A Double-Blind Replication Attempt of Offline 5Hz-rTUS-Induced Corticospinal Excitability

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43 Transcranial ultrasound stimulation (TUS) is a promising new form of non -44 invasive neuromodulation. As a nascent technique, replication of its effects on brain 45 function is important. Of particular interest is offline 5Hz repetitive TUS (5Hz -rTUS), 46 originally reported by Zeng and colleagues [1] to elicit lasting corticospinal excitability 47 increases, with large effect sizes. 48

and method 49 Here, we conducted a pre -registered ( https://osf.io/p5n4q) replication of this 50 protocol that benefitted from three additional features: double-blind application of TUS, 51 neuronavigation for consistent TMS positioning, and acoustic simulations to assess 52 M1 target exposure to TUS. Changes in resting motor thresholds (rMT), motor-evoked 53 potential (MEP) amplitude, short-interval intracortical inhibition (SICI) and intracortical 54 facilitation (ICF) in response to TUS (5Hz-rTUS vs. sham) were measured in the right 55 first dorsal interosseous (FDI), abductor digiti minimi (ADM) and abductor pollicis 56 brevis (APB) muscles, with unbiased selection of participants. Transducer location 57 was determined by the TMS-hotspot for motor representations of the right FDI , as in 58 the original work. 59

60 No significant effects of 5Hz -TUS (vs sham) were observed. Post -hoc 61 simulations showed considerable variability of the acoustic focus, which was outside 62 the anatomical M1 -hand area in 67% of participants – in line with the known poor 63 correspondence of TMS-hotspot location and M1-hand area. 64

65 Our results indicate that the effect sizes of the neuromodulatory effects of 5Hz-66 rTUS on M1 may be more variable than previously appreciated . We suggest that 67 double-blinding, neuronavigat ed TMS , individualised acoustic simulations for TUS 68 targeting and pre-registration will aid reproducibility across studies. 69 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 26, 2024. ; https://doi.org/10.1101/2024.11.25.625187doi: bioRxiv preprint 4

transcranial ultrasound stimulation (TUS), neuromodulation, corticospinal 70 excitability, neuronavigation 71 72 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 26, 2024. ; https://doi.org/10.1101/2024.11.25.625187doi: bioRxiv preprint 5

73 Transcranial ultrasonic stimulation (TUS) is a relatively novel technique for non-74 invasive neuromodulation in humans [2]. As a nascent approach for neuromodulation, 75 replication of the effects of TUS is a critical ingredient in progressing toward a mature 76 technology with clinical utility. One example illustrating this is the recent discovery that 77 the inhibitory effects of TUS on corticospinal excitability can be confounded by auditory 78 inputs [3]. In response, auditory masking and ramped pulsing have rapidly become 79 standard in the field [4-7] 80 Here we focus on 5Hz -rTUS (also referred to as theta -burst TUS; tbTUS), an 81 offline TUS protocol that has been reported to elicit strong facilitation of corticospinal 82 excitability (CSE), with large effects sizes, that outlasted sonication by up to 30 83 minutes [1]. The same research group has subsequently replicated these excitatory 84 effects in both healthy and clinical populations [8-14] 85 However, the opposite neuromodulatory effects of 5Hz -rTUS were recently 86 reported by an independent research group, instead showing inhibition (not excitation) 87 of CSE lasting up to 30 minutes following sonication [15]. In contrast with targeting M1 88 based on the TMS-hotspot location and only at a fixed depth of ~30mm [1], Bao and 89 colleagues personalised TUS application with acoustic simulations to ensure precise 90 targeting within M1 in each participant. The inhibitory effects were observed when 91 sonicating either the lip of precentral gyrus or deeper sections of M1. Simulations also 92 provided individual estimates of the acoustic intensity in the brain, as opposed to using 93 measurements in free-water which cannot account for individual variance in absorption 94 and refraction of ultrasound through the skull [16, 17]. Given the contrasting outcomes 95 of 5Hz -rTUS reported by separate research groups, and a dearth of independent 96 replication, at this early stage of offline human TUS application it is essential to obtain 97 better estimates of the likely effect sizes of 5Hz-rTUS and factors that may influence 98 these. This necessity is further underscored by the intended application of these 99 protocols in clinical populations [12]. 100 In the present study, we therefore conducted an independent replication of the 101 methodology initially reported by Zeng and colleagues [1]. We specifically focussed 102 on replicating the effect of 5Hz -rTUS on CSE, short-interval intracortical inhibition 103 (SICI), and intracortical facilitation (ICF). We employed the same targeting procedures, 104 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 26, 2024. ; https://doi.org/10.1101/2024.11.25.625187doi: bioRxiv preprint 6 stimulation protocol, and outcome measures as [1]. However, to guard against biases 105 and additional sources of variance, we incorporated double-blinding, neuronavigation 106 of TMS, more repetitions per condition, quantification of MEPs from adjacent hand 107 muscles, post-hoc acoustic simulations of TUS, and pre-registered the study. 108 We did not observe a significant effect of 5Hz -rTUS on CSE or intracortical 109 excitability. This result reappraises the likely effect sizes of 5Hz-rTUS, and we highlight 110 potential factors influencing these discrepancies. 111 112

