Dual transcranial electromagnetic stimulation of the precuneus-hippocampus network boosts human long-term memory

Reviewer #2 (Public review):

Summary:

The manuscript "Dual transcranial electromagnetic stimulation of the precuneus-hippocampus network boosts human long-term memory" by Borghi and colleagues provides evidence that the combination of intermittent theta burst TMS stimulation and gamma transcranial alternating current stimulation (γtACS) targeting the precuneus increases long-term associative memory in healthy subjects compared to iTBS alone and sham conditions. Using a rich dataset of TMS-EEG and resting-state functional connectivity (rs-FC) maps and structural MRI data, the authors also provide evidence that dual stimulation increased gamma oscillations and functional connectivity between the precuneus and hippocampus. Enhanced memory performance was linked to increased gamma oscillatory activity and connectivity through white matter tracts.

Strengths:

The combination of personalized repetitive TMS (iTBS) and gamma tACS is a novel approach to targeting the precuneus, and thereby, connected memory-related regions to enhance long-term associative memory. The authors leverage an existing neural mechanism engaged in memory binding, theta-gamma coupling, by applying TMS at theta burst patterns and tACS at gamma frequencies to enhance gamma oscillations. The authors conducted a thorough study that suggests that simultaneous iTBS and gamma tACS could be a powerful approach for enhancing long-term associative memory. The paper was well-written, clear, and concise.

Weaknesses:

(1) The study did not include a condition where γtACS was applied alone. This was likely because a previous work indicated that a single 3-minute γtACS did not produce significant effects, but this limits the ability to isolate the specific contribution of γtACS in the context of this target and memory function

(2) The authors applied stimulation for 3 minutes, which seems to be based on prior tACS protocols. It would be helpful to present some rationale for both the duration and timing relative to the learning phase of the memory task. Would you expect additional stimulation prior to recall to benefit long-term associative memory?

(3) How was the burst frequency of theta iTBS and gamma frequency of tACS chosen? Were these also personalized to subjects' endogenous theta and gamma oscillations? If not, were increases in gamma oscillations specific to patients' endogenous gamma oscillation frequencies or the tACS frequency?

(4) The authors do a thorough job of analyzing the increase in gamma oscillations in the precuneus through TMS-EEG; however, the authors may also analyze whether theta oscillations were also enhanced through this protocol due to the iTBS potentially targeting theta oscillations. This may also be more robust than gamma oscillations increases since gamma oscillations detected on the scalp are very low amplitude and susceptible to noise and may reflect activity from multiple overlapping sources, making precise localization difficult without advanced techniques.

(5) Figure 4: Why are connectivity values pre-stimulation for the iTBS and sham tACS stimulation condition so much higher than the dual stimulation? We would expect baseline values to be more similar.

(6) Figure 2: How are total association scores significantly different between stimulation conditions, but individual name and occupation associations are not? Further clarification of how the total FNAT score is calculated would be helpful.

Reviewer #3 (Public review):

Summary:

Borghi and colleagues present results from 4 experiments aimed at investigating the effects of dual γtACS and iTBS stimulation of the precuneus on behavioral and neural markers of memory formation. In their first experiment (n = 20), they found that a 3-minute offline (i.e., prior to task completion) stimulation that combines both techniques leads to superior memory recall performance in an associative memory task immediately after learning associations between pictures of faces, names, and occupation, as well as after a 15-minute delay, compared to iTBS alone (+ tACS sham) or no stimulation (sham for both iTBS and tACS). Performance in a second task probing short-term memory was unaffected by the stimulation condition. In a second experiment (n = 10), they show that these effects persist over 24 hours and up to a full week after initial stimulation. A third (n = 14) and fourth (n = 16) experiment were conducted to investigate the neural effects of the stimulation protocol. The authors report that, once again, only combined iTBS and γtACS increase gamma oscillatory activity and neural excitability (as measured by concurrent TMS-EEG) specific to the stimulated area at the precuneus compared to a control region, as well as precuneus-hippocampus functional connectivity (measured by resting-state MRI), which seemed to be associated with structural white matter integrity of the bilateral middle longitudinal fasciculus (measured by DTI).

