Female animals in biomedical research have traditionally been excluded from research studies due to the perceived added complexity caused by the estrus cycle. However, given the importance of sex differences in a variety of neurological disorders, testing female mice is critical to identifying sex-linked effects in diseases. To determine the susceptibility of simple behaviors to hormonal fluctuations in the estrus cycle, we studied the effects of sex and the estrus cycle on a variety of behavioral tasks commonly used in mouse phenotyping laboratories. Male and female C57BL/6J mice were tested in a small battery of short duration tests and, immediately on completion of each test, females were classified using cytology of vaginal lavages as sexually-receptive (proestrus and estrus) or non-receptive (NR; metestrus and diestrus). We showed that there was a significant difference in 3-chamber social interaction (SI) between female mice at different stages of their estrus cycle, with sexually-receptive mice showing no preferential interest in a novel female mouse compared with an empty chamber. NR female mice showed the same level of preference for a novel female mouse as male mice did for a novel male mouse. No differences between or within sexes were found for tests of anxiety elevated plus maze (EPM; Hole board), working memory [Novel object recognition (NOR)], and motor learning (repeated tests on rotarod). We conclude that the stage of the estrus cycle may impact SI between same-sex conspecifics, and does not impact performance in the elevated plus-maze, hole board, NOR, and rotarod.

Dispersal from the natal site or familial group is a core milestone of adolescent development in many species. A wild species of mouse, Mus spicilegus, presents an exciting model in which to study adolescent development and dispersal because it shows different life history trajectory depending on season of birth. M. spicilegus born in spring and summer on long days (LD) disperse in the first 3 months of life, while M. spicilegus born on shorter autumnal days (SD) delay dispersal through the wintertime. We were interested in using these mice in a laboratory context to compare age-matched mice with differential motivation to disperse. To first test if we could find a proxy for dispersal related behavior in the laboratory environment, we measured open field and novel object investigation across development in M. spicilegus raised on a LD 12 h:12 h light:dark cycle. We found that between the first and second month of life, distance traveled and time in center of the open field increased significantly with age in M. spicilegus. Robust novel object investigation was observed in all age groups and decreased between the 2nd and 3rd month of life in LD males. Compared to male C57BL/6 mice, male M. spicilegus traveled significantly longer distances in the open field but spent less time in the center of the field. However, when a novel object was placed in the center of the open field, Male M. spicilegus, were significantly more willing to contact and mount it. To test if autumnal photoperiod affects exploratory behavior in M. spicilegus in a laboratory environment, we reared a cohort of M. spicilegus on a SD 10 h:14 h photoperiod and tested their exploratory behavior at P60-70. At this timepoint, we found SD rearing had no effect on open field metrics, but led to reduced novel object investigation. We also observed that in P60-70 males, SD reared M. spicilegus weighed less than LD reared M. spicilegus. These observations establish that SD photoperiod can delay weight gain and blunt some, but not all forms of exploratory behavior in adolescent M. spicilegus.

Introduction: Dopamine has been increasingly recognized as a key neurotransmitter regulating fear/anxiety states. Nevertheless, the influence of sex and estrous cycle differences on the role of dopamine in fear responses needs further investigation. We aimed to evaluate the effects of sulpiride (a dopaminergic D2-like receptor antagonist) on contextual fear conditioning in females while exploring the influence of the estrous cycle. Methods: First, using a contextual fear conditioning paradigm, we assessed potential differences in acquisition, expression, and extinction of the conditioned freezing response in male and female (split in proestrus/estrus and metestrus/diestrus) Wistar rats. In a second cohort, we evaluated the effects of sulpiride (20 and 40 mg/kg) on contextual conditioned fear in females during proestrus/estrus and metestrus/diestrus. Potential nonspecific effects were assessed in motor activity assays (catalepsy and open-field tests). Results: No sex differences nor estrous cycle effects on freezing behavior were observed during the fear conditioning phases. Sulpiride reduced freezing expression in female rats. Moreover, females during the proestrus/estrus phases of the estrous cycle were more sensitive to the effects of sulpiride than females in metestrus/diestrus. Sulpiride did not cause motor impairments. Discussion: Although no sex or estrous cycle differences were observed in basal conditioned fear expression and extinction, the estrous cycle seems to influence the effects of D2-like antagonists on contextual fear conditioning.

