Peromyscus Californicus Research Articles

Comparative Transcriptional Analyses in the Nucleus Accumbens Identifies RGS2 as a Key Mediator of Depression-Related Behavior

BackgroundMajor depressive disorder is one of the most commonly diagnosed mental illnesses worldwide, with a higher prevalence in women than in men. Although currently available pharmacological therapeutics help many individuals, they are not effective for most. Animal models have been important for the discovery of molecular alterations in stress and depression, but difficulties in adapting animal models of depression for females has impeded progress in developing novel therapeutic treatments that may be more efficacious for women. MethodsUsing the California mouse social defeat model, we took a multidisciplinary approach to identify stress-sensitive molecular targets that have translational relevance for women. We determined the impact of stress on transcriptional profiles in male and female California mouse nucleus accumbens (NAc) and compared these results with data from postmortem samples of the NAc from men and women diagnosed with major depressive disorder. ResultsOur cross-species computational analyses identified Rgs2 (regulator of G protein signaling 2) as a transcript downregulated by social defeat stress in female California mice and in women with major depressive disorder. RGS2 plays a key role in signal regulation of neuropeptide and neurotransmitter receptors. Viral vector–mediated overexpression of Rgs2 in the NAc restored social approach and sucrose preference in stressed female California mice. ConclusionsThese studies show that Rgs2 acting in the NAc has functional properties that translate to changes in anxiety- and depression-related behavior. Future studies should investigate whether targeting Rgs2 represents a novel target for treatment-resistant depression in women.

Oxytocin receptor behavioral effects and cell types in the bed nucleus of the stria terminalis

Oxytocin is a neuropeptide that can produce anxiolytic effects and promote social approach. However, emerging evidence shows that under some conditions, oxytocin can instead induce anxiety-related behaviors. These diverse effects of oxytocin appear to be mediated by circuit-specific actions. Recent data showed that inhibition of oxytocin receptors (OTRs) in the bed nucleus of the stria terminalis (BNST) was sufficient to increase social approach and decrease social vigilance in female California mice (Peromyscus californicus) exposed to social defeat stress. As a member of the G-protein coupled receptor family, OTRs can induce distinct downstream pathways by coupling to different G-protein isoforms. We show that infusion of carbetocin, a biased OTR-Gq agonist, in the BNST reduced social approach in both female and male California mice. In both females and males, carbetocin also increased social vigilance. To gain insight into cell types that could be mediating this effect, we analyzed previously published single-cell RNAseq data from the BNST and nucleus accumbens (NAc). In the NAc, we and others showed that OTR activation promotes social approach behaviors. In the BNST, Oxtr was expressed in over 40 cell types, that span both posterior and anterior subregions of the BNST. The majority of Oxtr-expressing neurons were GABAergic. In the anterior regions of BNST targeted in our carbetocin experiments, Cyp26b1-expressing neurons had high average Oxtr expression. In the NAc, most Oxtr+ cells were D1 dopamine receptor-expressing neurons and interneurons. These differences in Oxtr cell type distribution may help explain how activation of OTR in BNST versus NAc can have different effects on social approach and social vigilance.

Comparative anatomical analysis of dopamine systems in Mus musculus and Peromyscus californicus.

Dopamine plays important roles in motivational and social behaviors in mammals, and it has been implicated in several human neurological and psychiatric disorders. Rodents are used extensively as experimental models to study dopamine function in health and disease. However, interspecies differences of dopamine systems remain incompletely characterized. Here, we assessed whether the commonly referenced anatomical organization of dopamine systems in Mus musculus differs from another rodent species, Peromyscus californicus, which exhibits unique social behaviors such as biparental care. We applied tyrosine hydroxylase immunofluorescence labeling and high-throughput microscopy to establish whole-brain maps of dopamine systems in P. californicus. By comparing these maps to those from M. musculus, we identified unexpected anatomical similarity and difference between these two species. A sex difference in dopamine neurons at the anteroventral periventricular nucleus of hypothalamus, which has been implicated in regulating the maternal behaviors of the uniparental M. musculus, is similarly present in the biparental P. californicus. In contrast, major interspecies differences from M. musculus are found in the ventral midbrain and striatum of P. californicus, including the expansion of midbrain dopamine neurons into the ventral substantia nigra and the presence of an internal capsule-like white matter tract that demarcates a dorsomedial area from the rest of the striatum. These features identified in P. californicus resemble the anatomical organization of the primate brain more closely compared to those in M. musculus. Our findings suggest that P. californicus is a unique model organism for studying the evolution of dopamine systems in mammals and the disorders of dopamine systems.