Strengths:

Combining non-invasive brain stimulation techniques is a novel, potentially very powerful method to maximize the effects of these kinds of interventions that are usually well-tolerated and thus accepted by patients and healthy participants. It is also very impressive that the stimulation-induced improvements in memory performance resulted from a short (3 min) intervention protocol. If the effects reported here turn out to be as clinically meaningful and generalizable across populations as implied, this approach could represent a promising avenue for the treatment of impaired memory functions in many conditions.

Methodologically, this study is expertly done! I don't see any serious issues with the technical setup in any of the experiments (with the only caveat that I am not an expert in fMRI functional connectivity measures and DTI). It is also very commendable that the authors conceptually replicated the behavioral effects of experiment 1 in experiment 2 and then conducted two additional experiments to probe the neural mechanisms associated with these effects. This certainly increases the value of the study and the confidence in the results considerably.

The authors used a within-subject approach in their experiments, which increases statistical power and allows for stronger inferences about the tested effects. They are also used to individualize stimulation locations and intensities, which should further optimize the signal-to-noise ratio.

Weaknesses:

I want to state clearly that I think the strengths of this study far outweigh the concerns I have. I still list some points that I think should be clarified by the authors or taken into account by readers when interpreting the presented findings.

I think one of the major weaknesses of this study is the overall low sample size in all of the experiments (between n = 10 and n = 20). This is, as I mentioned when discussing the strengths of the study, partly mitigated by the within-subject design and individualized stimulation parameters. The authors mention that they performed a power analysis but this analysis seemed to be based on electrophysiological readouts similar to those obtained in experiment 3. It is thus unclear whether the other experiments were sufficiently powered to reliably detect the behavioral effects of interest. That being said, the authors do report significant effects, so they were per definition powered to find those. However, the effect sizes reported for their main findings are all relatively large and it is known that significant findings from small samples may represent inflated effect sizes, which may hamper the generalizability of the current results. Ideally, the authors would replicate their main findings in a larger sample. Alternatively, I think running a sensitivity analysis to estimate the smallest effect the authors could have detected with a power of 80% could be very informative for readers to contextualize the findings. At the very least, however, I think it would be necessary to address this point as a potential limitation in the discussion of the paper.

It seems that the statistical analysis approach differed slightly between studies. In experiment 1, the authors followed up significant effects of their ANOVAs by Bonferroni-adjusted post-hoc tests whereas it seems that in experiment 2, those post-hoc tests where "exploratory", which may suggest those were uncorrected. In experiment 3, the authors use one-tailed t-tests to follow up their ANOVAs. Given some of the reported p-values, these choices suggest that some of the comparisons might have failed to reach significance if properly corrected. This is not a critical issue per se, as the important test in all these cases is the initial ANOVA but non-significant (corrected) post-hoc tests might be another indicator of an underpowered experiment. My assumptions here might be wrong, but even then, I would ask the authors to be more transparent about the reasons for their choices or provide additional justification. Finally, the authors sometimes report exact p-values whereas other times they simply say p < .05. I would ask them to be consistent and recommend using exact p-values for every result where p >= .001.

While the authors went to great lengths trying to probe the neural changes likely associated with the memory improvement after stimulation, it is impossible from their data to causally relate the findings from experiments 3 and 4 to the behavioral effects in experiments 1 and 2. This is acknowledged by the authors and there are good methodological reasons for why TMS-EEG and fMRI had to be collected in sperate experiments, but it is still worth pointing out to readers that this limits inferences about how exactly dual iTBS and γtACS of the precuneus modulate learning and memory.

There were no stimulation-related performance differences in the short-term memory task used in experiments 1 and 2. The authors argue that this demonstrates that the intervention specifically targeted long-term associative memory formation. While this is certainly possible, the STM task was a spatial memory task, whereas the LTM task relied (primarily) on verbal material. It is thus also possible that the stimulation effects were specific to a stimulus domain instead of memory type. In other words, could it be possible that the stimulation might have affected STM performance if the task taxed verbal STM instead? This is of course impossible to know without an additional experiment, but the authors could mention this possibility when discussing their findings regarding the lack of change in the STM task.

