Kilohertz Transcranial Magnetic Perturbation (kTMP): A New Non-invasive Method to Modulate Cortical Excitability

Reviewer #1 (Public Review):

Summary:

This paper reports the first results on the effects of a novel waveform for weak transcranial magnetic stimulation, which they refer to as "perturbation" (kTMP). The waveform is sinusoidal at kHz frequency with subthreshold intensities of 2V/m, instead of the suprathreshold pulses used in conventional TMS (~100V/m). The effect reported here concerns motor-evoked potentials (MEPs) elicited on the hand with single-pulse TMS. These MEPs are considered a marker of "corotico-spinal excitability. The manuscripts report that kTMP at 3.5kHz enhances MEPs with a medium effect size, and reports independent replications of this fining on 3 separate cohorts of subjects (N=16, 15, 16). This result is important for the field of non-invasive brain stimulation. The evidence in support of this claim is compelling.

Strengths:

• This is a novel modality for non-invasive brain stimulation.

• Knowing the history in this field, is likely to lead to a large number of follow-up studies in basic and clinical research.

• The modality cases practically no sensation which makes it perfectly suitable for control conditions. Indeed, the study itself used a persuasive double-blinding procedure.

• The replication of the main result in two subsequent experiments is very compelling.

• The effect size of Cohen's d=0.5 is very promising.

• It is nice the E-fields were actually measured on a phantom, not just modeled.

Weakness:

• The within-subject design may have carry-over effects, although a 2-day gap is probably enough for washout.

• It would have been nice to assess washout by comparing the per-conditions between days. Particularly problematic are the paired-pulse effects that are done within sessions in experiments 2 and 3 which could have carried over to the main metric of interest, which was the single pulse MEP.

• Statistical analysis combining Experiments 1, 2, and 3 is a little muddled.

• Related, the biorxiv version history of this work as experiments 1-3 came together to point to diverging results, and changing analysis methods. Specifically, an earlier version of the work claims that modulated kHz sinusoids are more effective than un-modulated sinusoids, yet the current version says that no differences were detected - which seems consistent with the data presented in this version. However, it does raise concerns about analytic methods, which seem to have shifted over time.

• While sensation has been documented nicely, it does not seem like blinding has been directly assessed, by asking participants at the end which group they thought to be in.

Reviewer #2 (Public Review):

Summary:

kTMP is a novel method of stimulating the brain using electromagnetic fields. It has potential benefits over existing technology because it is safe and easy. It explores a range of brain frequencies that have not been explored in depth before (2-5kHz) and thus offers new opportunities.

Strengths:

This work relied on standard methods and was carefully and conservatively performed.

Weaknesses:

The sham condition was prepared as well as could be done, but sham is always challenging in a treatment with sound and sensation and with knowledgeable operators. New technology, also, is very exciting to subjects and it is difficult to achieve a natural experiment. These difficulties are related to the technology, however, and not to the execution of these experiments.

Author Response

We provide here a provisional response to the Public Comments and main issues raised by the reviewers. We appreciate the opportunity to submit a revision and will give all of the reviewers’ comments careful consideration when modifying the manuscript.

(1) BioRxiv version history.

Reviewer 1 correctly noted that we have posted different versions of the paper on bioRxiv and that there were significant changes between the initial version and the one posted as part of the eLife preprint process. Here we provide a summary of that history.

We initially posted a bioRxiv preprint in November, 2021 (Version 1) that included the results of two experiments. In Experiment 1, we compared conditions in which the stimulation frequency was at 2 kHz, 3.5 kHz, or 5.0 kHz. In Experiment 2, we replicated the 3.5 kHz condition of Experiment 1 and included two amplitude-modulated (AM) conditions, with a 3.5 kHz carrier signal modulated at 20 Hz or 140 Hz. Relative to the sham stimulation, non-modulated kTMP at 2 kHz and 3.5 kHz resulted in an increase in cortical excitability in Experiment 1. This effect was replicated in Experiment 2.

In the original posting, we reported that there was an additional boost in excitability in the 20 Hz AM condition above that of the non-modulated condition. However, in re-examining the results, we recognized that the 20 Hz AM condition included an outlier that was pulling the group mean higher. We should have caught this outlier in the initial submission given that the resultant percent change for this individual is 3 standard deviations above the mean. Given the skew in the distribution, we also performed a log transform on the MEPs (which improves the normality and homoscedasticity of MEP distributions) and repeated the analysis. However, even here the participant’s results remained well outside the distribution. As such, we removed this participant and repeated all analyses. In this new analysis, there was no longer a significant difference between the 20 Hz AM and nonmodulated conditions in Experiment 2. Indeed, all three true stimulation conditions (nonmodulated, AM 20 Hz, AM 140 Hz) produced a similar boost in cortical excitability compared to sham. Thus, the results of Experiment 2 are consistent with those of Experiment 1, showing, in three new conditions, the efficacy of kHz stimulation on cortical excitability. But the results fail to provide evidence of an additional boost from amplitude modulation.