Effects of Rearing Experience and Sex on Neuroimmune Correlates of Paternal Care in a Biparental Mouse Model

Providing and receiving paternal care improves emotional regulation, stress-coping behaviors, and hippocampal structural plasticity in biparental species, like the California mouse (Peromyscus californicus). Conversely, paternal care absence results in sex-specific impairments in structural and functional plasticity of the hippocampus. Despite this knowledge, mechanisms underlying these behavioral and structural changes are understudied.

Testosterone pulses paired with a location induce a place preference to the nest of a monogamous mouse under field conditions.

Changing social environments such as the birth of young or aggressive encounters present a need to adjust behavior. Previous research examined how long-term changes in steroid hormones mediate these adjustments. We tested the novel concept that the rewarding effects of transient testosterone pulses (T-pulses) in males after social encounters alter their spatial distribution on a territory. In free-living monogamous California mice (Peromyscus californicus), males administered three T-injections at the nest spent more time at the nest than males treated with placebo injections. This mimics T-induced place preferences in the laboratory. Female mates of T-treated males spent less time at the nest but the pair produced more vocalizations and call types than controls. Traditionally, transient T-changes were thought to have transient behavioral effects. Our work demonstrates that in the wild, when T-pulses occur in a salient context such as a territory, the behavioral effects last days after T-levels return to baseline.

Specificity of California mouse pup vocalizations in response to olfactory stimuli.

To investigate flexibility in vocal signaling by rodent pups, we examined whether olfactory stimuli influence characteristics of pup calls and how these calls may be affected by sex and litter size in California mice (Peromyscus californicus). Pups were isolated and recorded during a 3-min baseline period followed by a 5-min exposure to bedding containing scent from their home cage, scent from the home cage of an unfamiliar family, coyote urine, or no scent (control). Latency to call, call rate, and call characteristics (duration, frequency, and amplitude) were compared between the baseline and scent-exposure periods and among olfactory conditions. Compared with the control condition, pups from two-pup litters called more quietly when exposed to odor from a predator, whereas pups from three-pup litters called more loudly. Additionally, pups showed nonsignificant tendencies to reduce call rates in response to odors from their home cage and to increase call rates when exposed to predator urine. Last, males produced higher-frequency calls and more ultrasonic vocalizations than females. These results indicate that pup calling behavior in this species can be influenced by acute olfactory stimuli as well as litter size and sex. The flexibility of pup calling in response to these three variables potentially increases the communication value of pup calls and helps shape the parents' responses.

Intranasal oxytocin reduces pre-courtship aggression and increases paternal response in California mice (Peromyscus californicus)

Oxytocin (OXT) is a neuropeptide that can facilitate prosocial behavior and decrease social stress and anxiety but can also increase aggression in some contexts. We investigated whether acute pulses of intranasal (IN) OXT influenced social behavior during social challenges that are likely to occur throughout the lifespan of a wild mouse. To test this, we examined the acute effects of IN OXT in the male California mouse (Peromyscus californicus), a monogamous, biparental, and territorial rodent, using a within-subjects longitudinal design. Social challenges included a pre-courtship male-female encounter conducted during the (1) initial aggressive and not the following affiliative phase of courtship, (2) same-sex resident intruder test, and (3) parental care test. Consecutive tests and doses were separated by at least two weeks. Males were treated with intranasal infusions of 0.8 IU/kg OXT or saline controls 5-min before each behavioral test, receiving a total of three treatments of either IN OXT or saline control. We predicted that IN OXT would 1) decrease aggression and increase affiliation during the pre-courtship aggression phase, 2) increase aggression during resident intruder paradigms, and 3) increase paternal care and vocalizations during a paternal care test. As predicted, during pre-courtship aggression with a novel female, IN OXT males displayed less contact aggression than control males, although with no change in affiliative behavior. However, post-pairing, during the resident intruder test, IN OXT males did not differ from control males in contact aggression. During the paternal care test, IN OXT males were quicker to approach their pups than control males but did not differ in vocalizations produced, unlike our previous research demonstrating an effect on vocalizations in females. In summary, during pre-courtship aggression and the paternal care test, IN OXT reduced antisocial behavior; however, during the resident intruder test, IN OXT did not alter antisocial behavior. These data suggest that IN OXT promotes prosocial behavior specifically in social contexts that can lead to affiliation.

