Parameter-Specific Effects of Low-Intensity Transcranial Focused Ultrasound Stimulation on Depression-Like Behaviors in a CUMS Mouse Model

Yan Zhang, Yaxing Zhang, Kaiming Zhang, Lan Wang, Dongyang Huang, Fengya Zhen, Ran Wang, Cuixia An

Neuropsychiatric Disease and Treatment 2026 · 10.2147/ndt.s586583

rodentdepressionbehaviourhistology molecular

Abstract

Purpose Depression is a multifactorial disorder involving neurotransmitter dysregulation, gut microbiota imbalance, and metabolic disturbances. Low-intensity transcranial focused ultrasound stimulation (LIFUS) holds promise for treating depression. However, the effects of different LIFUS parameter settings on depression-like behaviors, and their potential associations with gut microbiota and fecal metabolite changes, remain largely unexplored. This study aims to investigate the parameter-specific effects of LIFUS on depression-like behaviors in a chronic unpredictable mild stress (CUMS) mouse model, and to explore potential associations with changes in gut microbiota and fecal metabolites. Methods To establish a depression model, C57BL/6 mice were subjected to CUMS, while a separate cohort was kept as a control (CON) group. The CUMS-exposed mice were then randomly divided into four groups: CUMSpo, LIFUS1, LIFUS2 and SHAM. Depressive-like behaviors were evaluated using the sucrose preference test (SPT) and forced swim test (FST). The levels of neurotransmitters and Fecal concentrations of metabolites were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gut microbiota composition was analyzed by metagenomic sequencing, and α-diversity was assessed using the ACE, Chao1, and Shannon indices. Histopathology was assessed via HE staining. Results LIFUS at 1.5 kHz PRF, but not 300 Hz, significantly attenuated CUMS-induced depressive-like behaviors, evidenced by increased sucrose preference and reduced immobility time, without affecting locomotor activity. This behavioral effect was accompanied by a significant increase in cortical glutamate. LIFUS2 protocol was associated with a significant increase in tryptamine, alongside a concurrent trend towards restoring the abundance of Clostridia and enhancing gut microbiota α-diversity. HE staining confirmed protocol safety. Conclusion The antidepressant-like effects of LIFUS appear to be associated with multi-systemic alterations, including changes in cortical glutamate, modulation of the gut microbiota, and specific changes in tryptophan metabolism.

Abstract via europepmc.

Speciesmouse (C57BL/6)

Subjects8, 8swept animals

Sessions per subjectnot reported

Randomisedyes

Blindingnot reported

Sham / controlinactive transducer

Auditory controlother

Readout timingoffline

Anaesthesiaawake

Readoutsbehaviour, histology molecular — sucrose preference test; forced swim test; open field test; cortical neurotransmitters by LC-MS/MS; fecal metagenomics and tryptophan metabolites; HE staining

Direction of effectmixed or unclearProtocol 2 (PRF 1.5 kHz) increased sucrose preference, reduced FST immobility and raised cortical glutamate versus sham in CUMS mice; Protocol 1 (PRF 300 Hz) had no significant effect.

Adverse eventsnone observedHE staining after two weeks of stimulation showed no histopathological damage or hemorrhage in the prelimbic cortex (LIFUS2 protocol).

Exposures

Exposure 1: LIFUS1 (300 Hz PRF) and LIFUS2 (1.5 kHz PRF)

Target: ventromedial prefrontal cortex — “prelimbic cortex (PrL)”
Device: Olympus / Panametrics · Olympus · V301-SU ✓

Pulse timing
Waveform	pulsed
Fundamental frequency (kHz)	500	✓✓✓
Pulse duration (ms)	0.4	✓✓✓
Pulse repetition frequency (Hz)	300, 1,500swept	✓✓✓⚑
Duty cycle (%)	12, 60swept	✓✓✓
Sonication duration (s)	0.4	✓✓✓

Pressure and intensity, by domain
Free-field pressure (kPa)	not reported
Free-field Isppa (W/cm²)	not reported
Free-field Ispta (W/cm²)	not reported
In-situ estimate	not reported
In-situ pressure (kPa)	not reported
In-situ Isppa (W/cm²)	not reported
In-situ Ispta (W/cm²)	not reported
Ispta, domain unspecified (W/cm²)	0.26, 1.31swept	✓✓✓⚑

Protocol, in the paper’s words

Two protocols: Protocol 1 (PRF 300 Hz, PD 400 us, DC 12%, ISPTA 0.26 W/cm2) and Protocol 2 (PRF 1.5 kHz, PD 400 us, DC 60%, ISPTA 1.31 W/cm2). Both used a sonication duration of 400 ms with 3-second inter-stimulus intervals; each stimulation session lasted 15 minutes, daily, for about two weeks.

Flags from extraction

exposures[0].timing.pulse_repetition_frequency_hz — Second PRF (1.5 kHz) taken from Protocol 2 text; quote given is Protocol 1 sentence; Protocol 2 is in the PRF figure legend and the same paragraph as LaTeX.
exposures[0].unspecified_domain.ispta_w_cm2 — ISPTA given without stating water or in-brain domain.
n_subjects — n=8 per group for LIFUS1 and LIFUS2 (16 sonicated); sham group (n=8, no output) and CON/CUMS not counted; some assays had smaller n.
auditory_control — Sham was amplifier switched off, said to control for procedural noise and equipment sounds; no dedicated auditory control described.
n_sessions_per_subject — Daily 15-min sessions for 'two weeks' of stimulation; number of sessions not stated.
exposures[0].target.terms — Prelimbic cortex mapped to ventromedial_prefrontal_cortex (medial prefrontal).
direction_of_effect — Behavioural improvement with PRF 1.5 kHz only; direction of neural effect not directly assessed except cortical glutamate increase.