Medial anterior prefrontal cortex stimulation downregulates implicit reactions to threats and prevents the return of fear

  1. Eugenio Manassero
  2. Giulia Concina
  3. Maria Clarissa Chantal Caraig
  4. Pietro Sarasso
  5. Adriana Salatino
  6. Raffaella Ricci
  7. Benedetto Sacchetti

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    This study presents the useful observation that repetitive Transcranial Magnetic Stimulation (rTMS) over the medial prefrontal cortex (mPFC) is associated with immediate dampening effects of conditioned responses and generalization of these responses to similar cues. Additionally, the effects were still present one week later, in the absence of any stimulation. However, the evidence supporting the claims of the authors is incomplete. The main outcome data (skin conductance response) have been normalized and standardized in suboptimal ways and, most critically, no comparisons are being made with the strength of conditioned responses during acquisition. If the observations hold, when based on within-subject comparisons, the work will be of interest to psychologists and neuroscientists working on interventions into aberrant emotional memories.

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Abstract

Downregulating emotional overreactions toward threats is fundamental for developing treatments for anxiety and post-traumatic disorders. The prefrontal cortex (PFC) is critical for top-down modulatory processes, and despite previous studies adopting repetitive transcranial magnetic stimulation (rTMS) over this region provided encouraging results in enhancing extinction, no studies have hitherto explored the effects of stimulating the medial anterior PFC (aPFC, encompassing the Brodmann area 10) on threat memory and generalization. Here we showed that rTMS over the aPFC applied before threat memory retrieval immediately decreases implicit reactions to learned and novel stimuli in humans. These effects enduringly persisted 1 week later in the absence of rTMS. No effects were detected on explicit recognition. Critically, rTMS over the aPFC resulted in a more pronounced reduction of defensive responses compared to rTMS targeting the dorsolateral PFC. These findings reveal a previously unexplored prefrontal region, the modulation of which can efficiently and durably inhibit implicit reactions to learned threats. This represents a significant advancement toward the long-term deactivation of exaggerated responses to threats.

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  1. Version published to 10.7554/elife.85951 on eLife
  2. eLife

    Author Response

    Reviewer #2 (Public Review):

    Manassaro et al. present an extensive three-session study in which they aimed to change defensive responses (skin conductance; SCR) to an aversively conditioned stimulus by targeting medial prefrontal cortex (their words) using repetitive TMS prior to retrieval. They report that stimulating mPFC using TMS abolishes SCR responses to the conditioned stimulus, and that this effect is specific for the stimulated region and the specific CS-US association, given that SCR responses to a different modality US are not changed.

    I like how the authors have clearly attempted to control for several potential confounds by including multiple stimulation sites, measured SCR responses to several unconditioned stimuli, and applied the experiment in multiple contexts. However, several conceptual and practical issues remain that I think limit the value of potential conclusions drawn from this work.

    The first issue that I have with this study concerns the relationship between the TMS manipulation and the theoretical background the authors present in their rationale. In the introduction the authors sketch that what they call 'mPFC' is involved in regulation of threat responses. They make a convincing case, however, almost all of the evidence they present concerns the ventromedial part of the prefrontal cortex (refs 18-25). The authors then mention that no one has ever studied the effects of 'mPFC'-TMS on threat memories. That is not surprising given that stimulating vmPFC with TMS is very difficult, if not impossible. Simulation of the electrical field that develops as a consequence from the authors manipulation (using the same TMS coil and positioning the authors use) shows that vmPFC (or mPFC for that matter) is not stimulated. The authors then continue in the methods section stating that the region they aimed for was BA10. This region they presumably do stimulate, however, that does not follow logically from their argument. BA10 is anatomically, cytoarchitectonically and functionally a wholly different area than vmPFC and I wonder if their rationale would hold given that they stimulate BA10.