Acute inhibition of dopamine β-hydroxylase attenuates behavioral responses to pups in adult virgin California mice (Peromyscus californicus)

In biparental species, in which both parents care for their offspring, the neural and endocrine mediators of paternal behavior appear to overlap substantially with those underlying maternal behavior. Little is known, however, about the roles of classical neurotransmitters, such as norepinephrine (NE), in paternal care and whether they resemble those in maternal care. We tested the hypothesis that NE facilitates the initiation of nurturant behavior toward pups in virgin male and female California mice (Peromyscus californicus), a biparental rodent. Virtually all parents in this species are attracted to familiar and unfamiliar pups, while virgins either attack, avoid, or nurture pups, suggesting that the neurochemical control of pup-related behavior changes as mice transition into parenthood. We injected virgin males and females with nepicastat, a selective dopamine β-hydroxylase inhibitor that blocks NE synthesis (75 mg/kg, i.p.), or vehicle 2 h before exposing them to a novel pup, estrous female (males only), or pup-sized novel object for 60 min. Nepicastat significantly reduced the number of males and females that approached the pup and that displayed parental behavior. In contrast, nepicastat did not alter virgins' interactions with an estrous female or a novel object, suggesting that nepicastat-induced inhibition of interactions with pups was not mediated by changes in generalized neophobia, arousal, or activity. Nepicastat also significantly reduced NE levels in the amygdala and prefrontal cortex and increased the ratio of dopamine to NE in the hypothalamus. Our results suggest that NE may facilitate the initiation of parental behavior in male and female California mice.

Blocking olfactory input alters aggression in male and female California mice (Peromyscus californicus).

Olfactory input into the brain can be disrupted by a variety of environmental factors, including exposure to pathogens or environmental contaminants. Olfactory cues are often eliminated in laboratory rats and mice through highly invasive procedures like olfactory bulbectomy, which may also disrupt accessory olfactory pathways and detection of non-volatile odors. In the present study, we tested whether inducing anosmia through intranasal infusion of zinc gluconate alters aggression in a monogamous, biparental rodent species, the California mouse (Peromyscus californicus). This less invasive method of manipulating olfaction selectively targets the olfactory epithelium and reduces the detection of volatile odors. Treatment with zinc gluconate extended the time required for male and female California mice to find hidden pieces of apple and reduced the amount of time spent investigating bedding that was soiled by unfamiliar males. Moreover, inhibition of olfaction with zinc gluconate reduced aggressiveness in both sexes as demonstrated by an increased attack latency in the resident-intruder test among same-sex dyads from the same treatment group. These results suggest that volatile olfactory cues are necessary for agonistic responses in both male and female California mice. Therefore, even in species with complex social systems that include territorial aggression and monogamy, volatile olfactory cues modulate agonistic behavior.

104 Epigenetic Changes Due to Environmental Toxicants: An Animal Health Concern

Abstract Endocrine disrupting chemicals (EDCs) mimic natural hormones in the body, but they are not subject to normal homeostatic regulatory mechanisms. One such EDC that is particularly important in animal and human health is bisphenol A (BPA) that is an industrial chemical used to harden plastic, and thus, it is prevalent in many common household items. Notably, BPA, and likely other EDCs, persist in the environment. Besides binding to steroid and non-steroid receptors, BPA and other EDCs may induce epigenetic changes directly or by affecting gut bacteria that can promote such host changes. The objective of current studies was to determine whether developmental exposure to BPA and/or genistein, a phytoestrogen, induce persistent epigenetic and transcriptomic changes in various brain regions and the placenta. Additionally, the ability of these chemicals to alter gut microbiota and gut metabolites that may trigger such epigenetic alterations were investigated. Animal models used to examine for such effects included California mice (Peromyscus californicus), deer mice (Peromyscus maniculatus), laboratory mice (Mus musculus), and eastern painted turtles (Chrysemys picta). To link these ‘omics changes to actual phenotypic modifications, several behavioral domains were assessed in these species following developmental exposure to these compounds. Results across taxa clearly show that BPA and genistein leads to behavioral deficits, including cognitive and social impairments, anxiogenic behaviors, and reduced voluntary physical activity. Correspondingly, both chemicals transformed the epigenome and transcriptome in key brain regions and the placenta. Gut dysbiosis and stimulation of harmful bacterial metabolites ensued following early EDC exposure, and such effects persisted through adulthood. By using a one health medicine approached that evaluated various vertebrate animal species, there is solid evidence that perinatal exposure to BPA and genistein reprograms the epigenome and thereby lead to longstanding health consequences. Such findings have important veterinary and human health ramifications.