113 Unless stated otherwise, all elements of Experiment 2 by Zeng and colleagues 114 [1] were adhered to. 115 Design 116 Participants attended two sessions (one week apart) with either 5Hz-rTUS or 117 sham-TUS, in c ounterbalanced order across participants (Fig. 1A-C). In addition to 118 Zeng and co-workers [1], we used a double -blind procedure for TUS application to 119 reduce potential experimenter bias. MEP measures (MEP amplitude; SICI; ICF) were 120 recorded at baseline, and 5, 30, and 60 minutes after 5Hz-rTUS or sham (T5, T30, 121 T60; Fig 1D). Each session was conducted by two experimenters – one operating the 122 TMS and TUS, and the other assisting with neuronavigation and stimulation equipment 123 control. 124 Participants 125 We studied 15 healthy right-handed individuals (age: 31.3±12; five males; eight 126 Asian, six Caucasian, one African ) after written informed consent, all without 127 neurological or psychiatric diseases, no contraindications to brain stimulation, and no 128 medications known to affect brain excitability. The study was approved by the UCL 129 Research Ethics Committee (14233/003) and conducted in accordance with the 130 Declaration of Helsinki. 131 MRI 132 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 26, 2024. ; https://doi.org/10.1101/2024.11.25.625187doi: bioRxiv preprint 7 All participants had existing T1-weighted MRI scans obtained in one of UCL’s 133 neuroimaging facilities, and all consented to re -use of these images for this study. 134 While imaging sequence details varied for this reason, all images were acquired with 135 1mm isotropic resolution, covering the whole head. Note that for the simulation 136 conducted in this study, we focus on the location of the acoustic focus, not the actual 137 intensity values , which will not have been systematically influenced by small 138 idiosyncrasies in the MRI sequences across individuals. 139 140 141 Fig. 1. Experimental design and methodology 142 A. TMS-elicited motor-evoked potentials (MEPs) were measured from the primary muscle of 143 interest (FDI) and two adjacent hand muscles. 144 B. The 5Hz-rTUS protocol. PTD: pulse train duration; PD: pulse duration; PRI: pulse repetition 145 interval; DC: duty cycle; f Isppa_fw: free-water spatial-peak pulse-average intensity; Ispta_fw: free-146 water temporal-average spatial peak. 147 C. TUS positioning [1]: TMS coil position over the motor hotspot was marked on the scalp 148 (left), with the centre of the coil (middle) used to localise the TUS transducer on the scalp 149 (right). 150 D. Experimental design and procedure. 151 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 26, 2024. ; https://doi.org/10.1101/2024.11.25.625187doi: bioRxiv preprint 8 Transcranial magnetic stimulation 152 TMS was delivered with a Magstim 200 2 Monophasic stimulator (single-pulse) 153 and a Magstim Bistim2 system consisting of two 2002 monophasic stimulators (paired-154 pulse) connected to a D70 alpha F8 coil with an internal diameter of 70mm (Magstim 155 Co. Ltd, Whitland, Wales). Participants were seated in a comfortable chair with their 156 hands resting on a pillow on their lap. The TMS coil was placed over the left primary 157 motor cortex (M1), tangentially to the scalp, with the handle of the coil pointing 158 backwards at a 45-degree to the midline to induce an approximate posterior -anterior 159 current. The TMS-hotspot was defined as the area of the scalp where the largest and 160 most stable MEPs were observed in the right first dorsal interosseus (FDI) muscle [18] 161 (Fig. 1A). Apart from the setup in the original work [1], neuronavigation was used to 162 ensure precise and consistent placement of TMS both before and after 5Hz -163 rTUS/sham (Brainsight, Rogue Research, USA). We additionally recorded MEPs from 164 adjacent abductor pollicis brevis (APB) and abductor digiti minimi (ADM) muscles, to 165 capture any potential effects of the ultrasound beam on other intrinsic hand muscles 166 (Fig. 1A). 167 Our TMS procedures followed the original study [1]. Briefly, after identifying the 168 TMS hand motor hotspot, we determined the baseline rMT and the stimulator intensity 169 (SI) required to elicit a ~1mV MEP (SI1mV) [18, 19] . This baseline SI 1mV was the 170 intensity for all subsequent single -pulse MEP blocks to assess changes in CSE. For 171 the paired -pulse TMS block capturing intracortical excitability, the intensity of the 172 conditioning stimulus was set to 80% rMT for SICI (2 ms ISI) and ICF (10 ms ISI), and 173 the test stimulus was set to the SI 1mV measured in the beginning of the same block. 