Practical solutions for including sex as a biological variable (SABV) in preclinical neuropsychopharmacological research.

Recently, many funding agencies have released guidelines on the importance of considering sex as a biological variable (SABV) as an experimental factor, aiming to address sex differences and avoid possible sex biases to enhance the reproducibility and translational relevance of preclinical research. In neuroscience and pharmacology, the female sex is often omitted from experimental designs, with researchers generalizing male-driven outcomes to both sexes, risking a biased or limited understanding of disease mechanisms and thus potentially ineffective therapeutics. Herein, we describe key methodological aspects that should be considered when sex is factored into in vitro and in vivo experiments and provide practical knowledge for researchers to incorporate SABV into preclinical research. Both age and sex significantly influence biological and behavioral processes due to critical changes at different timepoints of development for males and females and due to hormonal fluctuations across the rodent lifespan. We show that including both sexes does not require larger sample sizes, and even if sex is included as an independent variable in the study design, a moderate increase in sample size is sufficient. Moreover, the importance of tracking hormone levels in both sexes and the differentiation between sex differences and sex-related strategy in behaviors are explained. Finally, the lack of robust data on how biological sex influences the pharmacokinetic (PK), pharmacodynamic (PD), or toxicological effects of various preclinically administered drugs to animals due to the exclusion of female animals is discussed, and methodological strategies to enhance the rigor and translational relevance of preclinical research are proposed.

Adult-born neurons maintain hippocampal cholinergic inputs and support working memory during aging.

Adult neurogenesis is reduced during aging and impaired in disorders of stress, memory, and cognition though its normal function remains unclear. Moreover, a systems level understanding of how a small number of young hippocampal neurons could dramatically influence brain function is lacking. We examined whether adult neurogenesis sustains hippocampal connections cumulatively across the life span. Long-term suppression of neurogenesis as occurs during stress and aging resulted in an accelerated decline in hippocampal acetylcholine signaling and a slow and progressing emergence of profound working memory deficits. These deficits were accompanied by compensatory reorganization of cholinergic dentate gyrus inputs with increased cholinergic innervation to the ventral hippocampus and recruitment of ventrally projecting neurons by the dorsal projection. While increased cholinergic innervation was dysfunctional and corresponded to overall decreases in cholinergic levels and signaling, it could be recruited to correct the resulting memory dysfunction even in old animals. Our study demonstrates that hippocampal neurogenesis supports memory by maintaining the septohippocampal cholinergic circuit across the lifespan.  It also provides a systems level explanation for the progressive nature of memory deterioration during normal and pathological aging and indicates that the brain connectome is malleable by experience.

CB1R blockade unmasks TRPV1-mediated contextual fear generalization in female, but not male rats.

Increasing evidence suggests that the neurobiological processes that govern learning and memory can be different in males and females, but many of the specific mechanisms underlying these sex differences have not been fully defined. Here we investigated potential sex differences in endocannabinoid (eCB) modulation of Pavlovian fear conditioning and extinction, examining multiple defensive behaviors, including shock responsivity, conditioned freezing, and conditioned darting. We found that while systemic administration of drugs acting on eCB receptors did not influence the occurrence of darting, females that were classified as Darters responded differently to the drug administration than those classified as Non-darters. Most notably, CB1R antagonist AM251 produced an increase in cue-elicited freezing and context generalization selectively in female Non-darters that persisted across extinction and extinction retrieval tests but was prevented by co-administration of TRPV1R antagonist Capsazepine. To identify a potential synaptic mechanism for these sex differences, we next employed biochemical and neuroanatomical tracing techniques to quantify anandamide (AEA), TRPV1R, and perisomatic CB1R expression, focusing on the ventral hippocampus (vHip) given its known role in mediating contextual fear generalization. These assays identified sex-specific effects of both fear conditioning-elicited AEA release and vHip-BLA circuit structure. Together, our data support a model in which sexual dimorphism in vHip-BLA circuitry promotes a female-specific dependence on CB1Rs for context processing that is sensitive to TRPV1-mediated disruption when CB1Rs are blocked.

