# Sex-specific difference on anxiety- and depressive-like behavior in neuronal growth regulator 1-knockout mice

## TL;DR

This study shows that removing the Negr1 gene causes different anxiety and depression-like behaviors in male and female mice, linked to sex-specific changes in gut and cellular stress responses.

## Contribution

The study reveals sex-specific behavioral and molecular effects of Negr1 deletion, emphasizing the role of gut-brain communication in psychiatric disorders.

## Key findings

- Female Negr1–/– mice showed depression-like behavior and impaired fear learning, while males showed heightened anxiety-like behavior.
- Male Negr1–/– mice had increased protective stress responses in peripheral tissues, whereas females showed increased cell death in the same tissues.
- Negr1–/– mice exhibited increased intestinal permeability and sex-specific patterns of Bdnf mRNA expression.

## Abstract

This study investigated how loss of the neuronal growth regulator 1 (Negr1) affects depression-related behaviors differently by sex in the Negr1 knockout mouse model. Both male and female mice in the absence of Negr1 exhibited reduced social interaction and impaired long-term learning abilities. However, the specific types of behavioral problems differed markedly by sex. Female Negr1–/– mice exhibited signs of depression, including increased immobility when suspended by their tails and difficulty learning to avoid fearful situations. In contrast, male Negr1–/– mice showed primarily anxiety-like behaviors, such as avoiding open spaces.

At the molecular level, we found opposite patterns between sexes in how their bodies respond to cellular stress. In peripheral organs like the liver and intestine, males activated protective stress-response pathways, while females showed reduced protective responses and increased cell death. Interestingly, these sex-specific differences were seen only in peripheral tissues, not in the brain itself.

These findings suggest that the gut–brain connection works differently in males and females when the Negr1 gene is absent. Males appear to mount protective responses in peripheral tissues while experiencing anxiety, whereas females show increased vulnerability to cellular damage in peripheral tissues alongside depression-like behaviors. This research highlights why understanding sex differences is crucial for developing more effective, personalized treatments for depression and anxiety disorders.

The online version contains supplementary material available at 10.1186/s13293-025-00816-2.

Sex differences are evident in anxiety and depression, and women more frequently present with comorbid anxiety and depression alongside gastrointestinal disturbances. This pattern suggests contributions from sex-specific biological mechanisms and gut–brain communication. Negr1, a molecule regulating neuronal growth and connectivity, has been linked to depression-relevant behaviors in animal models. However, its mechanisms and potential sex-specific effects remain unclear.

Behavioral tests were used to assess phenotypes related to depression, anxiety, and learning in male and female wild-type (WT) and Negr1–/– mice, and molecular assays were performed to evaluate endoplasmic reticulum (ER) stress and apoptosis in the brain, liver, and colon. Behavioral test data were analyzed using a three-way mixed repeated-measures analysis of variance (RM-ANOVA), and molecular data were analyzed using two-way or three-way ANOVA.

Negr1–/– mice exhibited sex-dependent phenotypes in both central and peripheral systems. Baseline analyses revealed increased intestinal permeability in Negr1–/– mice and sex-specific patterns of brain-derived neurotrophic factor (Bdnf) mRNA expression across multiple time points. Behaviorally, Negr1–/– mice showed increased anxiety-like behavior, decreased social interaction, and impaired spatial learning in the Morris water maze, regardless of sex. Female Negr1–/– mice displayed impaired fear learning and increased depression-like behavior, while male Negr1–/– mice exhibited heightened anxiety-like responses. At the molecular level, ER stress marker spliced X-box binding protein 1 (Xbp1s) mRNA was upregulated in peripheral tissues of males but downregulated in females. Apoptosis analysis revealed enhanced caspase-3 activation in peripheral tissues of female Negr1–/– mice, while males showed no significant changes. Brain tissue showed no significant apoptotic alterations in either sex.

This study demonstrates that, in Negr1–/– mice, both sexes show general behavioral alterations. However, female Negr1–/– mice exhibit greater vulnerability to fear learning impairments and depression-like behavior, whereas males Negr1–/– mice show heightened anxiety responses. These behavioral differences were associated with opposing ER stress responses and differential apoptotic signaling between the sexes in peripheral tissues. Our findings highlight the importance of considering sex as a biological variable in depression research and suggest that Negr1 plays a crucial role in the sex-specific pathophysiology of psychiatric disorders through complex mechanisms spanning central and peripheral systems.

The online version contains supplementary material available at 10.1186/s13293-025-00816-2.

Negr1–/– mice showed increased intestinal permeability, and sex-specific Bdnf mRNA expression across multiple time points.Female Negr1–/– mice showed impaired fear learning and increased depression-like behavior, while males Negr1–/– mice displayed stronger anxiety-like behavior.Xbp1s mRNA expression increased in peripheral tissues (liver and colon) of male Negr1–/– mice but decreased in females, with no changes in phosphorylated eukaryotic initiation factor 2α (p-eIF2α) or C/EBP homologous protein (Chop) expression.Female Negr1–/– mice exhibited enhanced caspase-3 cleavage in peripheral tissues (colon), while males showed no significant changes: brain tissue showed no apoptotic alterations in either sex.

Negr1–/– mice showed increased intestinal permeability, and sex-specific Bdnf mRNA expression across multiple time points.

Female Negr1–/– mice showed impaired fear learning and increased depression-like behavior, while males Negr1–/– mice displayed stronger anxiety-like behavior.

Xbp1s mRNA expression increased in peripheral tissues (liver and colon) of male Negr1–/– mice but decreased in females, with no changes in phosphorylated eukaryotic initiation factor 2α (p-eIF2α) or C/EBP homologous protein (Chop) expression.

Female Negr1–/– mice exhibited enhanced caspase-3 cleavage in peripheral tissues (colon), while males showed no significant changes: brain tissue showed no apoptotic alterations in either sex.

The online version contains supplementary material available at 10.1186/s13293-025-00816-2.