174 Paired-pulse trials were administered in pseudorandomized order. In contrast to Zeng 175 and co-workers` work [1], we acquired 25 trials for each measurement – originally 20 176 and 10 trials for each single- and paired-pulse metric, respectively – using an ISI of 5 177 seconds and a 10% jitter. All TMS measures were acquired at baseline , T5, T30 and 178 T60 (Fig. 1D). Each TMS block lasted approximately nine minutes. 179 Surface electromyography (EMG) signals were simultaneously recorded for 180 target FDI, APB and ADM with pairs of surface electrodes in a belly -tendon montage 181 (WhiteSensor 40713, AmbuR, Denmark; Fig. 1A). Signals were amplified with a gain 182 of 1000, bandpass filtered (5 Hz - 3000 Hz) by a Digitimer D360 amplifier (Digitimer 183 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 26, 2024. ; https://doi.org/10.1101/2024.11.25.625187doi: bioRxiv preprint 9 Ltd, Welwyn Garden City, Hers, UK), and digitised at 5000 Hz by a Power 1401 data 184 acquisition interface and Signal software version 7.01 (Cambridge Electronic Design 185 Ltd., Cambridge, UK). 186 Transcranial ultrasound stimulation 187 Transcranial ultrasound stimulation (TUS) was delivered using the NeuroFUS 188 system (manufacturer: Sonic Concepts Inc., Bothell, WA, USA; supplier/support: 189 Brainbox Ltd., Cardiff, UK) via a four -element 500 kHz annular array piezoelectric 190 transducer with a 64 mm radius of curvature and aperture diameter (CTX -500-025, 191 Sonic Concepts Inc., Bothell, WA, USA). A four -channel radiofrequency amplifier 192 (Transducer Power Output system; TPO) powered the transducer via a four -channel 193 electrical impedance matching network utilising a rectangular pulse shape. The 194 transducer was coupled with a 10 mm gel pad (Aquaflex, Parker, Laboratories, NJ, 195 USA). Ultrasound gel (Aquasonic 100, Parker Laboratories, NJ, USA) was centrifuged 196 to remove visible bubbles and applied between the gel pad and the transducer. We 197 defined our sonication depth as 33 mm in accordance with the acoustic field peak 198 reported previously [1, 12]. To reach 33 mm, the sonication depth on the TPO was set 199 at 43.5 mm to account for the additional distance added by ultrasound gel (0.5mm) 200 and gel pad (10mm). Table S1 and Figure S1 show the comparison of the acoustic 201 intensities and profiles between Zeng et al. (2022) and the current study. The applied 202 focal depth in the present study and the original study [1] matches the scalp-to-cortex 203 distance of ~30mm for the M1 omega formation. 204 The 5Hz-rTUS protocol was an 80 -second train of 20 -millisecond ultrasound 205 pulses repeated every 200 milliseconds (PRF 5Hz; 10% duty cycle), for a total of 400 206 pulses (Fig. 1B). The spatial-peak-pulse average intensity in free water (ISPPA) was set 207 to 10 W/cm2 (Table S1), similar to the original work. Applying an estimated 75% skull 208 attenuation relative to free -water measurements, our estimated transcranial I SPPA 209 (2.5W/cm2) was consistent with the original study (2.26W/cm2) [1]. Our actual 210 estimated transcranial ISPPA based on acoustic simulations was 1.20W/cm2 ± 0.43. 211 The TUS transducer location was determined by the TMS -hotspot, as 212 previously [1, 9-11, 13, 14]. The contours of the TMS coil were marked on the scalp 213 using a chinagraph pencil and the centre of the TMS coil was measured (Fig. 1C). We 214 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 26, 2024. ; https://doi.org/10.1101/2024.11.25.625187doi: bioRxiv preprint 10 additionally recorded the TUS transducer location using neuronavigation for post hoc 215 acoustic simulations. We also added a double -blind procedure, where both 216 experimenter and participant were blind to which TUS condition (5Hz-rTUS vs sham) 217 was administered, to minimise potential experimenter bias. To enable effective double-218 blinding, an independent researcher randomly assigned the TUS condition order 219 across participants, using a MATLAB script. TUS parameters were automatically input 220 into the TPO based on the defined participant and session number. Hair preparation 221 with centrifuged ultrasound gel was initially performed after threshold and hotspot 222 estimation, and finalised after the baseline TMS block to minimise preparation time 223 between baseline TMS and TUS application. During both 5Hz-rTUS and sham , 224 Gaussian white noise was played through bone -conductive headphones ( Sportz3, 225 AfterShokz, New York, USA) to maximise blinding of each condition [4]. Participants 226 selected the maximum volume for the white noise that they found acceptable. 227 Data preprocessing and analysis 228 Raw EMG data were exported from Signal (version 7.01; Cambridge Electronic 229 Design, UK) to MATLAB (version 9.7.0; R2019b). Peak -to-peak MEP amplitudes for 230 each muscle (FDI, APB, ADM) were calculated using a custom script. Data and code 231 to reproduce the results are provided here: https://doi.org/10.17605/OSF.IO/S5AG6. 