Hormones and Behavior

Physical activity offers myriad benefits to health and well-being, in humans and other animals as well. In rodents, voluntary wheel running can attenuate the effects of both physical and social stressors on rodent social behavior. Whether wheel running affects rodent social behaviors per se remains less well understood. We conducted the current study to test whether home cage access to running wheels impacts the social behaviors of adult, group-housed C57BL/6J female mice during same-sex interactions with novel females. Group-housed females were either given continuous home cage running wheel access or a standard paper hut starting at weaning, and as adults, social behaviors were measured during interactions with novel females. In two cohorts, we found that 5 weeks of running wheel access during adolescence reduced the time that subject females spent investigating a novel female and also tended to reduce total ultrasonic vocalizations produced during interactions. These effects were not reversed by a 2-week period of running wheel removal but were recapitulated in a different cohort by 2 weeks of running wheel access in adulthood. Unexpectedly, we found that these effects on female social behavior were not due to wheel running per se, because females raised from weaning with immobile running wheels also showed low rates of social behaviors during same-sex interactions in adulthood. Overall, we find that the presence of a running wheel in the home cage has an enduring inhibitory influence on female social behavior during same-sex interactions, a finding that has implications for the design of studies that include same-sex interactions between female mice.

To attract mating partners, female mammals communicate their reproductive status through one or multiple sensory modalities, providing redundant or complementary information. Chimpanzees (Pan troglodytes) are an excellent model for studying multimodal communication. Exaggerated sexual swellings of females serve as a visual proxy for ovulation but increased male mating interest during maximum swelling suggests that olfactory cues may pinpoint fertility more accurately than the swelling alone. Here, we combined gas chromatography-mass spectrometry, hormonal analyses, and bioassays to examine (1) whether chemical composition of female anogenital odours changes during the fertile period, and (2) whether males are able to detect these changes. Our results suggest that, in addition to prominent olfactory changes associated with swelling stages, chemical cues provide complementary information regarding the timing of the fertile window. These changes, however, are minor compared to those related to swelling stages. Male behavioural responsiveness in bioassays was too low to draw conclusions regarding their ability to detect these subtle shifts when presented with a chemical cue only. Overall, our findings support the existence of a multimodal fertility cue in chimpanzees, wherein visual signals are complemented by subtle olfactory changes indicating the timing of the fertile period.

Capturing and handling wild animals is essential for ecological and evolutionary research, yet their effects on physiology, behaviour, and reproductive success remain poorly understood. We investigated short- and longer-term consequences of a capture-handling-restraint protocol in wild great tits (Parus major) over three breeding seasons. To assess short-term responses, we measured circulating corticosterone, a metabolic hormone that responds to unpredictable challenges, and automatically recorded provisioning behaviour. We also explored whether environmental and individual traits were related to provisioning latency (i.e., time to resume provisioning after capture). To evaluate longer-term effects, we monitored provisioning in the days following capture and related it to reproductive success (fledgling number and body condition). We predicted that longer handling would increase stress-induced corticosterone and provisioning latency, that these variables would be positively correlated, and that higher corticosterone and longer latencies would be associated with lower reproductive success. After capture, great tits showed elevated corticosterone and delayed provisioning. Contrary to our predictions, handling duration was negatively associated with stress-induced corticosterone in males (but not females) and did not affect provisioning latency. Provisioning latency was unrelated to corticosterone, environmental, or individual variables. Following capture, parents resumed provisioning, and short-term responses had little influence on reproductive success. We show that parental behaviour and physiology are affected by capture restraint protocols on the short term, but offspring condition and survival are not. However, these results should be interpreted cautiously, as our study lacks an uncaptured control group. Our findings highlight that evaluating welfare impacts requires rigorous study design incorporating both immediate and longer-term behavioural and fitness effects.

