Two-way analysis of variance (ANOVA) was used to test for main effects of sex and rearing condition and three-way ANOVAs were used for tasks employing multiple days of testing. Follow-up one-way ANOVAs were performed with significant post hoc Bonferroni comparisons reported. For continuous home cage video monitoring, a general linear model repeated-measures ANOVA with 24 levels of hour and either two levels of treatment (control and ELS) and two levels of sex (male and female), or four levels of treatment (control, ELS, control post ketamine, and ELS post ketamine) were used to test for main effects of treatment and effect of time of day for each behavior. Alpha was set at 0.05.
During early adolescence and adulthood, no main effects of sex or rearing condition were detected between groups on either the OFT or the EPM, measures of anxiety-like behavior. Additionally, there were no effects of sex or rearing condition on locomotor activity, as measured by distance traveled during the task (Fig. 3a, b).
effect of animal sex on women
A detailed characterization of the effects of ELS on additional home cage behaviors in male and female mice can be found in Supplementary Information. Here we show a breakdown of the distribution of behaviors for control and ELS-exposed male and female mice over the course of 5 days of recording (Fig. 4d). Additional behaviors are analyzed by hour, and differences between groups are reported for drinking, eating, eating from hand, rearing, and hanging behaviors, as well as undefined movements (e.g. movements that could not be categorized into one of the other behavioral classes) (Supplementary Fig. 3).
Together, these data provide further support that ELS in the form of fragmented maternal care causes depressive-like outcomes selectively in female mice. We have shown that these sex-selective, stress-induced behaviors are rescued with the atypical antidepressant ketamine. Here we have evidence that home cage monitoring is an effective, noninvasive method for assessing these depressive-like behaviors, studying the potential mechanisms underlying stress-induced pathology, and for testing potential pharmacological interventions.
Here ELS in the form of limited bedding led to a developmentally emergent depressive-like phenotype that was present during early adolescence and became more robust in adulthood. ELS females showed symptoms of anhedonia; behavioral despair; as well as decreased motivation to approach a sweet reward in an anxiogenic environment (only in adulthood). While many of these effects were equally present in adolescent and adult mice, one noted difference was observed in the NIH task. Adolescent ELS females demonstrated a more innate anhedonic response to the sweet reward in their home cage, taking longer to approach than other groups; while in the novel cage environment, there were no observed effects of ELS or sex. In general, adolescent mice appeared to be more affected by the novel environment, with increased latencies and decreased time spent drinking in the new cage in comparison with adult mice (Supplementary Fig. 2). Alternatively, adult ELS females showed decreased motivation to approach the reward only in the novel cage, indicating that under home cage conditions, the reward was salient enough to similarly motivate these animals. Interestingly, depressive-like behaviors in ELS-exposed females were not comorbid with anxiety-like phenotypes. As measured in the EPM and OFT, ELS did not lead to anxiety-like behavior in either sex in early adolescence and caused only a slight decrease in exploratory behavior in adult animals, in both sexes. Effects seen in the depressive- and anxiety-like behavior tasks could not be explained by effects of ELS on locomotor activity. Further, using continuous home cage video monitoring, we detected additional, discrete depressive-like behaviors in ELS-exposed females. Specifically, we observed diminished self-care (grooming), decreased walking, and increased resting over the circadian cycle. Behavioral disturbances in ELS females mimicked core symptoms of depression in humans [50], and were rescued by the fast-acting antidepressant, ketamine. While we do anticipate some potential sex-selective shifts in the onset of puberty as a result of this ELS manipulation (Manzano-Nieves, in review), we did not directly assess effects of estrous cycle stage on anxiety or depressive-like behavior in this manuscript [41]. However, to account for such effects, a large number of animals from multiple litters were sampled to increase the probability of representation from multiple cycle stages in a given task. Additionally, adolescent animals were tested before the onset of estrous, and sex-selective effects of ELS were already observed in these mice. It is also possible that single housing associated with automated continuous monitoring served as a secondary mild stressor, magnifying the depressive-like phenotype in ELS female mice in home cage recording experiments. However, the presence of depressive-like behaviors in home cage monitoring, as well as across traditional behavioral testing, which were carried out in independent cohorts of group housed mice, indicates that the depressive-like phenotype in ELS females can be observed regardless of housing conditions.