232 Within each block, trials identified as significant outliers using Grubbs’ test (1.61%) 233 and trials with pre-contraction (1.78%) were excluded. Precontraction was defined as 234 the root mean square (RMS) of EMG activity in the 100ms prior to TMS exceeding the 235 block’s average by two standard deviations. All trials with an RMS exceeding a liberal 236 threshold of 0.045 were manually inspected (n = 10427/36000; 29%). Trials where 237 noise in the EMG signal prevented sufficient quantification of MEP amplitude were 238 excluded (0.55% across all muscles, 0% for FDI specifically). 239 Paired t -tests were conducted to assess baseline differences between 5Hz-240 rTUS and sham for rMT, SI1mV, MEP amplitude, SICI, and ICF. The MEP amplitudes 241 for paired-pulse measures were expressed as a ratio to the mean TS. 242 To assess main effects and interactions with maximal statistical power, linear 243 mixed models (LMMs) with a maximal random effects structure were fitted using the 244 lme4 package in R [20, 21]. Statistical significance was set at a two-tailed α=0.05 and 245 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 26, 2024. ; https://doi.org/10.1101/2024.11.25.625187doi: bioRxiv preprint 11 computed with t -tests using the Satterthwaite approximation of degrees of freedom. 246 Given the right -skewed nature of MEP amplitudes, trial -level square root corrected 247 MEP amplitudes were used for LMMs. The time course of MEP amplitudes, SICI, and 248 ICF was tested separately with models including TUS Condition (5Hz-rTUS/sham) and 249 Timepoint (Baseline/T5/T30/T60) as factors. TUS -induced changes in corticospinal 250 excitability were further tested for MEP amplitudes expressed as a ratio to the baseline 251 mean (i.e. baseline corrected), with TUS Condition (5Hz -rTUS/sham) and Timepoint 252 (T5/T30/T60) as factors. 253 In addition to LMMs, the previously implemented statistical procedures of the 254 original study [1] were replicated using two -way rm -ANOVA on raw data without 255 square root correction, with factors TUS Condition (5Hz -rTUS/sham) and Timepoint 256 (Baseline/T5/T30/T60; see Supplementary Results). 257 Acoustic Simulations 258 To determine the actual anatomical location targeted by TUS , we conducted 259 simulations of acoustic wave propagation for each individual, using k-Plan software, a 260 user interface for the pseudospectral time -domain solver k -Wave [22]. To generate 261 compatible skull images, a toolbox with pre-trained deep learning convolutional neural 262 networks was used to convert T1-weighted MRI scans to pseudo-CT images [23]. Next, 263 the four-element CTX500 transducer location was imported from the neuronavigation 264 TUS trajectory captured during the real 5Hz-rTUS session (exported as 3D 265 coordinates in Brainsight coordinate space). The simulation was run using six grid 266 points per wavelength for a single pulse duration to obtain the steady -state pressure 267 field. 268 Using a custom MATLAB script, the k-Plan pressure field and grid settings were 269 extracted using k -plan-matlab-tools (https://github.com/ucl -bug/k-plan-matlab-tools). 270 We resampled the simulation grid to have the same spacing as the anatomical scans 271 (i.e., T1w and pseudo-CT). Pulse average intensity was calculated from the simulated 272 acoustic pressure amplitude p, using the plane wave approximation I= p^2/2ρc, were 273 the density ρ was 1000 kg/m3 and the sound speed c was 1500 m/s. 274 We were primarily interested in determining the intersection between the 275 acoustic focus and the anatomical location of the M1 hand area within the vicinity of 276 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 26, 2024. ; https://doi.org/10.1101/2024.11.25.625187doi: bioRxiv preprint 12 the precentral gyrus. For this, we first segmented s tructural MRI scans into different 277 tissue types, using SPM12 (https://www.fil.ion.ucl.ac.uk/spm/software/spm12/). Grey 278 matter, white matter, and cerebrospinal fluid masks were merged to generate a binary 279 brain mask per subject. In native space, we identified the omega formation in the pre-280 central gyrus at a depth of ~30mm, and the lip of the precentral gyrus at a depth of 281 ~18mm [15, 24]. We extracted the location and value of the I SPPA for each individual 282 and calculated the Euclidean distance between the ISPPA location and these two 283 anatomical landmarks (Fig. 4E). We defined the acoustic focus as the volume where 284 the intensity values are equal to or higher than half of the intensity maximum in the 285 brain, equivalent to the full-width half-maximum intensity. Table S2 reports the 286 simulation of free water sonication and thermal simulation results, in line with recent 287 reporting guidelines [25]. K-Plan was also used to run a thermal simulation for one 288 representative participant for a full 80 seconds PTD with no cooling time. 289 For group -level plots, the T1 -weighted MRI scans and intensity maps were 290 resampled in MNI standard space at a 1 mm isometric resolution. The binary acoustic 291 focus maps in standard space were summed and overlaid on a standard brain with 292 MRIcroGL. To quantify targeting accuracy through spatial overlap between the 293 acoustic focus and the target , we required a volumetric definition of the M1 target. 294 Therefore, we first delineated M1 with a 15 mm radius spherical region of interest (ROI) 295 that covered both the lip of the precentral gyrus and the omega formation ( Fig. S2). 296 This ROI was chosen because stimulation of both these areas, and likely intermediate 297 regions as well, can elicit effects of TUS on CSE [15]. W e then calculated the 298 percentage of the acoustic focus that fell within the ROI, as well as the peak ultrasound 299 intensity within the ROI. 300 301

