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Increased Anatomical Specificity of Neuromodulation via Modulated Focused Ultrasound

Edin Mehić, Julia M. Xu, Connor J. Caler, Nathaniel K. Coulson, Chet T. Moritz, Pierre D. Mourad

PLoS ONE 2014, 9, e86939 · 10.1371/journal.pone.0086939

rodenthealthybehaviourhistology molecular

Abstract

Transcranial ultrasound can alter brain function transiently and nondestructively, offering a new tool to study brain function now and inform future therapies. Previous research on neuromodulation implemented pulsed low-frequency (250-700 kHz) ultrasound with spatial peak temporal average intensities (ISPTA) of 0.1-10 W/cm(2). That work used transducers that either insonified relatively large volumes of mouse brain (several mL) with relatively low-frequency ultrasound and produced bilateral motor responses, or relatively small volumes of brain (on the order of 0.06 mL) with relatively high-frequency ultrasound that produced unilateral motor responses. This study seeks to increase anatomical specificity to neuromodulation with modulated focused ultrasound (mFU). Here, 'modulated' means modifying a focused 2-MHz carrier signal dynamically with a 500-kHz signal as in vibro-acoustography, thereby creating a low-frequency but small volume (approximately 0.015 mL) source of neuromodulation. Application of transcranial mFU to lightly anesthetized mice produced various motor movements with high spatial selectivity (on the order of 1 mm) that scaled with the temporal average ultrasound intensity. Alone, mFU and focused ultrasound (FUS) each induced motor activity, including unilateral motions, though anatomical location and type of motion varied. Future work should include larger animal models to determine the relative efficacy of mFU versus FUS. Other studies should determine the biophysical processes through which they act. Also of interest is exploration of the potential research and clinical applications for targeted, transcranial neuromodulation created by modulated focused ultrasound, especially mFU's ability to produce compact sources of ultrasound at the very low frequencies (10-100s of Hertz) that are commensurate with the natural frequencies of the brain.

Abstract via europepmc.

Speciesmouse (C57BL/6)
Subjects3, 6, 3, 5, 5, 3swept animals
Sessions per subjectnot reported
Randomisednot reported
Blindingnot reported
Sham / controlnone
Auditory controlnot reported
Readout timingonline
Anaesthesiaanaesthetised
Readoutsbehaviour, histology molecularVideo-scored ordinal (0-3) motor-movement robustness/success-rate scale; hematoxylin-eosin and cresyl-violet brain histology
Direction of effectexcitatoryPlanar, FUS and mFU ultrasound each evoked motor movements (tail, paw, whisker) whose robustness scaled with temporal-average intensity, consistent with excitatory neuromodulation; no inhibitory effects were reported.
Adverse eventsnone observedHistological analysis (H&E, cresyl violet) after UNMOD trials showed no observable brain damage.

Exposures

Exposure 1: Planar (Ultran) ultrasound, midline sweep (500 kHz)

Target: whole brain or unfocused — “midline positions A (caudal), B (mid-sagittal), and C (rostral)
Device: Ultran · Ultran Group · GS500-D13

Pulse timing
Waveformpulsed
Fundamental frequency (kHz)500✓✓
Pulse duration (ms)200✓✓
Pulse repetition frequency (Hz)1,500✓✓
Duty cycle (%)not reportedpulse duration × PRF gives 30000%
Sonication duration (s)1✓✓
Pressure and intensity, by domain
Free-field pressure (kPa)not reported
Free-field Isppa (W/cm²)not reported
Free-field Ispta (W/cm²)0.15, 5.25swept✓✓
In-situ estimatenot 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

88 bursts of 500-kHz ultrasound, each of length 200 ms, at a pulse repetition frequency of 1.5 kHz in a 1-s interval, applied in a rostral-to-caudal sweep along the skull midline at three stable positions (A, B, C) 3 mm apart. One ultrasound trial consisted of ten applications of this protocol, completed in approximately 10 s; trials at the three positions were separated by ~5 min. A separate series of trials at position B varied ISPTA from 0.15 to 5.25 W/cm2 by changing peak pressure while keeping the temporal pattern fixed.

Consistency checks: pd exceeds period.

