The research, led by Ezequiel Monferrer and Rebeca Vidal, working under the senior guidance of Marta Rodríguez-Arias at the University of Valencia and M. Isabel Colado at the Complutense University of Madrid, tackled a long-standing problem in stress research: most animal studies of social stress have focused almost exclusively on males, leaving a serious gap in our understanding of how females respond. To close that gap, the team used a clever two-pronged approach. Male adolescent mice underwent the classical social defeat paradigm, in which they were repeatedly confronted by an aggressive resident mouse, while female adolescent mice underwent vicarious social defeat, a paradigm in which they witnessed the aggressive encounters without ever being physically attacked. By comparing these two models, the researchers could isolate the purely psychological and emotional component of social stress, common to both sexes, from the physical component of aggression that only the males experienced.

A total of 264 OF1 mice were enrolled in the study. Experimental animals arrived at the laboratory on postnatal day 21 and were exposed to stress between postnatal days 26 and 35, corresponding to early adolescence in the mouse. The males experienced four social defeat episodes, each lasting 25 minutes and structured in three phases: an initial protected confrontation behind wire mesh, a five-minute direct physical encounter with the aggressive resident, and a final period of continued social intimidation. Defeat was confirmed when the intruder adopted the characteristic submissive posture of upright position, limp forepaws, upwardly angled head and retracted ears. The females, in contrast, watched these encounters from behind a perforated barrier that allowed visual, olfactory and auditory cues to pass through but blocked physical contact, and they then remained housed near the resident male for 24 hours, separated by a methacrylate partition.

The critical question the researchers asked was not simply whether the mice were stressed, but which mice coped and which did not. Twenty-four hours after the final encounter, every animal underwent the social interaction test, a widely used behavioral assay in which the time a mouse spends near an unfamiliar social target is compared with the time it spends near the same empty cage. A ratio below 1, meaning the animal actively avoided the social target, classified the mouse as susceptible, while a ratio of 1 or above classified it as resilient. The results were telling: 40.7 percent of defeated males were classified as susceptible, whereas only 27.5 percent of vicariously defeated females showed susceptibility. This suggests that female adolescents possess a notable degree of protection against stress-induced social avoidance, even when they experience the emotional contagion of watching another animal be defeated.

Crucially, both forms of stress activated the hypothalamic-pituitary-adrenal axis to a similar degree in both sexes. Corticosterone, the principal rodent stress hormone, was elevated in resilient and susceptible animals alike, measured from blood samples taken on the first and last days of the stress protocol. This dissociation between hormone levels and behavior is one of the study’s most important messages: simply measuring a stress hormone cannot tell you whether an individual is coping well or badly. Resilient and susceptible mice bore the same endocrine burden, yet their brains and behaviors told very different stories.

Those behavioral stories diverged sharply. In the short term, all defeated males, whether resilient or susceptible, traveled greater distances in the open field test, indicating increased locomotor activity, and susceptible males also moved faster. Yet only resilient males showed heightened anxiety on the elevated plus maze, spending less time and making fewer entries into the open arms. Susceptible males, paradoxically, looked no more anxious than unstressed controls on this measure. The females showed a different pattern altogether: no locomotor changes, no adolescent anxiety, but a reduction in back grooming on the splash test, a sign of apathy and anhedonia that appeared in both resilient and susceptible females. In adulthood, most of these effects faded, but resilient adult females emerged as the one group with persistent anxiety-like behavior on the elevated plus maze, spending less time in the open arms than their unstressed counterparts. The tail suspension test, which measures immobility as an index of despair, showed no stress-related effects in either sex, though adult control females displayed more immobility than males, hinting at a baseline sex difference in depression-like tendencies.

Beyond behavior, the team turned to brain chemistry, focusing on the kynurenine pathway, the dominant metabolic route for the essential amino acid tryptophan. Approximately 95 percent of tryptophan is shunted through this pathway rather than into serotonin synthesis, and its activation is closely tied to inflammation and stress. The researchers dissected four brain regions, the hippocampus, cerebellum, limbic forebrain and striatum, 24 hours after the last social encounter, and quantified kynurenine, tryptophan and serotonin using high-performance liquid chromatography with fluorescence and ultraviolet detection. The kynurenine-to-tryptophan ratio served as an index of pathway activation.

The neurochemical results were as sex-divergent as the behavioral ones. Unstressed females already showed higher kynurenine levels and higher kynurenine-to-tryptophan ratios across nearly all regions examined, along with higher serotonin concentrations in the limbic forebrain and striatum. This baseline sex difference is striking because it was consistent across every structure measured, and the authors argue it may reflect greater HPA axis sensitivity and stronger immune signaling in females, potentially helping explain the higher prevalence of affective disorders in women. Among the stressed animals, only resilient females showed a significant change: a reduction in kynurenine levels in the striatum, a brain region central to reward processing. No comparable effect appeared in males.

In males, the most compelling neurochemical marker of susceptibility was the serotonin-to-tryptophan ratio. Susceptible adolescent males displayed a significantly elevated ratio in the cerebellum, limbic forebrain and striatum compared with both controls and resilient males. In other words, animals that avoided social contact had relatively more serotonin in relation to tryptophan in these regions, a biochemical signature that echoes earlier work from the same group in adult mice and a study in rats by Prakash and colleagues showing serotonergic plasticity in the dorsal raphe nucleus characterizing susceptible animals. The authors suggest that a lower relative serotonin level may characterize the resilient brain, although they caution that adolescent resilience is not the clean protective profile seen in adults.

Indeed, the adolescent picture proved messier and more complex than the adult one. Previous work by this group had shown that adult resilient mice maintain a stable protective phenotype, resisting both depressive-like effects of social defeat and the rewarding pull of cocaine and alcohol. In adolescents, by contrast, resilience appeared partial and fragmented: resilient males kept their social contacts intact and showed no anhedonia, yet they were the only group with heightened anxiety, and earlier studies have linked adolescent resilience to increased preference for cocaine and ethanol. The authors warn that translational conclusions drawn from adult resilience studies may simply not apply to younger brains.

The implications reach beyond the laboratory. Social stress during adolescence, whether from bullying, rejection or witnessing conflict, is a well-established risk factor for anxiety disorders, depression and substance use in humans. By identifying sex-specific biomarkers, an elevated serotonin-to-tryptophan ratio as a potential flag for susceptibility in males, and reduced striatal kynurenine alongside persistent anxiety in resilient females, the study points toward biological measures that could eventually help clinicians distinguish which young people are genuinely coping and which are silently at risk. It also reinforces the argument that preclinical stress research must include females, because assuming that findings in males generalize is now demonstrably untenable.

The authors acknowledge limitations, including the reduced sample sizes in the susceptible neurochemical groups, a constraint imposed by ethical animal-use requirements, and the impossibility of directly comparing defeated males and females given the different stress protocols. Nonetheless, the convergence of behavioral, endocrine and neurochemical data across 264 animals makes a strong case that adolescent social stress is processed differently in male and female brains, and that the kynurenine pathway sits at the heart of that divergence. Understanding how these metabolic routes shape vulnerability and resilience, the researchers conclude, could carry important translational benefits for the prevention and treatment of stress-related mental disorders in young people of both sexes.