302 None of the participants reported discomfort or adverse effects following 303 participation. Participants could not hear 5Hz-rTUS over the auditory mask, nor could 304 they distinguish between the 5Hz-rTUS and sham sessions. 305 No significant difference in baseline physiological measures 306 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 26, 2024. ; https://doi.org/10.1101/2024.11.25.625187doi: bioRxiv preprint 13 At baseline, no differences were observed for MEP amplitude (t(14)=0.837, p=.417, 307 d=0.216), SICI (t(14)=-0.619, p=0.546, d=-0.160), or ICF (t(14)=-0.724, p=0.481, d=-308 0.187; Fig. S3). Moreover, there were no differences between baseline 5Hz-rTUS and 309 sham conditions for rMT (t-tests; t(14)=0.811, p=0.431, d=0.209) or SI1mV (t(14)=1.353, 310 p=0.198, d=0.349; Fig. S3). 311 No significant effect of 5Hz-rTUS on corticospinal or intracortical excitability 312 To examine the time course of corticospinal excitability changes in the primary 313 muscle of interest (FDI), a linear mixed model was fitted predicting square root 314 corrected MEP amplitude by Condition ( 5Hz-rTUS/sham), Timepoint 315 (Baseline/T5/T30/T60) and their interaction. No significant effects were observed 316 (Timepoint: F(3,14)=1.757, p=0.201, η ₚ² =0.273; Condition: F(1,14)=1.855, p=0.195, 317 ηₚ² =0.117 Timepoint*Condition: F(3,14)=0.236, p=0.87, ηₚ² =0.048). When expressing 318 MEP amplitude as a ratio to baseline, we similarly found no evidence of 319 neuromodulation (Timepoint: F(2,14)=1.712, p=0.216, η ₚ² =0.195; Condition: 320 F(1,14)=0.687, p=0.421, η ₚ² =0.047; Timepoint*Condition: F(2,14)=0.013, p=0.988, 321 ηₚ² =0.002; Fig. 2). 322 As shown in F ig. 3 , t here were also no significant effects for either SICI 323 (Timepoint: F(3,14)=0.648, p=0.597, η ₚ² =0.12; Condition: F(1, 14)=0.187, p=0.672, 324 ηₚ² =0.013; Timepoint*Condition: F(3,18)=0.479, p=0.701, η ₚ² =0.075) or ICF 325 (Timepoint: F(3,15)=0.8, p=0.513, η ₚ² =0.141; Condition: F(1,14)=2.773, p=0.118, 326 ηₚ² =0.165 Timepoint*Condition: F(3,21)=0.099, p=0.96, η ₚ² =0.014). RM-ANOVAs 327 conducted on raw MEP amplitudes, following the same analyses as Zeng and co-328 workers, also did not reveal any significant effects [1] (see Supplementary Information 329 1). For the adjacent APB and ADM muscles , there was also no evidence for offline 330 excitatory effects of sonication (Supplementary Information 2 -3, Fig. S4-7). 331 Collectively, we did not observe any reliable effect of 5Hz-rTUS on CSE or intracortical 332 excitability. 333 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 26, 2024. ; https://doi.org/10.1101/2024.11.25.625187doi: bioRxiv preprint 14 334 Fig. 2. No significant effect of 5Hz-rTUS on MEP amplitude. 335 A. There was no significant effect of 5Hz-rTUS on corticospinal excitability. MEP amplitudes 336 are expressed as a ratio to baseline for each timepoint. Points and error bars represent 337 group mean ± standard error. 338 B. Participant-level data. 339 340 341 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 26, 2024. ; https://doi.org/10.1101/2024.11.25.625187doi: bioRxiv preprint 15 Fig. 3. No significant effect of 5Hz-rTUS on SICI or ICF 342 A. For both SICI and ICF, there are no significant differences between sham and TUS at 343 any time point. Data: Mean ± standard error. 344 B. Participant-level data. 345 346 No significant effect of 5Hz-rTUS on rMT or the stimulator intensity required to 347 elicit 1mV amplitude MEPs 348 There was no significant effect of sonication on resting motor thresholds 349 (Timepoint: F(3,42)=2.711, p=0.057, np2=0.162; Condition: F(1,14)=0.546, p=0.472, 350 np2=0.038; Timepoint*Condition: F(3,42)=0.319, p=0.812, np2=0.022 ) nor SI1mV 351 ( Timepoint: F(1.6,22)=0.68, p=0.485, np2=0.046; Condition: F(1,14)=0.303, p=0.591, 352 np2=0.021; Timepoint*Condition: F(2.1,29)=1.737, p=0.192, np2=0.11; Fig. S8). 353 Variable ultrasound targeting of M1 based on TMS hotspot location 354 It is possible that ultrasound targeting of M1 based on the scalp location of the 355 TMS hotspot is variable, and thereby may reduce the consistency and overall efficacy 356 of 5Hz-rTUS. To address this, we conducted post-hoc simulations of the sonication 357 target based on individual head models and assessed the degree of targeting variance 358 in our population. 359 First, we observed t hat previous reports may have overestimated acoustic 360 transmission, where the reported transcranial I SPPA (2.26-2.93 W/cm2) was estimated 361 by uniformly applying 75% attenuation from free-water ISPPA (9.04-11.72 W/cm2; Table 362 S1) [1, 9, 11, 13] . Here, we find a mean±sd transcranial ISPPA of 1.2±0.4 W/cm 2, 363 corresponding to a ~12% transmission rate, in line with empirically observed and 364 theoretical estimations of percentage intensity transmission at f=500 kHz [15, 16]. 