While the authors discuss the potential neural mechanisms by which the combined stimulation conditions might have helped memory formation, the psychological processes are somewhat neglected. For example, do the authors think the stimulation primarily improves the encoding of new information or does it also improve consolidation processes? Interestingly, the beneficial effect of dual iTBS and γtACS on recall performance was very stable across all time points tested in experiments 1 and 2, as was the performance in the other conditions. Do the authors have any explanation as to why there seems to be no further forgetting of information over time in either condition when even at immediate recall, accuracy is below 50%? Further, participants started learning the associations of the FNAT immediately after the stimulation protocol was administered. What would happen if learning started with a delay? In other words, do the authors think there is an ideal time window post-stimulation in which memory formation is enhanced? If so, this might limit the usability of this procedure in real-life applications.

Review #1 (Public Review):

Summary:

The authors employ a combination of repetitive transcranial magnetic stimulation (intermittent theta burst-iTBS) and transcranial alternating current stimulation (gamma tACS) as an approach aimed to improve memory in a face/name/profession task.

Strengths:

The paper has many strengths. The approach of stimulating the human brain non-invasively is potentially impactful because it could lead to a host of interesting applications. The current study aims to evaluate one such exciting application. The paper contains an unusual combination of noninvasive stimulation and brain imaging data, and includes independent replication samples.

Weaknesses:

(1) It remains unclear how this stimulation protocol is proposed to enhance memory. Memories are believed to be stored by precise inputs to specific neurons and highly tuned changes in synaptic strengths. It remains unclear whether proposed neural activity generated by the stimulation reflects the activation of specific memories or generally increased activity across all classes of neurons.

(2) The claim that effects directly involve the precuneus lacks strong support. The measurements shown in Figure 3 appear to be weak (i.e., Figure 3A top and bottom look similar, and Figure 3C left and right look similar). The figure appears to show a more global brain pattern rather than effects that are limited to the precuneus. Related to this, it would perhaps be useful to show the different positions of the stimulation apparatus. This could perhaps show that the position of the stimulation matters and could perhaps illustrate a range of distances over which position of the stimulation matters.

(3) Behavioral results showing an effect on memory would substantiate claims that the stimulation approach produces significant changes in brain activity. However, placebo effects can be extremely powerful and useful, and this should probably be mentioned. Also, in the behavioral results that are currently presented, there are several concerns:

a) There does not appear to be a significant effect on the STMB task.

b) The FNAT task is minimally described in the supplementary material. Experimental details that would help the reader understand what was done are not described. Experimental details are missing for: the size of the images, the duration of the image presentation, the degree of image repetition, how long the participants studied the images, whether the names and occupations were different, genders of the faces, and whether the same participant saw different faces across the different stimulation conditions. Regarding the latter point, if the same participant saw the same faces across the different stimulation conditions, then there could be memory effects across different conditions that would need to be included in the statistical analyses. If participants saw different faces across the different stimulus conditions, then it would be useful to show that the difficulty was the same across the different stimuli.

c) Also, if I understand FNAT correctly, the task is based on just 12 presentations, and each point in Figure 2A represents a different participant. How the performance of individual participants changed across the conditions is unclear with the information provided. Lines joining performance measurements across conditions for each participant would be useful in this regard. Because there are only 12 faces, the results are quantized in multiples of 100/12 % in Figure 3A. While I do not doubt that the authors did their homework in terms of the statistical analyses, it seems as though these 12 measurements do not correspond to a large effect size. For example, in Figure 3A for the immediate condition (total), it seems that, on average, the participants may remember one more face/name/occupation.

d) Block effects. If I understand correctly, the experiments were conducted in blocks. This is potentially problematic. An example study that articulates potential problems associated with block designs is described in Li et al (TPAMI 2021, https://ieeexplore.ieee.org/document/9264220). It is unclear if potential problems associated with block designs were taken into consideration.

e) In the FNAT portion of the paper, some results are statistically significant, while others are not. The interpretation of this is unclear. In Figure 3A, it seems as though the authors claim that iTBS+gtACS > iTBS+sham-tACS, but iTBS+gtACS ~ sham+sham. The interpretation of such a result is unclear. Results are also unclear when separated by name and occupation. There is only one condition that is statistically significant in Figure 3A in the name condition, and no significant results in the occupation condition. In short, the statistical analyses, and accompanying results that support the authors’ claims, should be explained more clearly.