In 2005, Holy and Guo advanced the idea that male mice produce ultrasonic vocalizations (USV) with some features similar to courtship songs of songbirds. Since then, studies showed that male mice emit USV songs in different contexts (sexual and other) and possess a multisyllabic repertoire. Debate still exists for and against plasticity in their vocalizations. But the use of a multisyllabic repertoire can increase potential flexibility and information, in how elements are organized and recombined, namely syntax. In many bird species, modulating song syntax has ethological relevance for sexual behavior and mate preferences. In this study we exposed adult male mice to different social contexts and developed a new approach of analyzing their USVs based on songbird syntax analysis. We found that male mice modify their syntax, including specific sequences, length of sequence, repertoire composition, and spectral features, according to stimulus and social context. Males emit longer and simpler syllables and sequences when singing to females, but more complex syllables and sequences in response to fresh female urine. Playback experiments show that the females prefer the complex songs over the simpler ones. We propose the complex songs are to lure females in, whereas the directed simpler sequences are used for direct courtship. These results suggest that although mice have a much more limited ability of song modification, they could still be used as animal models for understanding some vocal communication features that songbirds are used for.

Corticosterone facilitates behavioral adaptation to a novel experience in a coordinate manner via mineralocorticoid (MR) and glucocorticoid receptors (GR). Initially, MR mediates corticosterone action on appraisal processes, risk assessment and behavioral flexibility and then, GR activation promotes consolidation of the new information into memory. Here, we studied on the circular holeboard (CHB) the spatial performance of female mice with genetic deletion of MR from the forebrain (MR(CaMKCre)) and their wild type littermates (MR(flox/flox) mice) over the estrous cycle and in response to an acute stressor. The estrous cycle had no effect on the spatial performance of MR(flox/flox) mice and neither did the acute stressor. However, the MR(CaMKCre) mutants needed significantly more time to find the exit and made more hole visit errors than the MR(flox/flox) mice, especially when in proestrus and estrus. In addition, stressed MR(CaMKCre) mice in estrus had a shorter exit latency than the control estrus MR(CaMKCre) mice. About 70% of the female MR(CaMKCre) and MR(flox/flox) mice used a hippocampal (spatial, extra maze cues) rather than the caudate nucleus (stimulate-response, S-R, intra-maze cue) strategy and this preference did neither change over the estrous cycle nor after stress. However, stressed MR(CaMKCre) mice using the S-R strategy needed significantly more time to find the exit hole as compared to the spatial strategy using mice suggesting that the MR could be needed for the stress-induced strategy switch toward a spatial strategy. In conclusion, the results suggest that loss of MR interferes with performance of a spatial task especially when estrogen levels are high suggesting a strong interaction between stress and sex hormones.

Chronic stress drives behavioral and physiological changes associated with numerous psychiatric disease states. In rodents, the vast majority of chronic stress models involve imposition of external stressors, whereas in humans stress is often driven by internal cues, commonly associated with a sense of loss. We previously exposed groups of rats to environmental enrichment (EE) for a protracted period (1 month), followed by removal of enrichment (ER), to induce an experience of loss in male rats. ER enhanced immobility in the forced swim test (FST), led to hypothalamic pituitary adrenal (HPA) axis hypoactivity, and caused hyperphagia relative to continuously enriched (EE), single-housed (Scon) and pair-housed (Pcon) groups, most of which were reversible by antidepressant treatment (Smith et al., 2017). Here, we have applied the same approach to study enrichment loss in female rats. Similar to the males, enrichment removal in females led to an increase in the time spent immobile in the FST and increased daytime food intake compared to the single and pair-housed controls. Unlike males, ER females showed decreased sucrose preference, and showed estrus cycle-dependent HPA axis hyperactivity to an acute restraint stress. The increase in passive coping (immobility), anhedonia-like behavior in the sucrose preference test and HPA axis dysregulation suggest that enrichment removal produces a loss phenotype in females that differs from that seen in males, which may be more pronounced in nature.