CB1R blockade unmasks TRPV1-mediated contextual fear generalization in female, but not male rats.

Increasing evidence suggests that the neurobiological processes that govern learning and memory can be different in males and females, and here we asked specifically whether the endocannabinoid (eCB) system could modulate Pavlovian fear conditioning in a sex-dependent manner. Systemic (i.p.) injection of CB1R antagonist AM251 in adult male and female Sprague Dawley rats prior to auditory cued fear conditioning produced a female-specific increase in freezing that persisted across extinction and extinction retrieval tests but was prevented by co-administration of TRPV1R antagonist Capsazepine. Notably, AM251 also produced robust freezing in a novel context prior to auditory cue presentation the day following drug administration, but not the day of, suggesting that CB1R blockade elicited contextual fear generalization in females. To identify a potential synaptic mechanism for these sex differences, we next used liquid chromatography/tandem mass spectrometry, Western Blot, and confocal-assisted immunofluorescence techniques to quantify anandamide (AEA), TRPV1R, and perisomatic CB1R expression, respectively, focusing on the ventral hippocampus (vHip). Fear conditioning elicited increased vHip AEA levels in females only, and in both sexes, CB1R expression around vHip efferents targeting the basolateral amygdala (BLA) was twice that at neighboring vHip neurons. Finally, quantification of the vHip-BLA projections themselves revealed that females have over twice the number of neurons in this pathway that males do. Together, our data support a model in which sexual dimorphism in vHip-BLA circuitry promotes a female-specific dependence on CB1Rs for context processing that is sensitive to TRPV1-mediated disruption when CB1Rs are blocked.

Mouse spontaneous behavior reflects individual variation rather than estrous state.

Behavior is shaped by both the internal state of an animal and its individual behavioral biases. Rhythmic variation in gonadal hormones during the estrous cycle is a defining feature of the female internal state, one that regulates many aspects of sociosexual behavior. However, it remains unclear whether estrous state influences spontaneous behavior and, if so, how these effects might relate to individual behavioral variation. Here, we address this question by longitudinally characterizing the open-field behavior of female mice across different phases of the estrous cycle, using unsupervised machine learning to decompose spontaneous behavior into its constituent elements.1,2,3,4 We find that each female mouse exhibits a characteristic pattern of exploration that uniquely identifies it as an individual across many experimental sessions; by contrast, estrous state only negligibly impacts behavior, despite its known effects on neural circuits that regulate action selection and movement. Like female mice, male mice exhibit individual-specific patterns of behavior in the open field; however, the exploratory behavior of males is significantly more variable than that expressed by females both within and across individuals. These findings suggest underlying functional stability to the circuits that support exploration in female mice, reveal a surprising degree of specificity in individual behavior, and provide empirical support for the inclusion of both sexes in experiments querying spontaneous behaviors.

Adult-born neurons maintain hippocampal cholinergic inputs and support working memory during aging.

Adult neurogenesis is reduced during aging and impaired in disorders of stress, memory, and cognition though its normal function remains unclear. Moreover, a systems level understanding of how a small number of young hippocampal neurons could dramatically influence brain function is lacking. We examined whether adult neurogenesis sustains hippocampal connections cumulatively across the life span. Long-term suppression of neurogenesis as occurs during stress and aging resulted in an accelerated decline in hippocampal acetylcholine signaling and a slow and progressing emergence of profound working memory deficits. These deficits were accompanied by compensatory reorganization of cholinergic dentate gyrus inputs with increased cholinergic innervation to the ventral hippocampus and recruitment of ventrally projecting neurons by the dorsal projection. While increased cholinergic innervation was dysfunctional and corresponded to overall decreases in cholinergic levels and signaling, it could be recruited to correct the resulting memory dysfunction even in old animals. Our study demonstrates that hippocampal neurogenesis supports memory by maintaining the septohippocampal cholinergic circuit across the lifespan. It also provides a systems level explanation for the progressive nature of memory deterioration during normal and pathological aging and indicates that the brain connectome is malleable by experience.