In sex-role reversed species, females are socially polyandrous and compete for multiple mates, whereas males conduct the majority of parental care. To understand the extent to which physiological differences between females and males are shaped by sex roles, we examined sex differences in gene expression in sex-role reversed northern jacanas (Jacana spinosa). Given that females compete for mating opportunities, and males cycle between courtship and parental care, we predicted that transcriptomic profiles would be more similar between females and courting males, in contrast to female and parenting males. Leveraging a high quality de novo genome assembly, we conducted RNA-seq on two brain regions associated with the regulation of social behavior: the preoptic area of the hypothalamus and the nucleus taeniae. The majority of genes differentially expressed between the sexes were male-biased. Of these male-biased genes, the majority were located on the Z-chromosome. Contrary to our prediction, the greatest difference in autosomal gene expression was between females and courting males, in the preoptic area of the hypothalamus. Several differentially expressed genes related to elements of hormone signaling that are likely to be behaviorally salient, including higher expression of androgen receptor in females relative to parenting males, and higher expression of prolactin receptor in males, regardless of breeding stage. Some sex-associated gene networks were also associated with competitive traits, whereas others were associated with aggressive behaviors, regardless of sex. Few genes were differentially expressed between courting and parenting males, yet some nonetheless had connections to behavioral endocrinology, including prolactin, thyroid and insulin-like growth factor pathways. Our investigation of sex differences in gene expression can help to reveal the molecular mechanisms underlying female competition and male parental care in socially polyandrous species. We conclude that social polyandry is not a simple reversal in the direction of sex-biased gene expression in the brain, but rather a result of complex genetic and hormonal interactions that warrants further study.

Within species, male testosterone is often linked to mating competition and paternal care, suggesting that sex differences in endogenous testosterone values across mammals may covary with broader reproductive strategies. Using a structured literature search, we compiled 63 studies, spanning 31 non-human species and 9 human populations, reporting endogenous, non-experimentally manipulated testosterone values for both adult males and females within the same population and context. From these studies, we calculated male-to-female testosterone ratios, and analysed these data using Bayesian phylogenetic multilevel models. We tested whether testosterone dimorphism was associated with paternal care and sexual size dimorphism while accounting for sampling matrix, assay method, breeding context, and wild versus captive setting. Across non-human mammals, neither paternal care nor sexual size dimorphism (indexing competition) showed a clear association with testosterone ratios, and the same pattern emerged in the primate-only subset. By contrast, sampling matrix was consistently associated with testosterone dimorphism across all analyses, with lower male-to-female ratios in non-blood than in blood-based measures. In primates, testosterone ratios were also lower in captive than in wild populations, although this pattern was not clearly supported in the broader non-human dataset. In the human-only analysis, testosterone ratios did not clearly differ between industrialized and small-scale societies, whereas the matrix effect remained evident. Overall, our results suggest that sampling matrix is a major source of variation even for ratio-based measures, highlighting the need for caution when inferring between-species endocrine differences from studies using different substrates. More broadly, directly comparable, non-experimentally manipulated testosterone data for both sexes remain rare across mammals, limiting comparative inference.

