Comparative Evaluation of Corticosterone Administration, Chronic Restraint Stress, and Their Combination for Depression-like Behavioral and Molecular Alterations in Mice: A Multi-Domain Assessment

Major depressive disorder (MDD) involves dysregulation of the hypothalamic–pituitary–adrenal axis, neuroinflammation, serotonergic dysfunction, and impaired neurotrophic signaling. Whether combining corticosterone (CORT) with chronic restraint stress (CRS) produces a more comprehensive depression-like phenotype than either model alone remains unexplored. Male C57BL/6N mice were assigned to four groups—Sham (n = 7), CORT (20 mg/kg s.c.; n = 7), CRS (3 h/day; n = 7), and CORT + CRS (C+C; n = 8)—and evaluated by behavioral tests, hippocampal qRT-PCR, Western blot, LC-MS/MS metabolomics, and immunohistochemistry. All experimental groups showed elevated immobility in tail suspension and forced swim tests without inter-group differences. Two-way ANOVA revealed a behavioral–molecular dissociation: FST immobility showed CORT and CRS main effects without a significant interaction, whereas hippocampal LC-MS/MS analytes exhibited strong CORT × CRS interactions. The C+C group showed the strongest 5-HT1A receptor (HTR1A) upregulation, the greatest reductions in Trk-b and DCX, and unique decreases in serum dopamine, glutamine, and adenosine. Representative Western blot densitometry indicated the largest p-ERK/ERK reduction in C+C (54%), while p-CREB/CREB was most suppressed in CRS (52%). BDNF and NeuN proteins were lowest in C+C (68% and 61% reductions). Combined CORT + CRS produces the most comprehensive depression-related molecular profile, integrating multiple pathological domains.