Sounding the Alarm: Sex Differences in Rat Ultrasonic Vocalizations during Pavlovian Fear Conditioning and Extinction.

Pavlovian fear conditioning is a prevalent tool in the study of aversive learning, which is a key component of stress-related psychiatric disorders. Adult rats can exhibit various threat-related behaviors, including freezing, motor responses, and ultrasonic vocalizations (USVs). While these responses can all signal aversion, we know little about how they relate to one another. Here we characterize USVs emitted by male and female rats during cued fear acquisition and extinction, and assess the relationship between different threat-related behaviors. We found that males consistently emitted >22 kHz calls (referred to here as "alarm calls") than females, and that alarm call frequency in males, but not females, related to the intensity of the shock stimulus. Interestingly, 25% of males and 45% of females did not emit any alarm calls at all. Males that did make alarm calls had significantly higher levels of freezing than males who did not, while no differences in freezing were observed between female Alarm callers and Non-alarm callers. Alarm call emission was also affected by the predictability of the shock; when unpaired from a tone cue, both males and females started emitting alarm calls significantly later. During extinction learning and retrieval sessions, males were again more likely than females to emit alarm calls, which followed an extinction-like reduction in frequency. Collectively these data suggest sex dependence in how behavioral readouts relate to innate and conditioned threat responses. Importantly, we suggest that the same behaviors can signal sex-dependent features of aversion.

Automated classification of estrous stage in rodents using deep learning.

The rodent estrous cycle modulates a range of biological functions, from gene expression to behavior. The cycle is typically divided into four stages, each characterized by distinct hormone concentration profiles. Given the difficulty of repeatedly sampling plasma steroid hormones from rodents, the primary method for classifying estrous stage is by identifying vaginal epithelial cell types. However, manual classification of epithelial cell samples is time-intensive and variable, even amongst expert investigators. Here, we use a deep learning approach to achieve classification accuracy at expert level. Due to the heterogeneity and breadth of our input dataset, our deep learning approach ("EstrousNet") is highly generalizable across rodent species, stains, and subjects. The EstrousNet algorithm exploits the temporal dimension of the hormonal cycle by fitting classifications to an archetypal cycle, highlighting possible misclassifications and flagging anestrus phases (e.g., pseudopregnancy). EstrousNet allows for rapid estrous cycle staging, improving the ability of investigators to consider endocrine state in their rodent studies.

Darting across space and time: parametric modulators of sex-biased conditioned fear responses.

Pavlovian fear conditioning is a widely used behavioral paradigm for studying associative learning in rodents. Despite early recognition that subjects may engage in a variety of both conditioned and unconditioned responses, the last several decades have seen the field narrow its focus to measure freezing as the sole indicator of conditioned fear. We previously reported that female rats were more likely than males to engage in darting, an escape-like conditioned response that is associated with heightened shock reactivity. To determine how experimental parameters contribute to the frequency of darting in both males and females, we manipulated factors such as chamber size, shock intensity, and number of trials. To better capture fear-related behavioral repertoires in our animals, we developed ScaredyRat, an open-source custom Python tool that analyzes Noldus Ethovision-generated raw data files to identify darters and quantify both conditioned and unconditioned responses. We found that, like freezing, conditioned darting occurrences scale with experimental alterations. While most darting occurs in females, we found that with an extended training protocol, darting can emerge in males as well. Collectively, our data suggest that darting reflects a behavioral switch in conditioned responding that is a product of an individual animal's sex, shock reactivity, and experimental parameters, underscoring the need for careful consideration of sex as a biological variable in classic learning paradigms.