Early paternal retrieval experience influences the degree of maternal retrieval behavior in adult California mice offspring

In biparental species like the California mouse (Peromyscus californicus), paternal presence and care contributes to offspring survival with lasting consequences on brain development and social behavior. Paternal retrieval behavior may be particularly important since it protects young from dangers outside of the nest. We have previously shown that paternal retrievals influence social behavior of adult female and male offspring, as well as the expression of hormones associated with parental behavior. In male offspring, paternal retrieval influences future parenting, but whether paternal retrievals affect maternal behavior in adulthood is unclear. Here, we manipulated the experience of paternal retrieval during development and then assessed maternal behavior of adult female offspring. We did not detect group differences in maternal behavior during undisturbed observation or following pup displacement. However, following pup displacement we observed a moderate positive correlation between paternal retrievals experienced in development and maternal retrievals performed in adulthood. Further analysis revealed that the likelihood of females being a high or low retriever is influenced by their developmental experience. These findings suggest that although female California mice engage in similar levels of maternal behavior regardless of paternal care environment, there may be variation in retrieval behavior that is informed by paternal retrieval experience.

Intranasal oxytocin drives coordinated social approach

Coordinated responses to challenge are essential to survival for bonded monogamous animals and may depend on behavioral compatibility. Oxytocin (OT) context-dependently regulates social affiliation and vocal communication, but its role in pair members’ decision to jointly respond to challenge is unclear. To test for OT effects, California mouse females received an intranasal dose of OT (IN-OT) or saline after bonding with males either matched or mismatched in their approach response to an aggressive vocal challenge. Pair mates were re-tested jointly for approach response, time spent together, and vocalizations. Females and males converged in their approach after pairing, but mismatched pairs with females given a single dose of IN-OT displayed a greater convergence that resulted from behavioral changes by both pair members. Unpaired females given IN-OT did not change their approach, indicating a social partner was necessary for effects to emerge. Moreover, IN-OT increased time spent approaching together, suggesting behavioral coordination beyond a further increase in bonding. This OT-induced increase in joint approach was associated with a decrease in the proportion of sustained vocalizations, a type of vocalization that can be associated with intra-pair conflict. Our results expand OT’s effects on behavioral coordination and underscore the importance of emergent social context.

Pair-bonding leads to convergence in approach behavior to conspecific vocalizations in California mice (Peromyscus californicus).

Pair-bonding allows for division of labor across behavioral tasks such as protecting a territory, caring for pups or foraging for food. However, how these labor divisions are determined, whether they are simply intrinsic differences in the individual’s behavior or a coordinated behavioral response by the pair, remains unknown. We used the monogamous, biparental and territorial California mouse (Peromyscus californicus) to study how behavioral approach to an aggressive vocal stimulus in a novel environment was affected by pair-bonding. Using a three-chambered vocal playback paradigm, we first measured the amount of time individuals spent in close proximity to aggressive bark vocalizations. We found that animals could be categorized as either approachers or avoiders. We then paired individuals based on their initial approach behavior to an opposite sex individual who displayed either similar or different approach behaviors. These pairs were then retested for approach behavior as a dyad 10–11 days post-pairing. This test found that pairs showed convergence in their behavioral responses, such that pairs who were mismatched in their approach behaviors became more similar, and pairs that were matched remained so. Finally, we analyzed the ultrasonic vocalizations (USV) produced and found that pairs produced significantly more USVs than individuals. Importantly, increased USV production correlated with increasing behavioral convergence of pairs. Taken together, this study shows that pair-bonded animals alter their approach behaviors to coordinate their response with their partner and that vocal communication may play a role in coordinating these behavioral responses. Overall, our findings indicate that pair-bonding generates an emergent property in pairs, adjusting their combined approach behavior towards a new aggressive stimulus representing a potential challenge to the bonded pair. Such findings may be broadly important for social bonding in other social systems.

