General‐Purpose Ultrasound Neuromodulation System for Chronic, Closed‐Loop Preclinical Studies in Freely Behaving Rodents

Yehhyun Jo, Sang‐Mok Lee, Taesub Jung, Gijae Park, Chanhee Lee, Geun Ho Im, Seongju Lee, Jin Soo Park, Chaerin Oh, Geon Kook, Hyunggug Kim, Seongyeon Kim, Byung Chul Lee, Greg S.B. Suh, Seong‐Gi Kim, Jeongyeon Kim, Hyunjoo J. Lee

Advanced Science 2022, 9 · 10.1002/advs.202202345

rodenthealthyeeg megbehaviourhistology molecularautonomic physiology

Abstract

Transcranial focused ultrasound stimulation (tFUS) is an effective noninvasive treatment modality for brain disorders with high clinical potential. However, the therapeutic effects of ultrasound neuromodulation are not widely explored due to limitations in preclinical systems. The current preclinical studies are head-fixed, anesthesia-dependent, and acute, limiting clinical translatability. Here, this work reports a general-purpose ultrasound neuromodulation system for chronic, closed-loop preclinical studies in freely behaving rodents. This work uses microelectromechanical systems (MEMS) technology to design and fabricate a small and lightweight transducer capable of artifact-free stimulation and simultaneous neural recording. Using the general-purpose system, it can be observed that state-dependent ultrasound neuromodulation of the prefrontal cortex increases rapid eye movement (REM) sleep and protects spatial working memory to REM sleep deprivation. The system will allow explorative studies in brain disease therapeutics and neuromodulation using ultrasound stimulation for widespread clinical adoption.

Abstract via europepmc.

Speciesmouse (C57BL/6J)

Subjects87 animals

Sessions per subject1

Randomisednot reported

Blindingnot reported

Sham / controlinactive transducer

Auditory controlnot reported

Readout timingboth

Anaesthesiaawake

Readoutseeg meg, behaviour, histology molecular, autonomic physiology — EEG/EMG-based sleep-state scoring (WAKE/REM/NREM) and spectral power; spontaneous alternation behaviour (SAB) in a Y-maze (DeepLabCut pose tracking); c-Fos immunohistochemistry; serum corticosterone ELISA

Direction of effectmixed or unclearChronic closed-loop ultrasound stimulation of the medial prefrontal cortex during NREM sleep increased REM sleep duration and theta power and increased WAKE-to-REM and REM-to-REM transition probabilities relative to sham (inactive transducer); it also protected spatial working memory (Y-maze alternation rate) from REM sleep deprivation. Direct excitatory/inhibitory effect on mPFC neurons was not directly assessed (only EEG/behavioural proxies).

Adverse eventsnot reportedBioheat simulation (at a conservative 137.7 kPa peak pressure, more than double the actual 55 kPa device output) predicted a temperature rise of approximately 0.36°C at 300 s and <0.02°C at 2 s at the beam focus; transducer surface self-heating increased by 1.5°C over 1 min of continuous operation; the in vivo protocol delivered ultrasound for only 2 s at 6-s intervals.

Exposures

Exposure 1: Chronic closed-loop CMUT ultrasound stimulation of medial prefrontal cortex during NREM sleep

Target: ventromedial prefrontal cortex — “medial prefrontal cortex (mPFC)”
Device: custom-built · KAIST (custom MEMS/CMUT) · disk-type CMUT (384 cells, 8.1 mm diameter) ✓

Pulse timing
Waveform	pulsed
Fundamental frequency (kHz)	460	✓✓✓
Pulse duration (ms)	not reportedimplied by duty cycle ÷ PRF: 6 ms (not stated by the paper)	⚑
Pulse repetition frequency (Hz)	100	✓✓✓
Duty cycle (%)	60	✓✓✓
Sonication duration (s)	2	✓✓✓

Pressure and intensity, by domain
Free-field pressure (kPa)	55	✓✓✓
Free-field Isppa (W/cm²)	0.1	✓✓✓
Free-field Ispta (W/cm²)	not reported
In-situ estimate	measurementsingle value
In-situ pressure (kPa)	not reported
In-situ Isppa (W/cm²)	0.084	✓✓✓⚑
In-situ Ispta (W/cm²)	not reported

Protocol, in the paper’s words

A closed-loop system triggered a 460 kHz ultrasound burst (PRF 100 Hz, 60% duty cycle) for a 2-s stimulation period within each 6-s EEG/EMG analysis epoch whenever NREM sleep was detected online. Stimulation (STIM+) or sham (dummy device, STIM-) was delivered continuously over a single 10-h session (D2, 10:00-20:00) following a habituation day (D0) and a baseline recording day (D1); D3 was an observation-only day to assess chronic effects. A separate protocol added a 2-h stimulation period plus an 8-h sleep-deprivation protocol and Y-maze test on D3 for the memory experiment.

Flags from extraction

exposures[0].timing.pulse_duration_ms — PRF (100 Hz) and duty cycle (60%) are both stated, which would let pulse duration be computed by division; per instructions this was not calculated and pulse_duration_ms is left not_reported rather than derived.
direction_of_effect — Effect was assessed via EEG/behavioural proxies (REM sleep duration/theta power, Y-maze memory) rather than direct measurement of neuronal excitation or inhibition in the mPFC, so classified as mixed_or_unclear rather than excitatory/inhibitory.
exposures[0].in_situ.isppa_w_cm2 — The 84 mW/cm2 value is described as the beam intensity measured through an ex vivo mouse half-skull placed in front of the transducer (overall attenuation <30%), used here as the in-situ (measured-through-skull) value; free-field value (100 mW/cm2 estimated maximum center intensity) is from the same beam profile without the skull.