Bilaterian animals exhibit operational (functional) asymmetry--population-level, directional left-right differences in physiology and behavior, including responses to spatially symmetric environmental challenges. Whether such symmetry-to-asymmetry conversion can be driven at the systems level by neurohormonal regulators remains unclear. Here we tested whether a spatially symmetric neuroendocrine challenge--water deprivation (WD)--can elicit a directional left-right physiological response in rats using hindlimb postural asymmetry (HL-PA), a binary readout that quantifies left- versus right-sided hindlimb flexion. Twenty-four hours of WD induced robust HL-PA with right hindlimb flexion, revealed under anesthesia. The asymmetry persisted after complete thoracic spinal cord transection, suggesting that humoral signaling, rather than descending neural commands, may maintain the postural bias. Because dehydration recruits the hypothalamic-neurohypophysial arginine vasopressin (AVP) system, we next tested AVP receptor involvement. Both a V1B antagonist (SSR-149415) and a V1A/V2 antagonist (conivaptan) abolished WD-induced HL-PA, supporting an AVP-dependent mechanism that likely operates at least two anatomical sites. AVP signaling may involve pituitary V1B-dependent endocrine output and spinal V1A actions; consistent with the latter, expression of AVP V1A receptors is right-biased in lumbar spinal cord. Together, these findings identify WD as a symmetric systemic challenge capable of imposing a directional peripheral set-point, and implicate vasopressin signaling in symmetry breaking and left-right physiological regulation. Visual summary O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/708998v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@10d6b3corg.highwire.dtl.DTLVardef@1fb4b5aorg.highwire.dtl.DTLVardef@11003c0org.highwire.dtl.DTLVardef@66671c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Top down signaling from the cortex to the hypothalamus is critical to link cognitive and emotional processing to homeostasis and motivation. This study investigates signaling from the medial prefrontal cortex (mPFC) to the posterior hypothalamus (PH), a region that modulates endocrine and autonomic stress responses and motivated behaviors. The function and anatomy of this circuit was examined with patch clamp electrophysiology and mapping studies in male and female rats. Spontaneous firing properties of PH neurons were determined in a cell-type specific manner by combining a transgenic glutamic acid decarboxylase-Cre rat with Cre-dependent colorswitch virus to determine postsynaptic cell-type identity. Overall, PH neurons were more excitable in females compared to males and, in both sexes, data indicated tonic inhibition within the PH, with significantly greater inhibition in males. Using Channelrhodopsin-assisted circuit mapping to query the mPFC-PH circuit, we found that a majority of PH neurons received input from the mPFC and mPFC synapses targeted glutamatergic cells over GABAergic PH cells. Retrograde tracing revealed more PH-projecting neurons in females, specifically within the tenia tecta and infralimbic regions of the mPFC, with significantly more stress-activated PH-projecting cells in the female prelimbic cortex. Anterograde tracing revealed, surprisingly, no sex differences in mPFC presynaptic terminal density in the PH, despite more PH-projecting cell bodies in the female mPFC. These data help to elucidate the sexual divergence in cortical-hypothalamic signaling and how cognitive and emotional information from the prefrontal cortex may differentially regulate homeostasis and motivation between sexes. Significance StatementNeural signaling between the prefrontal cortex and the hypothalamus is important for maintaining homeostasis, particularly during contextual challenges such as stressors. Here we find multiple aspects of sex-specific organization and neurophysiology in this circuitry. Excitatory inputs from the medial prefrontal cortex target both excitatory and inhibitory neurons within the posterior hypothalamic nucleus in both sexes. However, there are sex differences in the number of stress-activated neurons in the prefrontal cortex that innervate the posterior hypothalamus, as well as differences in hypothalamic inhibitory signaling and estrous cycle-dependent effects on neuronal excitability. Altogether, these data suggest that organizational, synaptic, and hormonal factors may contribute to sex-specific behavioral and physiological integration.

Birth occurs during a sensitive period in brain development wherein hormones facilitate the dramatic shift in physiology that accomplishes the transition to extrauterine homeostasis. The surge in birth signaling hormones is abridged in cases of delivery by cesarean section (CS), which accounts for 32% of all births in the U.S. Epidemiological studies have associated birth via CS with increased risk of obesity in later life. Here, we sought to investigate this association using an experimental preclinical animal model, the prairie vole. Subjects were delivered either via vaginal delivery (VD) or CS and then cross-fostered. CS delivery led to increased body weight across development, which could be prevented with hormone rescue of oxytocin (OXT) and arginine vasopressin (AVP), delivered to neonates immediately after CS. This weight gain could not be attributed to differences in birth weight, parenting, food consumption, or thermoregulation; however, CS subjects moved slower than VD subjects, which hormone rescue reversed. Hormone rescue also reduced adiposity in adulthood among CS subjects. The dopamine system was dysregulated in the caudate/putamen of CS offspring, suggesting a neural mechanism for the decreased locomotion. Hormone rescue of CS neonates restored dopamine synthesis in the caudate/putamen and increased spontaneous locomotor activity. These findings suggest CS can lead to increased weight gain in part through a reduction of locomotion driven by long-lasting changes in striatal dopamine regulation, all of which can be prevented by treating CS neonates with a single peripheral administration of two birth-signaling hormones, OXT and AVP.