Despite the heightened female risk for both anxiety and depression, many ELS paradigms in rodents fail to replicate these sex effects [66]. This could be due to insensitivity of some traditional, short-term behavioral measures that are susceptible to differences in developmental stage and estrous cycle. To diagnose depression, a person must report (1) depressed mood or a loss of interest in daily activities for more than 2 weeks and (2) five of the nine core symptoms of MDD, nearly every day [67]. Yet, despite the complexity of MDD, measures of depressive-like behavior in animal models are acute measures (such as forced swim or tail suspension), while continuous measures such as changes in sleep and motor activity, self-care, motivation, or anxiety are often overlooked.
Here we have addressed the need for more robust methods to study depressive-like behaviors in mice, which are more relatable to the complex constellation of symptoms observed in human pathology. We supplemented traditional depressive- and anxiety-like behavior assays with the use of novel, noninvasive automated home cage continuous video monitoring. Using this system, we were able to detect robust behavioral markers of affective pathology over long spans of time and without the stress associated with typical behavioral methodologies. Using this combined approach (traditional and novel home cage assessment) we uncovered a female-selective depressive-like phenotype following ELS that emerged by early adolescence and strengthened in adulthood. Collectively, the data presented here provides a comprehensive picture of the effect of ELS on behavioral and affective outcomes in both male and female mice. The sex difference in risk for pathology opens up the possibility to leverage this model to understand sex differences in risk as well as possible factors mediating risk and resilience for stress-associated pathology.
Using this system, we find that ketamine has fast-acting antidepressant effects in ELS-exposed females; reversing home cage depressive-like behaviors, mirroring the rapid alleviation of core major depression symptoms following an intravenous dose of ketamine in otherwise treatment-resistant patients [68, 69]. The current findings are even more powerful as the video monitoring method allowed for a within-subject design, assessing the same metrics pre- and post treatment in the same animal. Use of traditional behavioral methods (forced swim, tail suspension, etc.), can only be employed a single time in a given animal, resulting in the need for either a cross-sectional approach or the use of different assays to assess outcomes from what were used prior to treatment. The novel home cage monitoring technique described here has the potential to be useful for depression screening in rodent models and long-term efficacy and safety testing of new pharmacological interventions, to identify better interventions for treatment-resistant MDD patients. By rescuing ELS-induced behavioral effects with a known antidepressant, we have revealed construct validity of this testing method for depression and drug screening. Quantifying home cage behaviors such as walking, sleeping, eating, and grooming over long periods of time could provide a significant benefit over the current gold standards of pharmacological testing. In addition to being one-dimensional, traditional behavioral tests (forced swim, tail suspension, etc.) have been shown to be sensitive to such external factors as the gender of the experimenter [70]. Continuous home cage monitoring eliminates effects of animal handling, time of day, temperature, estrous cycle, or user biases on testing outcomes, providing a more clean and thorough analysis.
Further research into the mechanism driving these sex-selective, ELS-induced depressive-like behaviors, is necessary. In previous work, we find that fragmented maternal care causes accelerated maturation of hippocampus by truncating neurodevelopment [16]. In males, ELS led to an earlier developmental silencing of cell proliferation and acceleration of parvalbumin interneuron cell maturation in the hippocampus and an accelerated development of contextual fear learning [16]. More work in females to assess ELS effects on the timing of regional brain maturation is necessary, as sex-selective disruption in neurodevelopmental timing may contribute to sex-selective development of depressive-like behaviors in female mice. Supporting this notion, we have found that ELS leads to sex disparities in cognitive outcomes, impairing spatial memory in male mice but sparing this behavior in females [39]. The current work lays the foundation for further exploration of the possible neurobiological underpinnings of these sex-selective effects on affective development, and mechanisms that underlie female risk for stress-associated mental illness. 2ff7e9595c
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