Hippocampal and striatal responses during motor learning are modulated by prefrontal cortex stimulation

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1. 

   eLife

   Jul 31, 2020

   ###Reviewer #3:

   The manuscript by Gann et al., investigates whether theta-burst TMS stimulation (TBS) of the DLPFC can alter hippocampal and striatal activity during a sequence-learning task (SRTT). Across two experiments, the authors provide a well-powered investigation of this question using MRS and fMRI. The first experiment describes a nice approach for selecting an anatomically accurate brain region, whilst the second experiment uses a robust 4-session within-subject design. The study is clearly written and has some very interesting results, with the authors concluding that it provides the first experimental evidence that brain stimulation can alter motor learning-related functional responses in the striatum and hippocampus. As described below, I believe the interpretation of these results is overstated and would be better framed …

   ###Reviewer #3:

   The manuscript by Gann et al., investigates whether theta-burst TMS stimulation (TBS) of the DLPFC can alter hippocampal and striatal activity during a sequence-learning task (SRTT). Across two experiments, the authors provide a well-powered investigation of this question using MRS and fMRI. The first experiment describes a nice approach for selecting an anatomically accurate brain region, whilst the second experiment uses a robust 4-session within-subject design. The study is clearly written and has some very interesting results, with the authors concluding that it provides the first experimental evidence that brain stimulation can alter motor learning-related functional responses in the striatum and hippocampus. As described below, I believe the interpretation of these results is overstated and would be better framed in the context of the other changes across the brain (it was not a specific effect between DLPFC and hippocampus/striatum) and also several clear negative effects (behaviour, MRS, fMRI).

   1. Interpretation of the results given the lack of a behavioural effect: I do appreciate the authors discussion of this, however the lack of a behavioural effect makes me wonder whether the imaging results are over interpreted. Generally, I found the negative results (behaviour, MRS, aspects of connectivity) to be understated and the positive results to be overstated, even though the positive results, linking DLPFC stimulation to changes in striatum and hippocampus, required a far more nuanced analysis of the data (Figure 6). The results seem to suggest that TBS has a general (and somewhat inconsistent) effect on connectivity across the brain rather than a specific effect on DLPFC-hippocampus/striatum connectivity. Given these issues, I believe a more cautious interpretation of the results are required in which it is not concluded multiple times that DLPFC brain stimulation can alter motor learning-related functional responses in the striatum and hippocampus without making clear this was in the context of other changes across the brain and also several clear negative effects. The manuscript would be improved if a more balanced picture were provided throughout.

   2. Use of iTBS vs cTBS: By using these TMS conditions, we do not know what the normal brain activation pattern is for the sequence task. Although the authors have provided a good attempt at trying to interpret these results, as a reader it was difficult to comprehend the somewhat inconsistent results across the two TMS conditions. The manuscript would have benefited from a sham/null TMS condition.

   3. Consistency of results for experiment 1: Although this experiment provides a nice mechanism to determine TMS location, the details of these results need to be more substantial. In particular, for the conjunction analysis, the reader needs to understand how consistent this effect (Figure 1B) was across participants. Does every participant show this conjunction map or what percentage of participants show this map? This feels like an important thing to report, and then possibly base a power analysis on for Experiment 2 i.e. how many participants do we expect to see the predicted TMS results given the % of participants who show this conjunction map?

   4. Clarity of results for experiment 2: I found it difficult to follow the results for experiment 2, especially from page 17. I appreciate the authors refer to the methods but I think a little more explanation of the methodology within the results is warranted. In addition, it got pretty difficult to follow the results relating task (seq vs random), stimulation (iTBS vs cTBS) and their interaction. Maybe more informative sub-titles would help?

   5. Interpretation of BOLD activity: Given recent work (https://elifesciences.org/articles/55241 ) could the authors discuss what increases and decreases in BOLD activity represent within a learning context? Is a decrease or increase beneficial?

   -Why do the authors keep using the term 'proof of concept'?

   -Figure 7B: which line is cTBS and iTBS?