Genetic disruption of the vomeronasal organ (VNO), an organ responsible for pheromone processing, drastically alters socio-sexual behavior in mice. However, it is not known whether the VNO has a role during the pubertal organizational period when sex-typical socio-sexual behaviors emerge, or if disruption of the organ in adulthood is sufficient to alter socio-sexual behavior. To bypass the lifelong VNO disruption of genetic knockout models, we surgically ablated the VNO of male and female mice either during the peripubertal period [postnatal day (PND) 28-30] or adulthood (PND 58-60), with sham controls at both ages. We ruled out anosmia via the buried food test and assessed sexual odor preferences by simultaneously exposing mice to same- and opposite-sex soiled-bedding. We then measured territorial aggression with the resident-intruder paradigm and assessed sexual behavior in response to an encounter with an estrus-induced female. Neural activity approximated by FOS-immunoreactivity along the VNO-accessory olfactory pathway was measured in response to opposite-sex odors. We found that peripubertal VNO ablation decreased sexual odor preferences and neural activity in response to opposite-sex odors, and drastically reduced territorial aggression in male mice. Conversely, adult VNO ablation resulted in subtle differences in sexual odor preferences compared with sham controls. Regardless of the VNO condition, mice displayed sex-typical copulatory behaviors. Together, these results suggest that puberty is a critical period in development whereby the VNO contributes to the sexual differentiation of behavior and neural response to conspecific odors.

Memory disruption commonly follows chronic stress, whereas acute stressors are generally benign. However, acute traumas such as mass shootings or natural disasters-lasting minutes to hours and consisting of simultaneous physical, social, and emotional stresses-are increasingly recognized as significant risk factors for memory problems and PTSD. Our prior work has revealed that these complex stresses (concurrent multiple acute stresses: MAS) disrupt hippocampus-dependent memory in male rodents. In females, the impacts of MAS are estrous cycle-dependent: MAS impairs memory during early proestrus (high estrogens phase), whereas the memory of female mice stressed during estrus (low estrogens phase) is protected. Female memory impairments limited to high estrogens phases suggest that higher levels of estrogens are necessary for MAS to disrupt memory, supported by evidence that males have higher hippocampal estradiol than estrous females. To test the role of estrogens in stress-induced memory deficits, we blocked estrogen production using aromatase inhibitors. A week of blockade protected male and female mice from MAS-induced memory disturbances, suggesting that high levels of estrogens are required for stress-provoked memory impairments in both males and females. To directly quantify 17β-estradiol in murine hippocampus we employed both ELISA and mass spectrometry and identified significant confounders in both procedures. Taken together, the cross-cycle and aromatase studies in males and females support the role for high hippocampal estrogens in mediating the effect of complex acute stress on memory. Future studies focus on the receptors involved, the longevity of these effects, and their relation to PTSD-like behaviors in experimental models.

Although an effective treatment for pediatric brain tumors, cranial radiation therapy (CRT) damages surrounding healthy tissue, thereby disrupting brain development. Animal models of pediatric CRT have primarily relied on visual tasks to assess cognitive impairment. Moreover, there has been a lack of sex comparisons as most research on the cognitive effects of pediatric CRT does not include females. Therefore, we utilized olfaction, an ethologically relevant sensory modality, to assess cognitive impairment in an animal model of CRT that included both male and female mice. Specifically, we used the novel odor recognition (NOdorR) task with social odors to test recognition memory, a cognitive parameter that has been associated with olfactory neurogenesis, a form of cellular plasticity damaged by CRT. In addition to odor recognition memory, olfactory ability or discrimination of non-social and social odors were assessed both acutely and 3 months after CRT. Magnetic resonance imaging (MRI) and histology were performed after behavioral testing to assess long-term damage by CRT. Long-term but not acute radiation-induced impairment in odor recognition memory was observed, consistent with delayed onset of cognitive impairment in human patients. Males showed greater exploration of social odors than females, but general exploration was not affected by irradiation. However, irradiated males had impaired odor recognition memory in adulthood, compared to non-irradiated males (or simply male controls). Female olfactory recognition memory, in contrast, was dependent on estrus stage. CRT damage was demonstrated by (1) histological evaluation of olfactory neurogenesis, which suggested a reduction in CRT versus control, and (2) imaging analyses which showed that the majority of brain regions were reduced in volume by CRT. Specifically, two regions involved in social odor processing (amygdala and piriform cortex) were damaged by cranial irradiation in males but not females, paralleling olfactory recognition findings.