Identifying the neural circuits engaged and reshaped by chronic stress is critical for understanding how adaptive responses shift to maladaptive behaviors that contribute to stress-related disorders. Our previous work demonstrates that chronic unpredictable stress (CUS) induces a persistent increase in the firing activity of proopiomelanocortin (POMC) neurons in the arcuate nucleus (ARC). This hyperactivity is due, in part, to a reduction in GABAergic synaptic transmission onto POMC neurons, indicating a disruption in inhibitory control. However, the sources of GABAergic inputs responsible for this effect of chronic stress are unknown. Although AgRP neurons provide local GABAergic input onto POMC neurons and are suppressed by chronic stress, chemogenetic activation of AgRP neurons during stress exposure failed to reduce POMC neuron hyperactivity. GABAergic projections originating from the dorsomedial hypothalamus (DMH) represent another source of inhibitory input to POMC neurons. We found that CUS decreased the firing activity of DMH GABAergic neurons with sex differences, with females exhibiting greater vulnerability to stress-induced suppression. Chemogenetic activation of these neurons during chronic stress markedly attenuated POMC neuron hyperactivity in both sexes, indicating that DMH GABAergic neurons function as a critical upstream regulator of POMC neuron activity under chronic stress. These findings suggest that reduced inhibitory input from DMH GABAergic neurons, rather than local GABAergic AgRP neurons, drives POMC neuron hyperactivity. The weakening of the DMHGABA[->]ARCPOMC circuit activity may represent a novel mechanism underlying maladaptive stress responses and a potential therapeutic target for stress-related disorders.

Dietary choice plays a critical role in metabolic and neurological health, yet the biological factors that shape macronutrient preference remain poorly understood. Evidence from both humans and rodents suggests potential sex differences in the attractiveness of specific nutrients, though findings have been inconsistent and often rely on self-report or diets with mixed macronutrient composition. The present study examined sex differences in macronutrient preference and food-directed behavior in mice using a controlled three-food choice paradigm. Adult male (n = 12) and female (n = 11) C57BL/6J mice were given simultaneous access to foods consisting of fat, sucrose, or a fat-carbohydrate combination across 14 days. Intake, latency to approach, and time spent near each food source were quantified, and estrous cycle stage was monitored in females. Female mice consumed significantly more food than males overall, driven by a selective increase in fat intake. Behavioral measures paralleled these results, with females spending more time in proximity to fat-associated food zones. In contrast, males preferentially consumed the fat-carbohydrate combination and showed weaker nutrient-specific engagement. Estrous cycle stage modestly influenced feeding behavior, with estrus associated with increased overall intake and greater consumption of combination diets, reflecting elevated carbohydrate intake. These findings demonstrate robust sex differences in macronutrient preference and suggest that hormonal state may selectively modulate nutrient-specific feeding behavior.

Male odor induces various behavioral and physiological responses across the reproductive cycle in female mice. Although male odor preference in females is reduced during pregnancy, how it changes across later stages of the reproductive cycle, including nursing and weaning, remains unclear. Here, we found that male odor preference is lost during pregnancy and nursing. To identify the olfactory systems involved in these changes, we examined neural activity using c-Fos immunohistochemistry. Male odor exposure during nursing increased neural activity in the accessory olfactory bulb and the posteroventral medial amygdala (MeApv), a key node of the accessory olfactory system, as well as in subdivisions of the central amygdala, but not in the ventromedial hypothalamus or the bed nucleus of the stria terminalis. Finally, lesions of the MeApv prevented the loss of male preference during nursing, indicating that the MeApv is required for suppression of male preference during this stage.