We posted a second bioRxiv preprint in May, 2023 (Version 2) with the corrected results for Experiment 2, along with changes throughout the manuscript given the new analyses.

Given the null results for the AM conditions, we decided to run a third experiment prior to submitting the work for publication. Here we used an alternative form of amplitude modulation (see Kasten et. al., NeuroImage 2018). In brief, we again observed a boost in cortical excitability in from non-modulated kTMP at 3.5 kHz, but no additional effect of amplitude modulation. This work is included in the third bioRrxiv preprint (Version 3), the paper that was submitted and reviewed at eLife.

(2) Statistical analysis.

Reviewer 1 raised a concern with the statistical analyses performed on aggregate data across experiments. We recognize that this is atypical and was certainly not part of an a priori plan. Here we describe our goal with the analyses and the thought process that led us to combine the data across the experiments.

Our overarching aim is to examine the effect of corticospinal excitability of different kTMP waveforms (carrier frequency and amplitude modulated frequency) matched at the same estimated cortical E-field (2 V/m). Our core comparison was of the active conditions relative to a sham condition (E-field = 0.01 V/m). We included the non-modulated 3.5 kHz condition in Experiments 2 and 3 to provide a baseline from which we could assess whether amplitude modulation produced a measurable difference from that observed with non-modulated stimulation. Thus, this non-modulated condition as well as the sham condition was repeated in all three experiments. This provided an opportunity to examine the effect of kTMP with a relatively large sample, as well as assess how well the effects replicate, and resulted in the strategy we have taken in reporting the results.

As a first step, we present the data from the 3.5 kHz non-modulated and sham conditions (including the individual participant data) for all three experiments in Figure 4. We used a linear mixed effect model to examine if there was an effect of Experiment (Exps 1, 2, 3) and observed no significant difference within each condition. Given this, we opted to pool the data for the sham and 3.5 kHz non-modulated conditions across the three experiments. Once data were pooled, we examined the effect of the carrier frequency and amplitude modulated frequency of the kTMP waveform.

(3) Carry-over effects

As suggested by Reviewer 1, we will examine in the revision if there is a carry-over effect across sessions (for the most part, 2-day intervals between sessions). For this, we will compare MEP amplitude in baseline blocks (pre-kTMP) across the four experimental sessions.

Reviewer 1 also commented that mixing the single- and paired-pulse protocols might have impacted the results. While our a priori focus was on the single-pulse results, we wanted to include multiple probes given the novelty of our stimulation method. Mixing single- and different paired-pulse protocols has been relatively common in the noninvasive brain stimulation literature (e.g., Nitsche 2005, Huang et al, 2005, López-Alonso 2014, Batsikadze et al 2013) and we are unaware of any reports suggested that mixed designs (single and paired) distort the picture compared to pure designs (single only).

(4) Sensation and Blinding

Reviewer 2 bought up concerns about the sham condition and blinding of kTMP stimulation. We do think that kTMP is nearly ideal for blinding. The amplifier does emit an audible tone (at least for individuals with normal hearing) when set to an intensity to produce a 2 V/m E-field. For this reason, the participants and the experimenter wore ear plugs. Moreover, we played a 3.5 kHz tone in all conditions, including the sham condition, which effectively masked the amplifier sound. We measured the participant’s subjective rating of annoyance, pain, and muscle twitches after each kTMP session (active and sham). Using a linear mixed effect model, we found no difference between active and sham for each of these ratings suggesting that sensation was similar for active and sham (Fig 8). This matches our experience that kHz stimulation in the range used here has no perceptible sensation induced by the coil. To blind the experimenters (and participants) we used a coding system in which the experimenter typed in a number that had been randomly paired to a stimulation condition that varied across participants in a manner unknown to the experimenter.

Reviewer 1 asked why we did not explicitly ask participants if they thought they were in an active or sham condition. This would certainly be a useful question. However, we did not want to alert them of the presence of a sham condition, preferring to simply describe the study as one testing a new method of non-invasive brain stimulation. Thus, we opted to focus on their subjective ratings of annoyance, pain, and finger twitches after kTMP stimulation for each experimental session.