    We would like to thank the Reviewer for highlighting this very important point. The Reviewer is right in stating that the Brodmann area 10 (BA 10) is anatomically, cytoarchitectonically, and functionally distinct from the ventromedial PFC. As we reported in the Methods section, the coil placement over the frontopolar midline electrode (Fpz) according to the international 10‒20 EEG coordinate system directly focused the stimulation over the medial portion of the BA 10. In the literature, the aPFC is also known as the “frontopolar cortex” or the “rostral frontal cortex” and encompasses the most anterior portion of the prefrontal cortex, which corresponds to the BA 10. In line with this observation, we have corrected “medial prefrontal cortex” (mPFC) with “medial anterior prefrontal cortex” (aPFC) throughout the manuscript. We also have corrected the theoretical background and the rationale in the Introduction section by mentioning several studies that: i) Reported the involvement of the aPFC in emotional down-regulation (Volman et al., 2013; Koch et al., 2018; Bramson et al., 2020). ii) Traced anatomical connections between the medial/lateral aPFC and the amygdala (Peng et al., 2018; Folloni et al., 2019; Bramson et al., 2020). iii) Detected functional connections between the aPFC and the vmPFC during fear down-regulation (Klumpers et al., 2010). iv) Found hypoactivation, reduced connectivity, and altered thickness of aPFC in PTSD patients (Lanius et al., 2005; Morey et al., 2008; Sadeh et al., 2015; Sadeh et al., 2016). v) Revealed that strong activation of the aPFC may promote a higher resilience against PTSD onset (Kaldewaij et al., 2021) and that enhanced aPFC activity and potentiated aPFC-vmPFC connectivity is detectable after effective therapy in PTSD patients (Fonzo et al., 2017). Furthermore, we discussed our results in light of this evidence in the Discussion section. We really thank the Reviewer for this key implementation of our study.

    The second concern I have is that although I think the authors should be praised for including both sham and active control regions, the controls might not be optimally chosen to control for the potential confounds of their condition of interest (mPFC-TMS). Namely, TMS on the forehead can be unpleasant, if not painful, whereas sham-TMS or TMS applied to the back of the head or even over dlPFC is not (or less so at the very least). Given that the SCR results after mPFC TMS show exactly the same temporal pattern as the sham-TMS but with a lower starting point, one could wonder whether a painful stimulation prior to the retrieval might have already caused habituation to painful stimulation observed in SCR in consequent CS presentations. A control region that would have been more obvious to take is the lateral part of BA10, by moving the TMS coil several centimeters to the left or right, circumventing all things potentially called medial but giving similar unpleasant sensations (pain etc).

    We would also like to thank the Reviewer for bringing to light this issue and allowing us to strengthen our results. The Reviewer is right in pointing out that rTMS application over the forehead can be subjectively perceived as unpleasant, relative to other head coordinates or sham stimulation. The question of whether an unpleasant stimulation prior to the retrieval might provoke habituation to discomfort sensations and lead to weaker SCRs in the consequent CS presentations is valid and reasonable. We also thank the Reviewer for advising us to stimulate the lateral part of BA 10 as an active control site. However, given the potential involvement of the lateral BA 10 in the fear network (see previous point) and the potential risks due to the anatomical proximity of lateral BA 10 with the temporal lobe, we reasoned to adopt an alternative approach to investigate whether “a painful stimulation prior to the retrieval might have already caused habituation to painful stimulation observed in SCR in consequent CS presentations”. We repeated the entire experiment in one further group (ctrl discomfort, n = 10) by replacing the rTMS procedure with a 10-min discomfort-inducing procedure over the same site of the forehead (Fpz) to mimic the rTMS-evoked unpleasant sensations in the absence of neural stimulation effects (see the new version of the Methods section). The electrical stimulation intensity was individually calibrated through a staircase procedure (0 = no discomfort; 10 = high discomfort). The shock amplitude was set at the current level corresponding to the mean rating of ‘4’ on the subjective scale because, in the new experiments that we performed targeting the aPFC with rTMS (n = 9), we collected participants’ rTMS-induced discomfort ratings obtaining a mean rating of 3.833 ± 0.589 SEM on the same scale. We found CS-evoked SCR levels not significantly different to those of the sham group during the test session as well as during the follow-up session, suggesting that the discomfort experienced during the rTMS procedure did not contribute to the reduction of electrodermal responses observed in the aPFC group. We reported the results of this experiment in the Results section and Figure 2-figure supplement 2.

    My final concern is that the main analyses are performed on single trials of SCR responses, which is a relatively noise measure to use on single trials. This is also done in relatively small groups (n=21). I would have liked to see both the raw or at least averaged timeseries SCR data plotted, and a rationale explaining how the authors decided on the current sample sizes, if that was based on a power analyses one must have expected quite strong effects.