The population of kisspeptin neurons located in the rostral periventricular area of the third ventricle (RP3V) is thought to have a key role in generating the GnRH surge that triggers ovulation. Using a modified GCaMP fibre photometry procedure, we have been able to record the in vivo population activity of RP3VKISS neurons across the estrous cycle of female mice. A marked increase in GCaMP activity was detected beginning on the afternoon of proestrus that lasted in total for 13{+/-}1 hours. This was comprised of slow baseline oscillations with a period of 91{+/-}4 min and associated with high frequency rapid transients. Very little oscillating baseline or transient activity was detected at other stages of the estrous cycle. Concurrent blood sampling showed that the peak of the LH surge occurred 3.5{+/-}1.1 h after the first baseline RP3VKISS neuron baseline oscillation on the afternoon of proestrus. The time of onset of RP3VKISS neuron oscillations varied between mice and across subsequent proestrous stages in the same mice. To assess the impact of estradiol on RP3VKISS neuron activity, mice were ovariectomized and given an incremental estradiol replacement regimen. Minimal patterned GCaMP activity was found in OVX mice, and this was not changed acutely by any of the estradiol treatments. However, on the afternoon of the expected LH surge, the same oscillating baseline activity with associated transients occurred for 7.1{+/-}0.5 h. These observations reveal an unexpected prolonged oscillatory pattern of RP3VKISS neuron activity that is dependent on estrogen and underlies the preovulatory LH surge as well as potentially other facets of reproductive behavior.

We assessed whether pigs provide consolation, referring to targeted affiliation that attenuates a partners stress, under experimental conditions that manipulated exposure to stressed partners. Using a within-subject design, 74 pigs were tested in three contexts: a helping task in which group members could observe and help a trapped focal pig to return to the group, a direct-reunion, in which group members were naive to the experience of a separated focal pig until reunion, and an undisturbed control. We measured affiliative and non-affiliative interactions, anxiety behaviours and changes in salivary cortisol. Only the helping context satisfied most consolation criteria: there were selective increases in unidirectional affiliative contacts from the observer to the focal pig, non-affiliative interactions remained at baseline, and focal pigs showed fewer anxiety behaviours. In contrast, direct-reunions triggered increases in affiliative and non-affiliative interactions and higher anxiety. Cortisol increased during both direct-reunions and helping, but its level was not linked to affiliation. Results add to growing evidence for consolation behaviour in pigs and suggest best practices for reintegrating pigs into groups. Graded reintroductions that allow observers to assess the emotional state of targets may promote social buffering, whereas abrupt regrouping may trigger more generalized arousal or personal distress.

Stress can cause or exacerbate psychiatric illness, and effects on the transcription factor CREB within the nucleus accumbens (NAc) are critically involved. In rodents, stress-induced activation of NAc CREB produces elevations in dynorphin (DYN), an endogenous opioid expressed in dopamine D1-receptor (D1R)-expressing medium spiny neurons (MSNs). In turn, elevated DYN signaling produces features of mood and anxiety disorders via actions at kappa-opioid receptors (KORs). Although individual differences in stress sensitivity have been described--with some appearing susceptible and others resilient--the contribution of NAc DYN to these phenotypes is unclear. Here we examined relationships between social behavior and DYN in D1R-expressing MSNs in mice exposed to chronic social defeat stress (CSDS). We used quantitative (q)RNAscope to assess co-expression of genes encoding CREB (Creb1), D1Rs (Drd1), and DYN (Pdyn) within the NAc. To leverage individual variability, we performed regression analyses across all mice, revealing negative correlations between social interaction behavior and expression of Drd1 and Pdyn, linking higher social avoidance with higher expression of these genes. There was no correlation with Creb1, suggesting stress-induced elevations in Pdyn depend on CREB activation (phosphorylation). These findings suggest that stress-induced elevations in D1R-associated DYN signaling within the NAc is a biomarker of susceptibility.

