Intranasal ketamine represents a rapid and minimally invasive antidepressant strategy, yet the molecular mechanisms underlying its antidepressant-like effects after intranasal administration remain incompletely understood. Male Sprague-Dawley rats were subjected to a 28-day chronic unpredictable mild stress (CUMS) paradigm and then received intranasal racemic ketamine at 8.58 mg/kg twice weekly for 14 days, with four administrations in total. Depressive-like behaviors were evaluated using the open-field, sucrose preference, and forced swimming tests. Hypothalamic histological alterations and synaptic and mitochondrial integrity were examined by Hematoxylin and eosin staining, Golgi-Cox staining, and transmission electron microscopy, and hypothalamic transcriptomics was combined with molecular validation. Intranasal ketamine improved CUMS-induced behavioral deficits, attenuated histological neuronal alterations in the hypothalamus, improved mitochondrial ultrastructure, increased synaptic density, and restored dendritic length and spine density. RNA sequencing highlighted oxidative phosphorylation-related changes and identified Ndufa10, encoding NADH:ubiquinone oxidoreductase subunit A10, as a ketamine-responsive mitochondrial complex I candidate; Real-time quantitative PCR and immunohistochemistry confirmed that, compared with the CUMS group, intranasal ketamine administration increased hypothalamic Ndufa10 mRNA and NDUFA10 protein levels. Pharmacological inhibition of mitochondrial complex I with rotenone attenuated ketamine-associated behavioral improvement and restoration of NDUFA10 expression, supporting a potential contribution of mitochondrial complex I activity. α-Synuclein (SNCA) protein abundance changed in parallel, whereas Snca mRNA levels remained unchanged, suggesting an accompanying protein-level alteration rather than a primary transcriptional response. Together, these findings suggest that hypothalamic mitochondrial complex I activity may contribute to the antidepressant-like effects of intranasal ketamine, with NDUFA10 emerging as a ketamine-responsive candidate molecule that requires further functional validation.