    Following the Reviewer’s suggestion, we decided to remove the analysis on single trials, and we apologize for not including SCR timeseries. To quantify the amount of effect induced by the rTMS protocol, we have now added within-group comparisons (through 2 × 2 mixed ANOVAs) that show, for each group, the amount of change in CS-evoked SCRs from the conditioning phase to the test phase, as well as from the conditioning phase to the follow-up phase. Furthermore, to directly and simply depict these changes, in addition to dot plots, we have also represented them with line charts (Figs. 2C, 2H, 4C, 4H, 5C, 5H). To estimate the sample size, we had previously performed a power analysis through G*Power 3.1.9.2 and it had resulted in n = 21 per experimental group. However, by correcting data pre-processing procedures (in accordance with Reviewer 1), we obtained data that were not normally distributed. Thus, we reasoned to enlarge our sample width by re-performing a power analysis (with the new suggested statistical analyses) and then repeating the experiments. For the main statistics, i.e. mixed ANOVA (within-between interaction) with two groups and two measurements, with the following input parameters: α equal to 0.05, power (1-β) equal to 0.95, and a hypothesized effect size (f) equal to 0.25, the new estimated sample size resulted in n = 30 per experimental group.

  3. eLife

    eLife assessment

    This study presents the useful observation that repetitive Transcranial Magnetic Stimulation (rTMS) over the medial prefrontal cortex (mPFC) is associated with immediate dampening effects of conditioned responses and generalization of these responses to similar cues. Additionally, the effects were still present one week later, in the absence of any stimulation. However, the evidence supporting the claims of the authors is incomplete. The main outcome data (skin conductance response) have been normalized and standardized in suboptimal ways and, most critically, no comparisons are being made with the strength of conditioned responses during acquisition. If the observations hold, when based on within-subject comparisons, the work will be of interest to psychologists and neuroscientists working on interventions into aberrant emotional memories.

  4. eLife

    Reviewer #1 (Public Review):

    In this manuscript, Manessero and colleagues argue that the prefrontal cortex (PFC), given its exquisite capability to down-regulate down-stream regions central in driving emotional responses to threat, maybe a promising target to stimulate in order to reduce aberrant fear memory responses. They aim to differ from previous studies that tested the strengthening of extinction learning, by merely focusing on the expression of threat memory without extinction learning. Given that other studies have often focused on the dorsolateral prefrontal cortex as promising target to regulate fear responses, they also ran experiments to directly compare effectiveness of targeting the mPFC and dlPFC in reducing fear memory responses. These aims are all focused on what the authors describe as "implicit memory", but they also test the effects of the interventions on "explicit memory" of the presented cues. However, in the introduction, the authors do not explicitly describe what their aim or theoretical rationale to implement these tests was. Likewise, the authors implemented generalisation stimuli (i.e., cues similar to the original CS) in the implicit memory tests, but the aim of these tests is also not explained.

    In order to test their hypotheses, the authors adopt a single-cue fear conditioning paradigm where participants learned to associate an auditory cue with the occurrence of short electrical stimulation across 15 repetitions of the CS-US pairing (80% reinforcement rate). One week later, for the second session, this cue was again presented 4 times, along with 2 types of generalization stimuli, that were each also presented four times. This test session took place in another environment. Conditioned skin conductance responses were measured as index of defensive responding. In the critical condition, during 10 minutes prior to these cue presentations, repetitive Transcranial Magnetic Stimulation (rTMS) was applied to specifically target the medial PFC. Another independent group of participants completed a two-alternative forced-choice (2AFC) explicit recognition test, to inquire to what extent they could recognize whether a given tone was presented during the conditioning phase (basically a source memory task). Finally, a two-alternative forced-choice (2AFC) perceptual discrimination test was presented, to ascertain that participants could discern the different tones presented. The second session was repeated yet another week later, but without any rTMS and in the original conditioning context again, to test whether any potential fear dampening effects were retained.

    The observations are quite straightforward: compared to sham and an active control group, mPFC stimulation prior to fear memory retrieval resulted in an immediate reduction of conditioned responses, a difference that was consistent across all 4 test trials. Also conditioned responses to the generalization cues were reduced upon mPFC stimulation. These effects seemed to be specific for memories, since responding to novel unconditioned cues (loud female scream) were not affected by prior mPFC stimulation. Likewise, measures of explicit memory were unaffected. In separate experiments, stimulation of the mPFC also outperformed stimulation of the dlPFC. This pattern of results was again observed during the tests a week later.