Birdsong mediates territory acquisition and mate choice. In agonistic interactions, local songs generally elicit stronger responses than songs from more distant populations. However, the molecular mechanisms associated with differential responses to local vs. foreign songs are poorly understood. We addressed this knowledge gap by combining behavioral assays in the field with blood transcriptomic analysis, using a within-subjects design to ask whether male song sparrows (Melospiza melodia) show differential gene expression when exposed to playback of local and foreign songs. Transcriptomic profiles reflected the difference in behavioral response to local vs. foreign songs, with individuals exposed to local songs showing greater expression of genes associated with song perception and production, anti-inflammatory responses and energy metabolism. Our study suggests that changes in expression of key molecular pathways correlate with behavioral responses to geographic song variation, providing insight into the potential mechanisms regulating signal recognition and response to social challenges. HighlightsO_LIGene expression in sparrow blood was measured after simulated territorial intrusion. C_LIO_LIStronger response to local songs was associated with differential gene expression. C_LIO_LISong-associated genes (FOXP2, NRXN1) had higher expression when birds heard local songs. C_LIO_LIGene expression in the blood contains potential biomarkers of song recognition. C_LI

Pregnancy leads to many adaptations in the maternal body, most of which are reversible. However, reproductive experience can also result in permanent effects. Here, we investigated how pregnancy influences the somatotrophic system and the lasting effects of reproductive experience on the maternal organism. Reproductive experience induced a pronounced increase in lean body mass and longitudinal growth in both wild-type and growth hormone (GH)-deficient mice compared with age-matched virgins. Body growth was primarily observed during the first pregnancy, whereas a second gestation was mostly associated with increased adiposity. Data from a cohort of women with isolated GH deficiency (IGHD) caused by a loss-of-function mutation in the GHRHR gene revealed that nulliparous women were 7 cm shorter than those with one or more pregnancies. Increased GH secretion was observed in pregnant wild-type mice but not in pregnant GHRHR-deficient mice. Pregnancy-induced body growth is preserved despite disruption of GH-, ghrelin-, and estrogen-related signaling pathways. In conclusion, reproductive experience induces permanent changes in the maternal organism, promoting body growth in models that allow this response. Pregnancy-induced body growth appears to be independent of GH action. These findings underscore the need for further studies to investigate the long-lasting consequences of reproductive experience in females.

Ultrasonic vocalizations (USVs) are widely used in rodent social communication, yet the functional significance of male-male vocal interactions in mice remains unclear. Here, we investigated whether USVs produced during specific social behaviors influence the behavior of conspecifics. Using playback experiments, we compared responses to vocalizations recorded during chasing and being chased in male-male interactions. We found that USVs emitted by chased intruders consistently elicited approach behavior in receiver mice, whereas those emitted by chasing individuals did not. Acoustic analyses revealed that these vocalizations differed in syllable composition, with intruder calls containing a higher proportion of upward frequency-modulated syllables and exhibiting higher mean frequencies. In addition, the temporal organization of syllables appeared to contribute to the behavioral response. Together, these results suggest that male mice respond selectively to certain USV patterns associated with specific social contexts, indicating that acoustic features and temporal structure may jointly influence social approach behavior in mice. HighlightsO_LIBehavioral context (chased vs. chasing) shapes the composition of USV syllable types C_LIO_LIMale mice selectively approach USVs from chased intruders, but not chasing residents C_LIO_LIThe approach response exhibits high temporal synchrony across individual receivers C_LIO_LITemporal organization of syllables modulates approach behavior based on acoustic features C_LI