365 Critically, our simulations reveal substantial variability in the location of the 366 acoustic focus across participants (Fig. 4A,B). The acoustic focus overlapped with the 367 M1 ROI in only 7 out of 15 participants. Only 33% of participants had more than 20% 368 of the acoustic focus volume in the M1 ROI. In those 33% of subjects, the maximum 369 intensity within the ROI was 1.0 ± 0.3 W/cm2 (Fig. 4B). 370 In native space, the Euclidean distance from the target omega formation seed 371 voxel to the peak intensity voxel (ISPPA) was 21.1±9.5 mm ( Fig. 4D). Along each 3D 372 axis, we performed one-sample t-tests and found a significant anteromedial shift of the 373 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 26, 2024. ; https://doi.org/10.1101/2024.11.25.625187doi: bioRxiv preprint 16 foci (Fig. 4C; anterior: t(14)=2.86, p=0.013; medial: t(14)=7.36, p=3.548). The degree 374 of anterior shift (10.5±14.2 mm) and the distribution of Euclidean distances correspond 375 with the well-known anterior shift of the TMS motor hotspot relative to the anatomical 376 hand motor area [26-30]. We furthermore observed a significant inferior shift relative 377 to the lip of the gyrus, which has a scalp -to-cortex distance of ~15 mm (t(14)=14.17, 378 p=1.079). However, there was no inferior shift relative to the targeted omega formation 379 at a depth of ~30 mm (t(14)=1.63, p=0.126 ; Fig. 4E,F). Taken together, our results 380 demonstrate that TMS motor hotspot -based targeting method for rTUS introduces 381 considerable dispersion around the intended target M1 location. While such 382 misalignment between the hotspot scalp location and underlying anatomy is known, it 383 quantifies the degree of bias and variability in directing sonication reliably to the same 384 brain target with this procedure. 385 386 Lack of e xcitatory offline 5Hz-rTUS effects when anatomical targeting is 387 accurate 388 5Hz-rTUS might have been effective in participants in whom sonication was 389 reliably directed to the presumed anatomical target region. We therefore identified 390 those participants where the acoustic focus overlapped with the M1 ROI by >20%. 391 However, in none of the 5 participants meeting this criterion did we observe an 392 excitatory effect of 5Hz -rTUS on single -pulse MEP amplitude (Fig. 4 G). Given the 393 previously reported effect sizes [1], one may expect these participants to show an 394 excitatory effect; this was not observed here. Finally, it is conceivable that there is a 395 systematic relationship between the targeting accuracy and the sonication effect. 396 Using linear models, we found no significant relationship between the percentage 397 acoustic focus overlap and the ratio of post -TUS MEPs to baseline MEPs for any 398 timepoint (Fig. 4H; T5: b=-0.002, t(14)=-1.483, p=0.162; T30: b=-4.194, t(14)=-0.175, 399 p=0.864; T60: b=6.948, t(14)=0.342, p=0.738). Taken together, even when sonication 400 is directed to M1 as intended, the excitatory effects of 5HzrTUS did not replicate. 401 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 26, 2024. ; https://doi.org/10.1101/2024.11.25.625187doi: bioRxiv preprint 17 402 Fig. 4. Targeting precision and offline excitatory effects of 5Hz-rTUS 403 A. Anatomical region -of-interest (15 mm M1 sphere) and variability in focus location 404 across subjects 405 B. The percentage of the acoustic focus in the M1 ROI exceed ed 20% in only 5/15 406 participants (dark grey). Maximum ultrasound intensities within the ROI are depicted 407 in light grey. 408 C. TMS motor hotspot-based targeting leads to an anteromedial shift of the ISPPA. 409 D. Distribution of Euclidean distances between the omega formation in the precentral 410 gyrus and the ISPPA in native space. 411 E. The lip/crown of the pre-central gyrus is at a scalp-to-cortex distance of ~18 mm, while 412 the omega formation we targeted in this study is at a depth of ~30 mm. 413 F. There is a significant inferior shift relative to the lip/crown of the gyrus. 414 G. Even when M1 was precisely targeted, there were no significant effects of sonication 415 on corticospinal excitability. 416 H. No significant association between targeting accuracy and MEP amplitude changes. 417 418 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 26, 2024. ; https://doi.org/10.1101/2024.11.25.625187doi: bioRxiv preprint 18

419 Neuromodulation with TUS holds promise for clinical interventions, due to its 420 spatial precision and potential for targeting deeper brain structures. Replication is an 421 essential process for scientific rigour and has been earmarked by the International 422 Transcranial Ultrasonic Stimulation Safety and Standards (ITRUSST) as crucial for 423 accelerating TUS toward an effective neuromodulation approach. There is indeed 424 growing attention to the rigorous experimental control required to demonstrate 425 replicability in the field of focused ultrasound [25, 31, 32] . This need is further 426 highlighted by examples from the fields of electrical and magnetic stimulation, where 427 initial reports of novel stimulation protocols have often been followed by more nuanced 428 appraisals of their efficacy [33, 34]. 429 In the present study, we sought to replicate recently published neuromodulatory 430 effects of offline 5Hz -rTUS directed to M1[1]. Our results did not reveal a significant 431 effect of 5Hz-rTUS on corticospinal or intracortical excitability, contrasting with the 14 432 out of 15 participants showing enhanced corticospinal excitability in response to 5Hz 433 rTUS in the original study[1], and the similarly large effect sizes of the same protocol 434 in subsequent studies by the same group [1, 8-14]. 435 Variable effects of 5Hz-rTUS 436 The absence of significant effects held for the targeted FDI and the adjacent 437 APB and ADM muscles. Here we initially assessed our results using linear mixed 438 models, which have greater statistical power than rm -ANOVAs. However, o ur null 439