    The authors conclude that, since these outcomes were observed in the absence of extinction training, the rTMS procedure directly modulated the defensive responses activated by the threat memory trace. The fact that defensive responses to novel unconditioned stimuli were not affected are in line with earlier observations that the mPFC seems critical for the expression of conditioned but not innate fear. Given that dlPFC stimulation seemed less effective, the mPFC may be the most suitable candidate for future therapeutic interventions.

    Major strengths:
    - Earlier work delving into the involvement of the prefrontal cortex in fear regulation has not only revealed a central role for the mPFC, but also for the dlPFC. An important strength of this study is that the authors therefore also directly compare groups that are targeted in either one of these regions, thereby revealing that even though stimulating the dlPFC results in some fear reduction, the effect is much stronger for mPFC. Another nice consequence of this extra group is that the earlier observations when targeting the mPFC are being replicated.

    - It is important to test novel avenues to achieve enduring fear dampening effects of interventions. An intervention that only exerts immediate but transient effects does not bring much clinical value. So the fact that this study incorporates a follow-up test and then shows that the acute fear dampening effects are retained in the absence of any TMS stimulation certainly is important.

    - It is only natural to show defensive responses to cues that previously have been paired with something aversive, like a shock. For this reason, generalized fear responses to cues that are similar to fear cues but in fact innocuous is considered maladaptive, and at the core of anxiety disorders. A strength of the paper is that the authors have added generalization tests in addition to (adaptive) fear retention, to ascertain that their intervention in fact also targets maladaptive responding.

    Major weaknesses
    - There are two major weaknesses in this paper, that can have a potentially detrimental consequence for the robustness of the results and conclusions. First of all, even though comparing the effect of mPFC stimulation with other groups that have been stimulated in other brain regions is important, another comparison - perhaps an even more essential one - is lacking: is there a significant reduction in conditioned fear responses after targeting the mPFC as compared to that group's own fear acquisition (or at least the final phase of acquisition)? Instead, the authors compare fear responses with responses PRIOR to conditioning, which is not meaningful. The same goes for the long-term follow-up: also here, a comparison with fear responding prior to the intervention is lacking. Such a reduction in conditioned fear responding should be larger than any reduction (e.g., due to habituation or forgetting) in fear responding in the (sham) control groups (i.e., an overall interaction between group and fear responding should be present). Whether this is the case is unfortunately unknown, since the fear acquisition data (neither raw, nor pre-processed) are not to be found in the manuscript, and are therefore also not included in any of the analyses. Since there is also no safe control stimulus, the crucial comparison is made entirely between-subjects, and for such a comparison groups of n~20 are quite modest.

    - Second, against commons practice, the authors commence by square root transforming all SCR data to normalize the data (while this should only be done in the final phase or preprocessing, if the variables entered in the statistical tests require so), only to then again normalize these obtained values by dividing by the unconditioned responses of the participants, that then are used to calculate differences scores with preconditioning. In these descriptions it is unclear which unconditioned stimulus it was (the original one, from conditioning?) and whether it was standardised to the highest response or an average of all the responses. Decisions that are taken in these early pre-processing steps can have a gigantic impact on the outcomes and conclusions, so this is not trivial. One may say that this cannot explain the group effects that have been observed, given the fact that all groups have been pre-processed in the same way. However, the mPFC group of interest seems to display relatively high unconditioned responses - standardising with these measures may result in relatively low conditioned responses in this particular group. This shortcoming is therefore closely related to the point made above: given that conditioning data would be standardised in the same vein, a test that included the within-subject comparison between acquisition and post-intervention is absolutely crucial to ascertain that the effects observed are not merely due to coincidences in pre-processing values and pre-existing group differences.

    - In addition to the above-described main analyses, some other potential weaknesses concern the analysis strategy applied to the generalization tests. Several ANOVAS are being run, one to test for the pattern of generalization responses within-subjects (i.e., the CS, NS1 and NS2), and several ones to compare each of these between the three groups. But such analyses are not warranted in the absence of an overall interaction between the within subject factors and group factor. Such overall omnibus tests however are lacking, and the high number of separate anovas risks false positives (i.e., these comparisons should have been made with planned contrasts). The fact that the included factors and levels are not being described, makes it generally hard to gauge what variables exactly have been entered in every analysis.