Considering Organismal Physiology in Laboratory Studies of Rodent Behavior.

Any experiment conducted in a rodent laboratory is done so against the backdrop of each animal's physiological state at the time of the experiment. This physiological state can be the product of multiple factors, both internal (e.g., animal sex, strain, hormone cycles, or circadian rhythms) and external (e.g., housing conditions, social status, and light/dark phases). Each of these factors has the potential to influence experimental outcomes, either independently or via interactions with others, and yet there is little consistency across laboratories in terms of the weight with which they are considered in experimental design. Such discrepancies-both in practice and in reporting-likely contribute to the perception of a reproducibility crisis in the field of behavioral neuroscience. In this review, we discuss how several of these sources of variability can impact outcomes within the realm of common learning and memory paradigms.

Response and recovery of endocrine, behavioral, and neuronal morphology outcomes after different traumatic stressor exposures in male rats.

Preclinical models of organismal response to traumatic stress (threat of death or serious injury) can be monitored using neuroendocrine, behavioral, and structural metrics. While many rodent models of traumatic stress have provided a glimpse into select components of the physiological response to acute and chronic stressors, few studies have directly examined the potential differences between stressors and their potential outcomes. To address this gap, we conducted a multi-level comparison of the immediate and longer-term effects of two types of acute traumatic stressors. Adult male rats were exposed to either underwater trauma (UWT), predator exposure (PE), or control procedural handling conditions. Over the next 7 days, yoked cohorts underwent either serial blood sampling for neuroendocrine evaluation across the circadian cycle, or repeated behavioral testing in the elevated plus maze. In addition, a subset of brains from the latter cohort were assessed for dendritic spine changes in the prefrontal cortex and basolateral amygdala. We observed stressor-dependent patterns of response and recovery across all measures, with divergence between endocrine responses despite similar behavioral outcomes. These results demonstrate that different stressors elicit unique behavioral, neuroendocrine, and neuro-structural response profiles and suggest that specific stress models can be used to model desired responses for specific preclinical applications, such as evaluations of underlying mechanisms or therapeutic candidates.

Considering sex as a biological variable will require a global shift in science culture.

For over half a century, male rodents have been the default model organism in preclinical neuroscience research, a convention that has likely contributed to higher rates of misdiagnosis and adverse side effects from drug treatment in women. Studying both sexes could help to rectify these public health problems, but incentive structures in publishing and career advancement deter many researchers from doing so. Moreover, funding agency directives to include male and female animals and human participants in grant proposals lack mechanisms to hold recipients accountable. In this Perspective, we highlight areas of behavioral, cellular and systems neuroscience in which fundamental sex differences have been identified, demonstrating that truly rigorous science must include males and females. We call for a cultural and structural change in how we conduct research and evaluate scientific progress, realigning our professional reward systems and experimental standards to produce a more equitable, representative and therefore translational body of knowledge.

The unique plasticity of hippocampal adult-born neurons: Contributing to a heterogeneous dentate.

The dentate gyrus (DG) of the hippocampus is evolutionarily conserved as one of the few sites of adult neurogenesis in mammals. Although there is clear evidence that neurogenesis is necessary for healthy hippocampal function, whether adult-born neurons are simply integrated into existing hippocampal networks to serve a similar purpose to that of developmentally born neurons or whether they represent a discrete cell population with unique functions remains less clear. In this review, we consider evidence for discrete cellular, synaptic, and structural features of adult-born DG neurons, suggesting that neurogenesis contributes to the formation of a heterogeneous DG. We therefore propose that hippocampal neurogenesis creates a specialized neuronal subpopulation that may play a key role in hippocampal functions like episodic memory. We note critical gaps in this extensive body of work, including a general failure to include female animals in relevant research and a need for more precise consideration of intrahippocampal neuroanatomy.

Sex-dependent effects of endocannabinoid modulation of conditioned fear extinction in rats.