were the same when employing the statistical procedures as in the original 440 study [1]. Additional qualitative assessment of individual data indicated that in only 2 441 out of 15 participants of the current study were the observed MEP changes consistent 442 with the originally reported effects at T5 and T30. These results suggest the effects of 443 5Hz-rTUS protocol across different cohorts are likely more nuanced and less robust 444 than originally appreciated. 445 This conclusion aligns with a recent replication from another group showing the 446 opposite, i.e., inhibitory rather than excitatory effects lasting approximately 30 minutes 447 after offline 5Hz-rTUS [15]. How the same TUS protocol directed to the same neural 448 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 26, 2024. ; https://doi.org/10.1101/2024.11.25.625187doi: bioRxiv preprint 19 structure with the same overall approach can lead to completely opposite 449 neuromodulatory effects is currently unclear. One major difference was that Bao and 450 colleagues (2024) targeted TUS based on structural landmarks with a priori 451 simulations at two focal depths - the lip/crown of the pre -central gyrus and one 452 targeting the deeper, omega-shaped formation of the pre -central gyrus . Inhibitory 453 effects of 5Hz-rTUS were observed at both stimulation depths. 454 While one cannot rule out that relatively subtle differences in applied sonication 455 intensity between the two studies explain the opposite effects on excitability , a 456 complete reversal of the neuromodulatory effects with such subtle intensity variation 457 raises concerns about the clinical utility of such protocols, and certainly provide s a 458 mandate for further independent replication. Moreover, other neuromodulatory 459 protocols used in both animal work and human research, including the 5Hz -rTUS 460 protocol, have typically required intensities that were at least ~300% higher (>30 461 W/cm2; 5Hz-rTUS: [35-40]. Regardless of the mechanistic explanation for how 5Hz -462 rTUS at very low intensities might elicit such strong neuromodulatory effects, our 463 findings here and those by Bao and colleagues (20024) suggest that across different 464 cohorts, the effects of low-intensity 5Hz-rTUS are more variable and potentially even 465 orthogonal to the ones initially reported. 466 467 Variability in targeting with TMS motor hotspot-based transducer placement 468 One factor that will determine the effects of TUS is the precision and 469 consistency of its targeting. To target the primary motor cortex in combined TUS-TMS 470 experiments, the TMS motor hotspot is commonly used as a heuristic to determine the 471 TUS transducer location on the scalp that sonicates M1 [1, 3, 41-43]. However, this 472

poses obvious risks of poor target exposure to TUS. 473 First, when using TMS over M1, the location of the largest electrical fields and 474 neural activation thresholds depend on several factors, including cortical folding, tissue 475 type, and induced current direction [44, 45]. Furthermore, cortical areas closer to the 476 lateral surface will always experience stronger electrical fields than deeper parts, due 477 to the rapid decay of the induced electric field with increasing distance from the TMS 478 coil [44, 45]. Therefore, the direct axial projection from the geometrical centre of the 479 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 26, 2024. ; https://doi.org/10.1101/2024.11.25.625187doi: bioRxiv preprint 20 TMS coil (marked on the scalp), which is often used to determine the scalp position of 480 the TUS transducer , is unlikely to be a reliable marker of either a specific neural 481 structure, or the anatomical site of effective stimulation with TMS. Considering that the 482 width of the sonication beam is considerably smaller than the spatial specificity of TMS 483 [41, 42], even small deviations of the TUS transducer in the axial plane will lead to 484 mis-targeting. Compared to TMS, where an altogether much larger region of the brain 485 is targeted, this will increase the probability of directing sonication to different cortical 486 targets across individuals. 