Depression remains one of the most prevalent psychiatric conditions worldwide, and epidemiological evidence has long shown that early-life adversity is a powerful risk factor for psychopathology in adulthood. Women are diagnosed with stress-related mood disorders roughly twice as often as men, yet the biological mechanisms underlying this sex bias remain only partially understood. Animal models have repeatedly implicated the hippocampus—a brain region essential for memory, emotion regulation, and one of the few adult brain regions where new neurons continue to be born—as a key site where stress leaves lasting traces. But most previous studies examined single outcomes in isolation or focused on one sex. The Málaga team instead adopted a two-hit experimental framework: a first hit of stress during the juvenile, peripubertal period, and a second hit of acute stress in adulthood, testing whether the early experience changes the way the adult brain responds.

The researchers exposed juvenile mice to stress and then, later in life, subjected the same animals to an acute adult stressor, creating four comparison groups for each sex: no stress, juvenile stress only, adult stress only, and combined juvenile-plus-adult stress. They then measured an unusually broad battery of endpoints. Behaviorally, they assessed saccharin preference, a classic measure of anhedonia, or loss of interest in rewards, together with social exploratory behavior toward an unfamiliar mouse. At the cellular level, they analyzed microglial morphology—the shape and branching complexity of the brain’s resident immune cells—along with cell proliferation and the abundance of immature neurons in the dentate gyrus, the hippocampal subregion where adult neurogenesis occurs. They also quantified inflammatory cytokines and performed an unbiased proteomic analysis of hippocampal tissue to capture the full molecular fingerprint of each stress condition.

The results that emerged were strikingly selective, the authors report. Effects depended on sex, on the timing and combination of stress, on the endpoint measured, and even on the hippocampal subregion examined. In males, stress did not clearly alter saccharin preference or the social behaviors assessed, suggesting that the behavioral readouts of depression-like states were largely spared. At the molecular level, however, adult acute stress increased hippocampal interferon-gamma, a signaling molecule of the immune system, and was associated with selective changes in microglial morphology. Proliferation and neurogenic effects in males were limited and confined to specific cell populations and subregions. When juvenile and adult stress were combined, males showed proteomic differences concentrated in proteins related to synaptic signaling and metabolic processes—changes that, notably, were not accompanied by overt behavioral alterations.

In females, the picture was different in almost every respect. Juvenile stress was associated with sex-dependent alterations in saccharin preference, although the authors emphasize that it did not produce a consistent, wholesale loss of sweet-reward interest. Juvenile stress and the combined stress condition both affected specific components of social exploratory behavior, pointing to subtle but measurable disruptions in how females approach and investigate novel conspecifics. The cellular consequences in females were subtle and region-specific, yet the proteomic changes were broader and more densely interconnected than in males, particularly after combined stress, which produced the widest hippocampal protein response observed in the study.

Perhaps the most important takeaway is a methodological one: the different biological levels did not move in lockstep. Most cytokines were unchanged across conditions, microglial morphology was shaped mainly by sex itself rather than by stress exposure, and neurogenic effects were restricted to particular cell populations and hippocampal subregions. Behavioral changes, when they appeared, were not always mirrored by corresponding cellular or molecular alterations, and molecular signatures did not always predict behavioral outcomes. This decoupling across levels of analysis challenges the widespread assumption that depression-like behavior in animal models must be accompanied by uniform, measurable changes in neurogenesis, neuroinflammation, or brain immune cells, and it cautions researchers against relying on any single biomarker as a proxy for stress vulnerability.

From a technical standpoint, the study’s strength lies in its breadth and its factorial design. By crossing stress timing with sex and measuring everything from cytokine concentrations to protein interaction networks, the authors could show that the female hippocampus responds to combined stress with a broad, coordinated proteomic reorganization, whereas the male hippocampus responds with narrower, more compartmentalized changes in immune signaling and synaptic-metabolic proteins. The selective increase of interferon-gamma in stressed males hints at a neuroimmune contribution to the stress response that is not shared, or at least not expressed the same way, in females. Meanwhile, the finding that microglial morphology differed primarily between the sexes rather than between stress conditions suggests that baseline sexual differentiation of the brain’s immune landscape may set the stage on which stress later acts, rather than being a direct consequence of it.

The authors are careful about interpretation. The study was conducted in mice, and depression-like behaviors in rodents—anhedonia measured by sweet preference, social withdrawal measured by exploration—are imperfect proxies for the human syndrome. Many of the cellular effects were subtle, and the absence of overt behavioral change in males despite clear molecular signatures underscores that biological perturbation can occur silently, without manifesting as measurable behavior. Still, the two-hit model captures a clinical reality: early adversity rarely acts alone, but instead appears to calibrate the brain’s later responses to stress. Identifying precisely which molecular pathways are reprogrammed in each sex during that calibration could ultimately guide sex-specific preventive strategies or treatments for people with histories of childhood adversity.

The work also adds to a growing recognition in neuroscience that sex must be treated as a biological variable rather than a nuisance factor. Funding bodies and journals increasingly mandate the inclusion of both sexes, and this study demonstrates why: pooling males and females, or studying only one, would have obscured entirely distinct patterns of hippocampal response. The Málaga group’s finding that combined stress produces the broadest proteomic response in females aligns with the epidemiological observation of greater female vulnerability to stress-related disorders, and it provides concrete molecular targets—synaptic and metabolic proteins, immune signaling components, neurogenic populations—that future research can interrogate. As the authors conclude, males and females respond differently depending on when stress occurs and which outcome is examined, and understanding those distinct trajectories may help explain why the same stressful world leaves such different imprints on different brains.

Subject of Research: Sex-specific hippocampal responses to peripubertal and adult acute stress involving microglia, neurogenesis, proteomics, and depression-like behavior in mice.

Article Title: Sex-specific hippocampal trajectories induced by peripubertal and adult acute stress: proteomic profile, microglial alterations, neurogenic dysregulation, and depression-like behaviors

Article References: Sex-specific hippocampal trajectories induced by peripubertal and adult acute stress: proteomic profile, microglial alterations, neurogenic dysregulation, and depression-like behaviors. (n.d.). https://doi.org/10.1186/s13293-026-00979-6

Image Credits: AI Generated

DOI: 10.1186/s13293-026-00979-6

Keywords: stress, hippocampus, sex differences, depression, microglia, neurogenesis, proteomics, juvenile stress, cytokines, anhedonia, neuroinflammation, two-hit model