Parameter Specific Modulation of Neuronal Firing and Extracellular Action Potential Dynamics by Transcranial Focused Ultrasound Stimulation in Rat Motor Cortex

Haorun Huang, Charlotte Smets, Liyi Chen, Myles Mc Laughlin

2026 · 10.2139/ssrn.6360139

rodenthealthyinvasive electrophysiology

Abstract

BackgroundTranscranial focused ultrasound stimulation (TUS) has emerged as a promising tool for noninvasive neuromodulation, however, the specific effects of stimulation parameters on neural activity remain incompletely understood.ObjectiveTo investigate how TUS pulse repetition frequency (PRF) and pulse duration (PD) influence neural activity in the rat motor cortex.MethodsIn vivo extracellular recordings were performed in anesthetized rats using 32-channel silicon probes while TUS was applied over the motor cortex. We systematically varied PRF (25, 50, 500 and 2000 Hz) and PD (50, 100, 200, 400 and 800 µs) and quantified changes in spike rate and extracellular action potential (EAP) shape. Control experiments included sonication of the visual cortex (V1) and the electrode holding bar. Effects were analyzed using linear mixed-effects models with Bonferroni corrected post hoc testing.ResultsTUS caused a significant increase in spike rate at 500 Hz PRF when the PD was set at 200 µs. When PRF was set at 50 Hz, spike rate increase at 800 µs was significantly higher than at 50 µs and 100 µs. Analysis of the EAP revealed significant parameter-dependent changes in action potential shape. When the most effective excitatory parameters (500 Hz PRF, 800 µs PD) were combined, a larger spike rate increase was observed in the motor cortex as was a significant change in action potential shape. The same combination applied to control conditions caused a smaller spike rate increase in V1 but no significant effect in the bar control, and no EAP changes were detected in either control condition.ConclusionThese findings offer direct single-unit evidence that TUS modifies both spike rate and EAP characteristics in a parameter-dependent and region-specific manner. Optimizing PRF and PD is essential for reproducible neuromodulation and for moving TUS toward translational applications in systems and clinical neuroscience.

Abstract via crossref.

Speciesrat (Sprague-Dawley)

Subjects26 animals

Sessions per subject1

Randomisednot reported

Blindingnot reported

Sham / controlactive control site, other

Auditory controlcontrol experiment

Readout timingonline

Anaesthesiaanaesthetised

Readoutsinvasive electrophysiology — 32-channel silicon-probe single-unit recordings (spike sorting with Kilosort 4.0); extracellular action potential waveform features (peak/trough amplitude, half-width at trough, half-width at peak)

Direction of effectbidirectionalSpike rate increase was strongest at 500 Hz PRF (fixed 200 µs PD) and at 800 µs PD (fixed 50 Hz PRF), with a non-significant decrease at the shortest 50 µs PD. Extracellular action potential half-width at trough decreased (shortened) at higher PRFs (50 and 2000 Hz) but increased (broadened) at the longest pulse duration (800 µs), i.e. the direction of the waveform effect depended on which parameter was varied.

Adverse eventsnot reported

Exposures

Exposure 1: PRF and pulse-duration sweep over motor cortex (M1)

Target: motor cortex — “motor cortex”
Device: Ultran · Ultran Group Inc. · GS500-D13-P25 ✓

Pulse timing
Waveform	pulsed
Fundamental frequency (kHz)	500	✓✓✓
Pulse duration (ms)	0.05, 0.1, 0.2, 0.4, 0.8swept	✓✓✓
Pulse repetition frequency (Hz)	25, 50, 500, 2,000swept	✓✓✓
Duty cycle (%)	not reported	⚑
Sonication duration (s)	0.5	✓✓✓

Pressure and intensity, by domain
Free-field pressure (kPa)	1,320	✓✓✓⚑
Free-field Isppa (W/cm²)	58.1	✓✓✓
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

Protocol, in the paper’s words

Trigger pulses were generated at PRFs of 25, 50, 500 and 2000 Hz for a total pulse train duration of 500 ms, with a pulse train repetition interval of 1 s. PRF was varied (25, 50, 500, 2000 Hz) at a fixed pulse duration of 200 µs, and pulse duration was varied (50, 100, 200, 400, 800 µs) at a fixed PRF of 50 Hz. For each parameter set, a 3-minute pre-TUS period (stimulation OFF) was followed by a 3-minute TUS stimulation period (stimulation ON). A second cohort of 8 rats then compared the combined strongest parameters (500 Hz PRF, 800 µs PD; 'M1 - Exp2') applied to motor cortex against the same parameters applied to the visual cortex (V1) and to the electrode holding bar.

Flags from extraction

n_subjects — Methods state 26 rats were used overall, but Results report 21 rats for the PRF/PD sweep plus a separate cohort of 8 rats for the control experiments (21+8=29), which does not reconcile with the stated total of 26.
exposures[0].free_field.pressure_kpa — Text states the measured peak calibration pressure was 1.32 MPa, but the Figure 1C legend states the maximum pressure at the focus was approximately 1.627 MPa; the two values disagree.
exposures[0].timing.duty_cycle_pct — Duty cycle is not stated as a percentage for any PRF/PD combination; it would require computing PD x PRF, which was not done here.
sham_type — V1 sonication (real ultrasound to a different brain region) is recorded as active_control_site; the electrode-holding-bar sonication is a non-anatomical mechanical/vibration control not covered by the vocabulary and is recorded as other.