487 Indeed, our post -hoc acoustic simulations demonstrate such inter-individual 488 variability in the location of the acoustic focus relative to M1 , where only 5/15 of 489 participants showed a substantial degree of overlap between sonication and the M1 490 target. Scalp-based transducer placement can thus introduce inter-individual variation 491 in the specific targeted pre- or postcentral elements . Such variation aligns with the 492 known complexity of mapping the TMS-hotspot on the scalp to a specific anatomical 493 target, e.g., a specific section of the omega -shaped hand knob [26-30]. Interestingly, 494 the only study that used personalised targeting to eliminate this variability reported 495 inhibitory effects of 5Hz-rTUS [15]. 496 Neuronavigation and double-blinding may explain failure to replicate 497 While differences in transducer model may come to mind as a possible 498 explanation for the different findings , w e would argue that the collapse from 499 neuromodulatory effects in 14/15 participants [1] to non -significance cannot be 500 explained solely by a difference in transducers. The axial intensity profiles between 501 our four-element transducer and the two -element transducer used previously [1] are 502 similar, apart from a near-field peak at 12 mm for the original two-element transducer 503 that is too superficial to reach the brain ( Fig. S1). If the neuromodulatory effects of 504 5Hz-rTUS were indeed entirely dependent on these very small variations in the 505 pressure fields, it would require extraordinary precision in targeting the same neural 506 structures consistently in every subject. This seems unlikely. 507 The key differences between the original work [1] and the present study were 508 the inclusion of TMS neuronavigation and double-blinding. Using only markings on the 509 scalp, precise TMS repositioning is challenging, particularly in terms of orientation . 510 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 26, 2024. ; https://doi.org/10.1101/2024.11.25.625187doi: bioRxiv preprint 21 Without neuronavigation, slight trial -by-trial shifts in TMS position can bias MEP 511 amplitudes [46, 47]. Further, the approach is prone to circular reasoning if the MEPs, 512 the dependent variable, are themselves used to determine repositioning of the TMS 513 coil. In combination with unblinded researchers, th e lack of neuronavigation and 514 double-blinding risks introduction of unconscious confirmation bias . The changes to 515 the original work we introduced minimise such bias. It would seem prudent to suggest 516 that inclusion of neuronavigation for TMS positioning and TUS targeting, and double-517 blinding should become standard ingredients of future TUS-TMS investigations. 518 Novel approaches to neuromodulation require realistic assessment of their 519 efficacy, to develop methodological standards, direct further development, and 520 ultimately help accelerate clinical use. A key component for this is independent 521 replication. Our replication results here reappraise the effect sizes of excitatory 522 neuromodulation of 5Hz -rTMS to M1 previously reported , but also suggest avenues 523 toward consistent and reproducible evaluation of novel TUS protocols. 524 525 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 26, 2024. ; https://doi.org/10.1101/2024.11.25.625187doi: bioRxiv preprint 22 Data Statement 526 Data and code to reproduce the results reported in this study are available at: 527 https://doi.org/10.17605/OSF.IO/S5AG6 528 529 Declaration of competing interest 530 No conflicts of interest. 531 532

533 We thank Paul Hammond for their help and assistance. 534 Funding 535 Author Funder Grant Reference Number John C Rothwell Medical Research Council UK MR/P006671/1 Benjamin Kop, Lennart Verhagen Nederlandse Organisatie voor Wetenschappelijk Onderzoek VIDI 18919 Carys Evans, Lennart Verhagen European Innovation Council Pathfinder 101071008 Carys Evans, Sven Bestmann Dunhill Medical Trust RPGF1810\93 536 Contributions 537 Po- Yu Fong: investigation, conceptualisation, methodology, data curation, writing – 538 original draft, writing – review & editing , Resources, Supervision (the investigation 539 team). 540 Benjamin Kop: conceptualisation, methodology, software, formal analysis, data 541 curation, writing – original draft, writing – review & editing, visualisation. 542 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 26, 2024. ; https://doi.org/10.1101/2024.11.25.625187doi: bioRxiv preprint 23 Carys Evans: conceptualisation, methodology, writing – original draft, writing – review 543 & editing, visualisation. 544 Vidya Gani Wijaya: investigation 545 Yongling Lin: investigation 546 Drew Cappotto: investigation 547 Jenny S. A. Lee: investigation 548 Anna Latorre: investigation 549 Joy Song: investigation 550 Bradley Treeby: writing – Resources, review & editing 551 Eleanor Martin: writing – Resources, review & editing 552 John Rothwell: Funding acquisition, Resources, Supervision 553 Lennart Verhagen : Methodology, Writing - review & editing, Funding acquisition, 554 Conceptualisation, Supervision 555 Sven Bestmann: Supervision, conceptualisation, methodology, writing – original draft, 556 writing – review & editing 557 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 26, 2024. ; https://doi.org/10.1101/2024.11.25.625187doi: bioRxiv preprint 24

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