Navigating complex decisions and considering their relative risks and rewards is an important cognitive ability necessary for survival. However, use of and dependence on illicit drugs can result in long-lasting changes to this risk/reward calculus in individuals with substance use disorder. Recent work has shown that chronic exposure to cocaine causes long-lasting increases in risk taking in male and female rats, but there are still significant gaps in our understanding of the relationship between cocaine use and changes in risk taking. For example, it is unclear whether the magnitude of cocaine intake dictates the extent to which risk taking is altered. To address this, male and female Sprague-Dawley rats underwent cocaine (or sucrose) self-administration and, following a period of abstinence, were trained and tested in a rodent model of risky decision making. In this behavioral task, rats made discrete-trial choices between a lever associated with a small food reward (i.e., "safe" option) and a lever associated with a larger food reward accompanied by a variable risk of footshock delivery (i.e., "risky" option). Surprisingly, and in contrast to prior work in Long-Evans rats, there were no effects of cocaine self-administration on choice of the large, risky reward (i.e., risk taking) during abstinence in males or females. There was, however, a significant relationship between cocaine intake and risk taking in female rats, with greater intake associated with greater preference for the large, risky reward. Relative to their sucrose counterparts, female rats in the cocaine group also exhibited irregular estrous cycles, characterized by prolonged estrus and/or diestrus phases. Collectively, these data suggest that there may be strain differences in the effects of cocaine on risk taking and highlight the impact that chronic cocaine exposure has on hormonal cyclicity in females. Future work will focus on understanding the neural mechanisms underlying cocaine's intake-dependent effects on risk taking in females, and whether this is directly related to cocaine-induced alterations in neuroendocrine function.

In female rodents, sexual receptivity is coordinated with cyclic changes in the release of gonadal hormones. Increases in estradiol (E) and progesterone (P) during proestrus and estrus not only induce ovulation but also modulate behaviors that increase the likelihood that the female will find a mate and reproduce. This includes changes in receptive behaviors, such as lordosis, as well as changes in appetitive or proceptive behaviors, including motivation. Interestingly, the direction of these changes in motivation is dependent on the type of reward that is being pursued. While induction of sexual receptivity by E and P increases motivation for access to a male, motivation for a palatable food reward is decreased. These concurrent changes may facilitate adaptive choice across the estrous cycle; females bias their choice for sex when fertilization is most likely to occur, but for food when copulation is unlikely to result in impregnation. In order to test this hypothesis, we developed a novel paradigm to measure the motivated choice between a palatable food reward and access to a male conspecific. Ovariectomized, hormone primed females were trained to operantly respond for both food and sex on a fixed interval (FI) schedule. After training, unprimed and primed females were tested in a chamber that allows them to choose between food and sex while still requiring responding on the FI schedule for reach reward. From this we can not only determine the impact of hormone priming on female choice for food or sex, but also how this is reflected by changes in motivation for each specific reward, as measured by the average number of responses made during each fixed interval. Induction of sexual receptivity by hormone priming biases choice toward sex over food and this change is accompanied by an increase in motivation for sex but a decrease in motivation for food. This work provides evidence in support of a novel framework for understanding how the release of ovarian hormones over the course of the estrous cycle modulates adaptive behavioral choice in females by directly assessing motivation via operant responding when multiple rewards are available.

Adult male mice emit large number of complex ultrasonic vocalizations (USVs) when interacting with adult females. Call numbers and call categories differ greatly among inbred mouse strains. Little is known about USV emissions when the social partner departs. To investigate whether call repertoires and call rates are different when the male is interacting with a female and after the removal of the female, we designed a novel male-female social interaction test in which vocalizations were recorded across three phases. During phase 1, the male subject freely interacts with an unfamiliar estrus female mouse in a clean cage for 5 min. During phase 2, the female is removed while the male remains in the cage for 3 min. During phase 3, the same female is returned to the cage to rejoin the male subject mouse for 3 min. C57BL/6J (B6), FVB.129P2-Pde6b(+) Tyr(c-ch)/Ant (FVB), and BTBR T+ tf/J (BTBR) male subject mice were tested in this paradigm. All three strains emitted USVs during their initial interaction with the female partner. When the female was reintroduced in phase 3, numbers of USVs were similar to the initial introductory phase 1. Strain comparisons indicated fewer calls in pairs of BTBR males and stimulus females than in pairs of B6 males and stimulus females and pairs of FVB males and stimulus females. In the absence of the female, all FVB males vocalized, while only one third of B6 males and one third of BTBR males vocalized. In all three strains, changes in call category repertoires were detected after the female was removed. Call categories reverted to the phase 1 pattern when the female was returned in phase 3. Present findings indicate that males of commonly used inbred strains emit USVs when a partner female leaves the testing arena, suggesting that removing a salient social stimulus may be a unique approach to elicit USVs from mice. Our three-phase paradigm may also be useful for studying attention to social cues, and qualitative differences in vocalizations when a social partner is present vs. suddenly absent.