BACKGROUND AND PURPOSE: Women are twice as likely as men to develop post-traumatic stress disorder (PTSD) making the search for biological mechanisms underlying these gender disparities especially crucial. One of the hallmark symptoms of PTSD is an alteration in the ability to extinguish fear responses to trauma-associated cues. In male rodents, the endocannabinoid system can modulate fear extinction and has been suggested as a therapeutic target for PTSD. However, whether and how the endocannabinoid system may modulate fear expression and extinction in females remains unknown. EXPERIMENTAL APPROACH: To answer this question, we pharmacologically manipulated endocannabinoid signalling in male and female rats prior to extinction of auditory conditioned fear and measured both passive (freezing) and active (darting) conditioned responses. KEY RESULTS: Surprisingly, we found that acute systemic inhibition of the endocannabinoid anandamide (AEA) or 2-arachidonoyl glycerol (2-AG) hydrolysis did not significantly alter fear expression or extinction in males. However, the same manipulations in females produced diverging effects. Increased AEA signalling at vanilloid TRPV1 receptors impaired fear memory extinction. In contrast, inhibition of 2-AG hydrolysis promoted active over passive fear responses acutely via activation of cannabinoid1 (CB1 ) receptors. Measurement of AEA and 2-AG levels after extinction training revealed sex- and brain region-specific changes. CONCLUSION AND IMPLICATIONS: We provide the first evidence that AEA and 2-AG signalling affect fear expression and extinction in females in opposite directions. These findings are relevant to future research on sex differences in mechanisms of fear extinction and may help develop sex-specific therapeutics to treat trauma-related disorders.

Controllable stress elicits circuit-specific patterns of prefrontal plasticity in males, but not females.

Actual or perceived behavioral control during a traumatic event can promote resilience against future adversity, but the long-term cellular and circuit mechanisms by which this protection is conferred have not been identified. Clinical outcomes following trauma exposure differ in men and women, and, therefore, it is especially important in preclinical research to dissect these processes in both males and females. In male adult rats, an experience with behavioral control over tail shock ("escapable stress", ES) has been shown to block the neurochemical and behavioral outcomes produced by later uncontrollable tail shock ("inescapable stress", IS), a phenomenon termed "behavioral immunization". Here, we determined whether behavioral immunization is present in females. Unlike males, the stress-buffering effects of behavioral control were absent in female rats. We next examined the effects of ES and IS on spine morphology of dorsal raphe nucleus (DRN)-projecting prelimbic (PL) neurons, a circuit critical to the immunizing effects of ES in males. In males, IS elicited broad, non-specific alterations in PL spine size, while ES elicited PL-DRN circuit-specific spine changes. In contrast, females exhibited broad, non-specific spine enlargement after ES but only minor alterations after IS. These data provide evidence for a circuit-specific mechanism of structural plasticity that could underlie sexual divergence in the protective effects of behavioral control.

Stress: Influence of sex, reproductive status and gender.

Emerging evidence from the preclinical and human research suggests sex differences in response to different types of stress exposure, and that developmental timing, reproductive status, and biological sex are important factors influencing the degree of HPA activation/function. Here we review data regarding: i) sex differences in behavioral and neural responses to uncontrollable and controllable stressors; ii) distinct trajectories of behavioral development and HPA-axis function in male and female rats following adolescent stress exposure; iii) normative changes in behavior and dopamine function in early postpartum rats; iv) aberrant HPA-axis function and its link to abnormal behaviors in two independent, preclinical mouse models of postpartum depression; and, v) data indicating that gender, in addition to sex, is an important determinant of stress reactivity in humans. Based on these findings, we conclude it will be important for future studies to investigate the short and long-term effects of a wide variety of stressors, how these effects may differ according to developmental timing and in relation to gonadal function, the relationship between aberrant HPA-axis activity during the postpartum and mood disorders, and influences of both sex and gender on stress reactivity in humans.