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Weak Ultrasound Contributes to Neuromodulatory Effects in the Rat Motor Cortex

Po-Chun Chu, Chen-Syuan Huang, Pi-Kai Chang, Rou-Shayn Chen, Ko-Ting Chen, Tsung-Hsun Hsieh, Hao-Li Liu

International Journal of Molecular Sciences 2023, 24, 2578 · 10.3390/ijms24032578

rodenthealthyemg mephistology molecularcellular imaging

Abstract

Transcranial focused ultrasound (tFUS) is a novel neuromodulating technique. It has been demonstrated that the neuromodulatory effects can be induced by weak ultrasound exposure levels (spatial-peak temporal average intensity, I SPTA 2 ) in vitro. However, fewer studies have examined the use of weak tFUS to potentially induce long-lasting neuromodulatory responses in vivo. The purpose of this study was to determine the lower-bound threshold of tFUS stimulation for inducing neuromodulation in the motor cortex of rats. A total of 94 Sprague-Dawley rats were used. The sonication region aimed at the motor cortex under weak tFUS exposure (I SPTA of 0.338-12.15 mW/cm 2 ). The neuromodulatory effects of tFUS on the motor cortex were evaluated by the changes in motor-evoked potentials (MEPs) elicited by transcranial magnetic stimulation (TMS). In addition to histology analysis, the in vitro cell culture was used to confirm the neuromodulatory mechanisms following tFUS stimulation. In the results, the dose-dependent inhibitory effects of tFUS were found, showing increased intensities of tFUS suppressed MEPs and lasted for 30 min. Weak tFUS significantly decreased the expression of excitatory neurons and increased the expression of inhibitory GABAergic neurons. The PIEZO-1 proteins of GABAergic neurons were found to involve in the inhibitory neuromodulation. In conclusion, we show the use of weak ultrasound to induce long-lasting neuromodulatory effects and explore the potential use of weak ultrasound for future clinical neuromodulatory applications.

Abstract via europepmc.

Speciesrat (Sprague-Dawley)
Subjects94 animals
Sessions per subjectnot reported
Randomisedyes
Blindingnot reported
Sham / controlinactive transducer
Auditory controlnot reported
Readout timingoffline
Anaesthesiaanaesthetised
Readoutsemg mep, histology molecular, cellular imagingTMS-evoked motor-evoked potentials (MEPs); GFAP, c-Fos and GAD-65 immunostaining; Fluo-4 calcium imaging in primary cortical neurons
Direction of effectinhibitoryWeak tFUS (ISPTA 0.338-12.15 mW/cm2) decreased MEP amplitude for up to 30 min in a dose-dependent manner; the lowest intensity tested (0.014 mW/cm2) had no effect. Decreased c-Fos and increased GAD-65 expression indicated suppression of cortical activity and increased inhibitory interneuron activity.
Adverse eventsnone observedGFAP immunohistochemistry showed no significant astrogliosis or difference from sham after ISPTA=50 mW/cm2 sonication, suggesting the tFUS intensities used did not damage brain tissue.

Exposures

Exposure 1: Weak tFUS intensity sweep to left primary motor cortex

Target: primary motor cortex — “left primary motor cortex (M1)
Device: custom-built

Pulse timing
Waveformpulsed
Fundamental frequency (kHz)700✓✓
Pulse duration (ms)0.8✓✓
Pulse repetition frequency (Hz)100✓✓
Duty cycle (%)8pulse duration × PRF gives 8%✓✓
Sonication duration (s)300✓✓
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 estimatenot 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)0.000014, 0.000338, 0.00304, 0.0122, 0.05swept?
Protocol, in the paper’s words

MEPs were recorded for 10 min before tFUS as baseline, tFUS was delivered for a total sonication duration of 5 min at one of five mechanical-index/ISPTA levels (group 1 sham; groups 2-5: MI 0.003-0.081, ISPTA 0.014-12.15 mW/cm2), and MEP recording continued for 30 min afterward to assess post-tFUS effects. A separate safety cohort received ISPTA=50 mW/cm2 tFUS for histological (GFAP) analysis.

Flags from extraction

  • n_subjectsThe paper states a single total (94 rats) for the whole study; this includes the MEP dose-response groups (n=15/group per Fig. S1), the ISPTA=50 mW/cm2 GFAP safety cohort, and animals used only as a source of embryos for the in vitro primary-neuron experiments, which were not themselves exposed to tFUS. The breakdown exposed-vs-not is not fully reconciled in the main text.
  • auditory_controlPaper argues (Discussion) that using pulsed mode and low PRF 'eliminated' the confounding auditory effect, but this is a post-hoc argument, not a masking/behavioural auditory control condition actually implemented; classified as other.
  • exposures[0].unspecified_domain.ispta_w_cm2Domain (free-field vs in-brain) of the stated ISPTA values is not specified by the paper; recorded as unspecified_domain. Values converted from stated mW/cm2 to W/cm2.