Social isolation refers to an extreme form of social deprivation that has enduring effects on the brain and behavior. Adolescents show selective vulnerability to such heightened social stress, displaying aberrant behavior and psychiatric ailments. The post-weaning social isolation rodent model has been widely used to recapitulate such behavioral anomalies and delineate their mechanistic bases. Here, we aim to identify how prolonged social isolation during adolescence affects neuroimmune responses in both sexes and the implications for behavioral outcomes, particularly aggression. While males subjected to adolescent isolation were hyper-aggressive with pathological signs, females showed reduced social exploration and inactivity. Cytokine profiling in core brain regions implicated in aggression revealed reduced interleukin 6 (IL6) levels, specifically in the hypothalamus, in both sexes. Other proinflammatory cytokines, including interferon-gamma and interleukin-1beta, were unaltered. IL6-responsive genes, SOCS3 and TIMP1, were also downregulated in the hypothalamus of both socially isolated males and females. The hypothalamus is crucial for stress responsiveness and the expression of excessive aggression. Despite behavioral dimorphism, reduced IL6 levels in both sexes may indicate differences in downstream signaling and roles beyond classical immune responses. Our findings suggest that hypothalamic IL6 may be a key mediator of adolescent social isolation, which is associated with aberrant behavior, including aggression.

IntroductionAdolescence is a sensitive developmental period during which chronic stress can induce lasting adaptations in corticolimbic circuits involved in stress regulation, cognition, and emotional behavior. We examined the long-term behavioral, endocrine, and molecular consequences of adolescent chronic variable stress (CVS) in male and female rats, focusing on the infralimbic cortex (IL) and basolateral amygdala (BLA) MethodsSprague Dawley rats of both sexes were exposed to CVS during late adolescence and evaluated in adulthood after an extensive recovery period. Behavioral testing included cued fear conditioning and extinction recall, delayed spatial win-shift, novel object recognition, Morris water maze, three-chamber social behavior, and passive avoidance. HPA-axis reactivity to acute restraint was assessed. Targeted qPCR was used to measure stress-related gene expression in the IL and BLA immediately after stress or after a 5-week recovery period ResultsAdolescent CVS did not cause generalized cognitive impairment, but instead produced selective, sex-specific effects. Females had reduced HPA responses to acute stress and mild deficits in delayed spatial win-shift performance, together with long-term IL changes in genes related to adrenergic signaling, plasticity, and GABA clearance. Males showed enhanced Morris water maze probe retention, weaker novel object discrimination, altered passive avoidance with marked inter-individual variability, and enhanced social preference. At the molecular level, males exhibited long-term upregulation of Fkbp5 in IL and downregulation of PACAP, 1D adrenergic receptor, and proenkephalin in BLA, whereas females showed delayed PACAP upregulation in BLA DiscussionAdolescent CVS induces persistent, sex- and region-specific recalibration of corticolimbic function, supporting distinct patterns of vulnerability and resilience, rather than uniform stress pathology.

Animals produce different vocalization types, which differ in their acoustic features and are produced in different behavioral contexts. How vocalization-related brain circuits are organized to enable the production of different vocalization types remains poorly understood. The nucleus retroambiguus is a hindbrain premotor region that regulates the production of both ultrasonic vocalizations (USVs) and distress calls (squeaks) in adult mice, but whether distinct or overlapping populations of RAm neurons are recruited during the production of these two vocalization types is unknown. In the current study, we used Fos immunohistochemistry to compare the counts and spatial distributions of Fos-positive RAm neurons in males and females that produced USVs and females that produced courtship squeaks. We also combined in vivo activity-dependent (TRAP2) labeling with Fos immunohistochemistry to directly compare Fos expression associated with the production of USVs and courtship squeaks in the same females. Our findings suggest that RAm contains three vocalization-related populations of neurons: squeak-related neurons, USV-related neurons, and shared neurons that are recruited during both vocalization types. These findings refine current models of the premotor control of vocalization and set the stage for future work to explore anatomical and functional heterogeneity within RAm.