy and androgen administration on the fighting behavior of adult female mice. A dose of T previously shown to hyperstimulate accessory organ growth in adult male castrates was sufficient to induce the complete behavioral repertoire of male-like aggression in females never before treated with exogenous androgen. As determined by radioimmunoassay, blood levels of T produced by implants containing an aggression-inducing dose of T (10-mg implant) were within the range of T concentrations observed in intact males. Following treatment with a 10-mg T implant, the aggressive behavior of ovariectomized females could be fully maintained with a dose of T (0.3-mg implant) that failed to maintain weight of the accessory organs in adult male castrates. In fact, females “androgenized” were subsequently more responsive to the aggression-activating properties of T than were males castrated after prenatal and perinatal androgen exposure.