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2. 

   eLife

   Jul 31, 2020

   ###Reviewer #2:

   Gann and colleagues report the effect of iTBS and cTBS of DLPFC on GABA+, BOLD-activity, and functional connectivity during sequence learning (SRTT). Despite finding no difference between the brain stimulation conditions on behavioral performance, the authors report widespread differences in BOLD-activity and functional connectivity between intermittent and continuous TBS. The key result (reported in Figure 6), is a complex difference between the stimulation conditions and the GABA+ change in DLPFC with i) the learning-related activity in hippocampus and ii) the learning-related changes in functional connectivity between DLPFC and putamen. The authors affirm that these results are important, as they are the first to show an interaction between DLPFC stimulation, learning-related changes in MRS, and BOLD-activity change …

   ###Reviewer #2:

   Gann and colleagues report the effect of iTBS and cTBS of DLPFC on GABA+, BOLD-activity, and functional connectivity during sequence learning (SRTT). Despite finding no difference between the brain stimulation conditions on behavioral performance, the authors report widespread differences in BOLD-activity and functional connectivity between intermittent and continuous TBS. The key result (reported in Figure 6), is a complex difference between the stimulation conditions and the GABA+ change in DLPFC with i) the learning-related activity in hippocampus and ii) the learning-related changes in functional connectivity between DLPFC and putamen. The authors affirm that these results are important, as they are the first to show an interaction between DLPFC stimulation, learning-related changes in MRS, and BOLD-activity change in the hippocampus and striatum.

   The authors have undertaken a mammoth effort in running this study. The targeted brain stimulation appears to have been conducted in an exemplary fashion, and the integration of multiple MR modalities is impressive. Nonetheless, I feel that the lack of a control group in the study design is a major concern and makes interpreting the study's results challenging. In addition, I have several reservations regarding the analysis that should be addressed before this work is suitable for publication.

   General comments:

   1. This study does not include a control group, and all conclusions are drawn based on the comparison between inhibitory and excitatory TBS protocols. A control condition is necessary to put the difference between the i/cTBS differences in perspective. Without this perspective, it is challenging to interpret the directionality and magnitude of the effects reported in this study.

   2. The authors stress that the major contribution of this work is revealing the effects of DLPFC stimulation on fMRI/MRS signals during/after learning (e.g., Abstract: line 43-45; Introduction: lines 102-104; Discussion: lines 498-500, 741-743). As it is written, this work is primarily interesting to the brain stimulation community. The article would be of substantially broader interest if the authors discussed their results with respect to the contribution of DLPFC to sequence learning, rather than as an exploratory investigation into the effect of brain stimulation.

   Methodological/Analytical comments:

   1. The small volume correction analysis and reporting has several issues. Throughout the results, the authors report analyses plotted on the whole brain, and do not make reference to any small volume correction being used (except for the results reported in Table 2). However, in the methods section, the authors report that analyses were conducted using small volume corrections (10mm spheres drawn around the points reported in Supplementary table 7). There are several problems here.

   i) Most importantly, the authors use (by my count) 87 separate small volumes, and reconstructing the spheres from the coordinates in Supplemental table 7 shows that this mask covers a substantial portion of the brain. This seems highly unusual to me. However, it is not clear whether all of these small volumes were considered together, or whether they were each considered as an independent volume. In either case, the authors should report whether any of their results survive whole brain correction. Additionally, if the authors tested 87 regions independently, multiple corrections should be applied to account for all regions tested (0.05/87 = 0.00057 for the new FWE-corrected p-threshold). Alternatively, if the authors used this single mask for correction, they should provide a justification for using an analytical mask restricted in this way, which, again, seems highly unusual.

   ii) In the main text and figures, the authors should note when small volume correction is being applied.

   iii) In the whole brain figures, it should be made clear what voxels were considered for the analysis (e.g., by shading the brain that was outside the small volume).

   1. The authors appear to have done two instances of spatial smoothing (8mm before fitting the GLM (line 1184), and 6mm on the resulting statistical map (line 1224)). Again, this seems highly unusual, and given that the majority of results are conducted within small volumes, it seems smoothing to this extent would introduce unwanted levels of spatial blurring. The authors should report the total smoothness of the image for all subjects (AFNI's 3dFWHMx can do this) and consider performing the group analyses without additional smoothing applied to the statistical maps.

   2. Although Study 1 is obviously important to the manuscript, I think it is perhaps overstated and makes the present work difficult to parse. Specifically, it does not seem to be important for interpreting the effects of the stimulation during learning; rather, Study 1 is a means to localize a brain stimulation target (i.e., a methodological point). Further, in the methods section, the authors reveal that they constrained their search for a conjunctive target (connected to both Hippocampus and Striatum) to the superior frontal gyrus and middle frontal gyrus. Thus, the authors seemed to have "found what they were looking for", because they restricted their search to a fairly well circumscribed region before running this study. Taken together, the authors might consider moving these details entirely to the Methods section, and removing Study 1 and associated figure from the main text.

   3. Are there any relationships between Glx levels and fMRI effects?

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3. 

   eLife

   Jul 31, 2020

   ###Reviewer #1:

   This is a very ambitious and interesting study that uses a state-of-the art combination of multiple methods to provide new insights into functional network interactions during motor learning. However, I have several major concerns against the design and analyses that may have contributed to the overall very weak effects that are reported (mainly null effects in standard measures at the behavioural and neural network level). I also think that some of the conclusions are not justified given the partly non-significant and overall weak effects.