    Further remarks:
    - There is a possibility that a re-analysis of the data using properly preprocessed SCR data along with analyses that include comparisons with the conditioned responses during acquisition reveal a different pattern of results. Therefore, whether the authors truly achieved their aims and whether the results support their conclusions is as of yet undecided.

    - Even if the pattern of results holds, then the claim that the long-term follow-up reductions of fear were achieved in the absence of any extinction cannot be made with confidence: after all, upon mPFC stimulation during the second session, the CS was presented four times, and so were each of the two generalization stimuli. So perhaps extinction was not complete, but almost certainly some extinction has taken place: it is well-known that the strongest extinction-learning typically takes place in the first trials (e.g., due to higher prediction errors). The authors do not give any alternative theoretical explanation for the enduring reduction of fear reduction, which would be interesting to learn their thoughts on.

    - If the results hold and satisfiable reasons are provided as to why the effects remain visible in the follow-up, this study could be a valuable contribution to the field: it may refocus future studies to the mPFC as major target to not only promote acute fear regulation, but perhaps even more importantly form a clinical perspective, a route for enduring fear reductions.

  5. eLife

    Reviewer #2 (Public Review):

    Manassaro et al. present an extensive three-session study in which they aimed to change defensive responses (skin conductance; SCR) to an aversively conditioned stimulus by targeting medial prefrontal cortex (their words) using repetitive TMS prior to retrieval. They report that stimulating mPFC using TMS abolishes SCR responses to the conditioned stimulus, and that this effect is specific for the stimulated region and the specific CS-US association, given that SCR responses to a different modality US are not changed.

    I like how the authors have clearly attempted to control for several potential confounds by including multiple stimulation sites, measured SCR responses to several unconditioned stimuli, and applied the experiment in multiple contexts. However, several conceptual and practical issues remain that I think limit the value of potential conclusions drawn from this work.

    The first issue that I have with this study concerns the relationship between the TMS manipulation and the theoretical background the authors present in their rationale. In the introduction the authors sketch that what they call 'mPFC' is involved in regulation of threat responses. They make a convincing case, however, almost all of the evidence they present concerns the ventromedial part of the prefrontal cortex (refs 18-25). The authors then mention that no one has ever studied the effects of 'mPFC'-TMS on threat memories. That is not surprising given that stimulating vmPFC with TMS is very difficult, if not impossible. Simulation of the electrical field that develops as a consequence from the authors manipulation (using the same TMS coil and positioning the authors use) shows that vmPFC (or mPFC for that matter) is not stimulated. The authors then continue in the methods section stating that the region they aimed for was BA10. This region they presumably do stimulate, however, that does not follow logically from their argument. BA10 is anatomically, cytoarchitectonically and functionally a wholly different area than vmPFC and I wonder if their rationale would hold given that they stimulate BA10.

    The second concern I have is that although I think the authors should be praised for including both sham and active control regions, the controls might not be optimally chosen to control for the potential confounds of their condition of interest (mPFC-TMS). Namely, TMS on the forehead can be unpleasant, if not painful, whereas sham-TMS or TMS applied to the back of the head or even over dlPFC is not (or less so at the very least). Given that the SCR results after mPFC TMS show exactly the same temporal pattern as the sham-TMS but with a lower starting point, one could wonder whether a painful stimulation prior to the retrieval might have already caused habituation to painful stimulation observed in SCR in consequent CS presentations. A control region that would have been more obvious to take is the lateral part of BA10, by moving the TMS coil several centimeters to the left or right, circumventing all things potentially called medial but giving similar unpleasant sensations (pain etc).

    My final concern is that the main analyses are performed on single trials of SCR responses, which is a relatively noise measure to use on single trials. This is also done in relatively small groups (n=21). I would have liked to see both the raw or at least averaged timeseries SCR data plotted, and a rationale explaining how the authors decided on the current sample sizes, if that was based on a power analyses one must have expected quite strong effects.

  6. Version published to 10.1101/2023.02.06.527256v1 on bioRxiv