Transmission of paternal retrieval behavior from fathers to sons in a biparental rodent

Transmission of maternal behavior across generations occurs, but less is known about paternal behavior. In biparental species like the California mouse (Peromyscus californicus), paternal care contributes to the well-being of offspring with lasting consequences on the brain and behavior. Paternal huddling/grooming behavior can be passed on to future generations, but whether paternal retrieval, which removes young from potential harm, is transmitted independently is unclear. We manipulated paternal retrieval experience through pup displacement manipulations, then examined whether males exposed to higher levels of paternal retrieval in development altered their adult retrieval behavior with their offspring. Males exposed to heightened paternal retrievals, as compared to reduced retrievals, retrieved their offspring more often but huddled/groomed offspring less during undisturbed natural observations. No differences were observed following a pup displacement challenge. The high paternal retrieval group also exhibited more physical activity and stereotypy. Our results are consistent with the hypothesis that paternal retrieval levels are transmitted across generations and may function via mechanisms separate from huddling/grooming. One modifying factor may be anxiety because increased activity and stereotypy occurred in the high retrieval group. We speculate how the transmission of paternal retrievals may inform a protective parenting style.

Enriched laboratory housing increases sensitivity to social stress in female California mice (Peromyscus californicus)

Domesticated mice and rats have shown to be powerful model systems for biomedical research, but there are cases in which the biology of species is a poor match for the hypotheses under study. The California mouse (Peromyscus californicus) has unique traits that make it an ideal model for studying biological mechanisms underlying human-relevant behaviors such as intra-female aggression, biparental care, and monogamy. Indeed, peer-reviewed scientific publications using California mouse as a model for behavioral research have more than doubled in the past decade. Critically, behavioral outcomes in captive animals can be profoundly affected by housing conditions, but there is very limited knowledge regarding species-specific housing needs in California mice. Currently, California mouse investigators have to rely on guidelines aimed for more common laboratory species that show vastly different physiology, behavior, and/or ecological niche. This not only could be suboptimal for animals’ welfare, but also result in lack of standardization that could potentially compromise experimental reproducibility and replicability across laboratories. With the aim of assessing how different housing systems can affect California mouse behavior both in the home cage as well as the open field and social interaction tests before and after social defeat stress, here we tested three different caging systems: 1. Standard mouse cage, 2. Large cage, and 3. Large cage + environmental enrichment (EE), which focused on increasing vertical complexity based on observations that California mice are semiarboreal in the wild. We found that the effects of housing were largely sex specific: compared to standard cages, in females large + EE reduced home cage stereotypic-like backflipping and rearing behaviors, while large cage increased social interactions. In males, the large + EE cage reduced rearing and digging but did not significantly affect backflipping behavior. Interestingly, while there were no significant differences in the open field and social interaction pre-stress behaviors, large and large + EE housing increased the sensitivity of these tests to detect stress induced phenotypes in females. Together, these results suggest that increasing social and environmental complexity affects home cage behaviors in male and female California mice without interfering with, but rather increasing the magnitude of, the effects of defeat stress on the open field and social interaction tests.

Reproductive success diminished following mate loss for females but not males in a monogamous rodent

The strictly monogamous California mouse (Peromyscus californicus) forms life-long pair bonds and mates exclusively with a single partner. While studies in the wild indicate that individuals may re-pair with a new partner following mate loss, the preponderance of this behavior and subsequent reproductive outcomes following re-pairing are understudied. To examine reproductive outcomes following re-pairing and to look for sex-specific differences following mate loss, birth records of 584 California mouse pairs from our laboratory were analyzed. Of these pairs, 59 pairs were identified as re-pairs and used for further descriptive analysis. We found that 50/59 (84.7 %) of re-paired animals gave birth, indicating that reproduction with a new mate is not only possible, but perhaps more common than previously described for this species. Additionally, we found that when re-paired, females took significantly longer to birth a subsequent litter as compared to original breeding pairs. Overall findings from the current study provide evidence for sex differences in reproductive outcomes following repairing and for greater flexibility in mating strategy for a species described as strictly monogamous.

