Optimized ultrasound neurostimulation of ventromedial prefrontal cortex reverses depression behaviours and normalizes brain metabolism

Ultrasound neurostimulation (USNS) offers non-invasive access to deep mood-regulating circuits, but most protocols rely on arbitrary parameters and lack mechanistic evaluation. We first optimised single-pulse USNS in healthy mice by mapping pressure–response curves during motor cortex stimulation with behavioural scoring, electromyography and hydrophone measurements to define a focal, reproducible setting. We then applied repeated USNS to the infralimbic cortex, the rodent homologue of ventromedial prefrontal cortex, for five days during unpredictable chronic mild stress. Behavioural outcomes (nest-building, open-field exploration), whole-brain 18F-FDG microPET and targeted metabolomics in infralimbic cortex, amygdala and hippocampus were assessed. Optimised infralimbic USNS selectively activated local neurons, reversed self-care and anxiety-like deficits to near baseline and outperformed chronic fluoxetine. PET revealed sustained hypermetabolism in vmPFC, dorsal hippocampus, periaqueductal grey and raphe nuclei, while metabolomics showed coordinated down-regulation of glutamate-centred pathways, supporting USNS as a precise, non-destructive antidepressant-like intervention.