Exposure 2: Focused ultrasound (FUS), single 2 MHz carrier, parietal grid

Target: parietal lobe — “six 3x3 mm regions spanning bregma to lambda, emphasising the parietal region (54-square grid, 1 mm resolution)
Device: Sonic Concepts · Sonic Concepts · H-148

Pulse timing
Waveformpulsed
Fundamental frequency (kHz)2,000✓✓
Pulse duration (ms)200✓✓
Pulse repetition frequency (Hz)1,500✓✓
Duty cycle (%)not reportedpulse duration × PRF gives 30000%
Sonication duration (s)1✓✓
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
Protocol, in the paper’s words

The focused UNMOD (FUS) protocol was designed to overlap the planar protocol's temporal structure (88 bursts, PRF 1.5 kHz, each burst as stated 200 ms, within a 1-s interval); both elements of the dual-element transducer were driven at 2 MHz. One trial consisted of ten applications completed in ~10 s. The transducer was moved in 1-mm steps through a 54-square grid (six 3x3 mm regions) spanning bregma to lambda; FUS was applied by equalising the carrier frequencies of the two elements.

Consistency checks: pd exceeds period.

Exposure 3: Modulated focused ultrasound (mFU), 500 kHz difference frequency, parietal grid

Target: parietal lobe — “six 3x3 mm regions spanning bregma to lambda, emphasising the parietal region (54-square grid, 1 mm resolution)
Device: Sonic Concepts · Sonic Concepts · H-148

Pulse timing
Waveformpulsed
Fundamental frequency (kHz)1,750, 2,250swept✓✓
Pulse duration (ms)200✓✓
Pulse repetition frequency (Hz)1,500✓✓
Duty cycle (%)not reportedpulse duration × PRF gives 30000%
Sonication duration (s)1✓✓
Pressure and intensity, by domain
Free-field pressure (kPa)not reported
Free-field Isppa (W/cm²)not reported
Free-field Ispta (W/cm²)0.1, 5swept?
In-situ estimatenot 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

One element of the focused transducer was driven at 1.75 MHz and the other at 2.25 MHz, producing a difference (modulation) frequency of 500 kHz at the focus; otherwise the mFU parameters mimicked the planar protocol (88 bursts, PRF 1.5 kHz, each burst as stated 200 ms, in a 1-s interval; one trial = ten applications in ~10 s). In a separate intensity-sweep experiment, mFU intensity was decreased from a suprathreshold level by reducing the number or duration of pulses; motor responses were induced until reaching 1 W/cm2 ISPTA.

Consistency checks: pd exceeds period.

Flags from extraction

  • exposures[0].timing.pulse_duration_msText states 88 bursts at a PRF of 1.5 kHz (period 0.67 ms) each lasting 200 ms; these are dimensionally inconsistent (a 200 ms pulse cannot repeat at 1.5 kHz within a 1-s interval), likely a units typo (e.g. 200 microseconds) in the source; recorded as stated ('200 ms').
  • exposures[1].timing.pulse_duration_msSame burst-timing arithmetic inconsistency as the planar protocol, which this FUS protocol was designed to mimic; recorded as stated.
  • exposures[2].timing.pulse_duration_msSame burst-timing arithmetic inconsistency as the planar protocol, which the mFU parameters mimicked; recorded as stated.
  • exposures[1].free_field.ispta_w_cm2Unit label is garbled in the source ('varied by intensity (0.1-5.0 I )'), missing the ISPTA subscript; value is inferred to be W/cm2 ISPTA given the paper's consistent use of ISPTA elsewhere, and the sentence describes both mFU and FUS jointly.
  • exposures[2].free_field.ispta_w_cm2Unit label is garbled in the source ('varied by intensity (0.1-5.0 I )'), missing the ISPTA subscript; value is inferred to be W/cm2 ISPTA given the paper's consistent use of ISPTA elsewhere. A separate sentence states motor responses were induced 'until reaching 1 W/cm2' as mFU intensity was reduced in a dedicated three-mouse experiment.
  • exposures[2].fundamental_frequency_khzmFU's 500 kHz is the vibro-acoustography 'difference frequency' produced by two transducer elements driven at 1.75 MHz and 2.25 MHz, not a single literal carrier frequency; recorded as the paper's own stated effective/fundamental frequency for mFU.
  • n_subjectsPaper reports several independent group sizes across sub-experiments without ever stating a study-wide total: 3 mice (planar intensity sweep), 6 mice (planar spatial sweep), 3 mice (mFU variable-intensity), 5 mFU mice and 5 FUS mice (separate cohorts), 3 mice (mFU & FUS applied to the same animals). It is not stated whether any of these cohorts overlap. Note also that the Figure 10 legend states 'ten mFU and ten FUS mice', which conflicts with the main text's statement of five mice per group for that experiment; the main-text value (5 and 5) was used per the rule that figures alone do not count.