The Effects of Regulator of G-Protein Signaling 2 (RGS2) on Depression-Like Behavior in Female California Mice

Stressful experiences are a well-known risk factor for the development of psychiatric disorders such as depression (MDD). Although many pharmaceutical therapies have been developed, over half of individuals that seek treatment do not respond positively, indicating a dire need for new treatments and thus new molecular targets.

An acute dose of intranasal oxytocin rapidly increases maternal communication and maintains maternal care in primiparous postpartum California mice.

Maternal-offspring communication and care are essential for offspring survival. Oxytocin (OXT) is known for its role in initiation of maternal care, but whether OXT can rapidly influence maternal behavior or ultrasonic vocalizations (USVs; above 50 kHz) has not been examined. To test for rapid effects of OXT, California mouse mothers were administered an acute intranasal (IN) dose of OXT (0.8 IU/kg) or saline followed by a separation test with three phases: habituation with pups in a new testing chamber, separation via a wire mesh, and finally reunion with pups. We measured maternal care, maternal USVs, and pup USVs. In mothers, we primarily observed simple sweep USVs, a short downward sweeping call around 50 kHz, and in pups we only observed pup whines, a long call with multiple harmonics ranging from 20 kHz to 50 kHz. We found that IN OXT rapidly and selectively enhanced the normal increase in maternal simple sweep USVs when mothers had physical access to pups (habituation and reunion), but not when mothers were physically separated from pups. Frequency of mothers' and pups' USVs were correlated upon reunion, but IN OXT did not influence this correlation. Finally, mothers given IN OXT showed more efficient pup retrieval/carrying and greater total maternal care upon reunion. Behavioral changes were specific to maternal behaviors (e.g. retrievals) as mothers given IN OXT did not differ from controls in stress-related behaviors (e.g. freezing). Overall, these findings highlight the rapid effects and context-dependent effect a single treatment with IN OXT has on both maternal USV production and offspring care.

Impacts of Timber Harvest on Communities of Small Mammals, Ticks, and Tick-Borne Pathogens in a High-Risk Landscape in Northern California.

Timber harvest may impact tick-borne disease by affecting small mammal and tick community structures. We assessed tick and small mammal populations in older second-growth redwood (Sequoia sempervirens (D. Don) Endl) habitat at two harvested sites in Santa Cruz County, California, where local risk of tick-borne disease is high and determined the prevalence of tick-borne pathogens in ticks. After single-tree removal harvest in 2014, there was a modest reduction in canopy, primarily toward the end of the study. Harvested sites showed strong reductions in California mouse (Peromyscus californicus, (Gambel)) captures 2-yr after harvest, resolving such that treatments and controls were comparable by the end of the study. Following harvest, treated sites experienced a transient decreased tick infestation while control plots experienced an increase. Ixodes angustus (Neumann) infestation probability on harvested plots decreased immediately after harvest, increasing with time but remaining lower than control plots, whereas I. pacificus (Cooley and Kohls) prevalence was higher shortly after the harvest on harvested plots, and continued to increase. Mean abundance of ticks on vegetation increased on control plots. We detected Borrelia burgdorferi ((Johnson et al.) Baranton) and Anaplasma phagocytophilum ((Foggie 1949) Dumler) in 3.8 and 3.1% of ticks on rodents, but no differences were associated with harvest. Impacts of forest harvest on tick-borne disease depend on removal practice and intensity, whether or not hosts are habitat specialists, and whether or not ticks are host specialists.

Sex-specific effects of social defeat stress on miRNA expression in the anterior BNST

Women are more likely to suffer from stress-related affective disorders than men, but the underlying mechanisms of sex differences remain unclear. Previous works show that microRNA (miRNA) profiles are altered in stressed animals and patients with depression and anxiety disorders. In this study, we investigated how miRNA expression in the anterior bed nucleus of stria terminalis (BNST) was affected by social defeat stress in female and male California mice (Peromyscus californicus). We performed sequencing to identify miRNA transcripts in the whole brain and anterior BNST followed by qPCR analysis to compare miRNA expression between control and stressed animals. The results showed that social defeat stress induced sex-specific miRNA expression changes in the anterior BNST. Let-7a, let-7f and miR-181a-5p were upregulated in stressed female but not male mice. Our study provided evidence that social stress produces distinct molecular responses in the BNST of males and females.