   1. My main concern is that no baseline stimulation condition (sham TBS) was included. The authors address this in the discussion but I cannot agree with their argumentation. Without a baseline, it is impossible to assess whether each stimulation protocol had a significant impact on the …

   ###Reviewer #1:

   This is a very ambitious and interesting study that uses a state-of-the art combination of multiple methods to provide new insights into functional network interactions during motor learning. However, I have several major concerns against the design and analyses that may have contributed to the overall very weak effects that are reported (mainly null effects in standard measures at the behavioural and neural network level). I also think that some of the conclusions are not justified given the partly non-significant and overall weak effects.

   1. My main concern is that no baseline stimulation condition (sham TBS) was included. The authors address this in the discussion but I cannot agree with their argumentation. Without a baseline, it is impossible to assess whether each stimulation protocol had a significant impact on the outcome measures. For instance, it would be plausible that both protocols had opposite effects (which is also hypothesized by the authors) which were, however, only slightly or not significant from baseline. If cTBS slightly decreases connectivity and iTBS slightly increases it, this could result in a difference between both protocols that might not be observed when contrasting each protocol against baseline. Put differently, how do we know that these changes are meaningful and significantly different from zero (baseline)? I think this is especially important in the present study since the overall effects are weak and there is no significant modulation of behavior - so the functional / behavioral relevance of the observed modulation remains unclear. I think that without the inclusion of a baseline (sham), it is very hard to interpret the data.

   2. Another main concern is that the reported effects are very weak and not properly corrected for multiple comparisons. I don't think that it is justified to apply small volume corrections for large-scale network effects and it seems that some of the results are at threshold. Given the weak effects in these analyses, in combination with the absence of any modulation in the "standard" analyses (fMRI, connectivity, behaviour, MRS only significant in exploratory post-hoc tests which are not well justified), I am not sure if the reported results are really reflecting any stimulation-induced modulations at all or mainly show some noise added by the TMS protocols. This of course affects the conclusions that can be drawn from the study.

   3. There are a number of issues with the design that might have contributed to the weak findings. These include data loss (e.g. no MRS data for the hippocampal voxel) and somehow arbitrary sample sizes that are not well justified. I am also not sure why cortical excitability measures (MEPs) were performed after TBS because this is of minor importance and delayed the start of the fMRI sessions. Given that TBS effects are expected to decrease over time, I am not sure if this was necessary. Was the potential change of the TBS effects across session taken into account (e.g., by using a parametric modulation of the TMS effect)?

   4. Given the overall weak effects the conclusions should be toned down. The discussion would further benefit from including additional work that demonstrated changes in remote subcortical regions and effective connectivity after TMS over a frontal area (e.g. Herz et al., J Neurosci 2014).

   5. There is no modulatory effect of TBS on behaviour, which is surprising in light of previous neurostimulation studies on motor learning. I think the way this is sold in the discussion is a bit odd. I guess that initially, one would have expected a behavioural modulation that should ideally be correlated with any TBS induced changes in functional connectivity (or with the MRS data). If not, how would you be able to claim behavioural relevance? In the discussion, the absence of a behavioural modulation is sold as an advantage, I think this is not justified and should be toned down. Moreover, since the authors speculate about potential influences of TBS on motor consolidation, I was wondering if consolidation was assessed (which seems to be a relevant parameter here)?

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4. 

   eLife

   Jul 31, 2020

   ##Preprint Review

   This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.

   ###Summary:

   The main concerns with the study were three-fold. First, the absence of a control group makes it hard to draw conclusions about the effects of inhibitory and excitatory TBS protocols separately, limiting the appeal of the study. Secondly, the control for multiple statistical tests is not adequate - conducting 87 independent "small volume corrected" tests will lead to an inflated family-wise error rate. Finally, all reviewers were unanimous in judging the effects to be somewhat interesting, but rather weak - …

   ##Preprint Review

   This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.

   ###Summary:

   The main concerns with the study were three-fold. First, the absence of a control group makes it hard to draw conclusions about the effects of inhibitory and excitatory TBS protocols separately, limiting the appeal of the study. Secondly, the control for multiple statistical tests is not adequate - conducting 87 independent "small volume corrected" tests will lead to an inflated family-wise error rate. Finally, all reviewers were unanimous in judging the effects to be somewhat interesting, but rather weak - with the absence of an effect on behaviour and simpler standard fMRI and connectivity measures being as remarkable as the positive effects reported.

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5. 

   Version published to 10.1101/2020.06.05.136531 on bioRxiv

   Jun 6, 2020