Making memories last using the peripheral effect of direct current stimulation

  1. Alison M Luckey
  2. Lauren S McLeod
  3. Yuefeng Huang
  4. Anusha Mohan
  5. Sven Vanneste

  • Curated by eLife

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    This paper will be of fundamental interest to many sub-disciplines of neuroscience, ranging from cognitive neuroscientists to cellular neuroscience. It provides compelling and substantial brain and behavioral evidence of a novel intervention that can boost long-term memory. The key claims of the manuscript are generally well supported by the data, though the correlational nature of the data in different types of experiments raises some issues about interpretation.

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Abstract

Most memories that are formed are forgotten, while others are retained longer and are subject to memory stabilization. We show that non-invasive transcutaneous electrical stimulation of the greater occipital nerve (NITESGON) using direct current during learning elicited a long-term memory effect. However, it did not trigger an immediate effect on learning. A neurobiological model of long-term memory proposes a mechanism by which memories that are initially unstable can be strengthened through subsequent novel experiences. In a series of studies, we demonstrate NITESGON’s capability to boost the retention of memories when applied shortly before, during, or shortly after the time of learning by enhancing memory consolidation via activation and communication in and between the locus coeruleus pathway and hippocampus by plausibly modulating dopaminergic input. These findings may have a significant impact for neurocognitive disorders that inhibit memory consolidation such as Alzheimer’s disease.

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  1. Version published to 10.7554/elife.75586 on eLife
  2. eLife

    Author Response

    Reviewer #2 (Public Review):

    Weaknesses (major)

    1. Adding control groups (sham stimulation) to Experiment 5 and Experiment 8 would be needed to increase confidence that NITESGON's memory-enhancing effects do not depend on sleep but do depend on dopamine receptor activity.

    Thank you for highlighting this major weakness within our research; we will be sure to include control groups in future research if we conduct replication studies. Additionally, upon review of your comment, we have addressed the lack of control/sham groups in Experiment 5 and 8 in the Discussion section when acknowledging the limitations of the research.

    Please see the newly added text from the Discussion section on pages 21-22 below:

    “Moreover, it must also be acknowledged that Experiments 5 and 8 did not include a control-sham stimulation group, thus limiting the interpretation of these two experimental findings. Control-sham stimulation groups would increase our confidence in our findings that NITESGON’s memory-enhancing effects depend not on sleep but on DA receptor activity.”

    1. Task order in the interference study in Experiment 4 was randomized during the first visit for task training as well as during the memory test, however, the word-association and spatial navigation tasks used in Experiments 3 and 4 were not counterbalanced during training or memory testing. Thus, the authors cannot rule out the possibility of order effects.

    Upon reading your comment and reviewing the paper, we have decided to add a limitations paragraph to the paper which highlights the concern of Experiments 3 and 4 not being counterbalanced during training or memory testing. Additionally, the new section provides an explanation of how not counterbalancing Experiments 3 and 4 introduced the possibility of order effects being present in the results.

    Please see the new addition from the Discussion section on page 21 below:

    “When interpreting the current findings, it must be considered that some limitations exist within the research; limitations on experimental design are noted below, followed by a discussion of utilizing indirect proxy measures. The task order for Experiment 4 was randomized during the first visit for training and the recall-only memory test 7-days later; however, the word association and spatial navigation task used in Experiments 2 and 3 were not counterbalanced; therefore, the findings of Experiments 2 and 3 could have been impacted by a potential order effect.”

    1. It is unclear how Experiment 3 and Experiment 4 differ. Percent of words recalled is the measure of memory performance, however, there is not a clear measure of interference in Experiment 4 (i.e., words recalled during Memory task II that were from Memory task I).

    Thank you for highlighting the difficulty in distinguishing the differences between Experiment 3 and Experiment 4. To clarify what the differences are between Experiment 3 and Experiment 4, we explained in Experiment 4’s introductory paragraph that the object-location task used in Experiment 3 was replaced with a Japanese-English verbal associative learning task in Experiment 4.

    Please see the paragraph from the Experiment 4 subsection on page 10 below:

    “Experiments 2 and 3 revealed both retroactive and proactive memory effects 7-days after initial learning of the two tasks. To further explore if NITESGON is linked to behavioral tagging and evaluate if interference impacts NITESGON as the strong stimulus, Experiment 4 removed the object-location task used in Experiments 2 and 3 and replaced it with a Japanese-English verbal associative learning task similar to the Swahili-English verbal associative task. Considering how memory formation and persistence are susceptible to interference occurring pre-and post-encoding(37-39) and are heavily influenced by commonality amongst the learned and intervening stimuli(40); it is believed that conducting two consecutive, like-minded word-association (i.e., Swahili-English and Japanese-English) tasks will result in one’s consolidation process interfering with that of the other(41). Considering how our previous experiments suggest the effect obtained by NITESGON improves the consolidation of information via behavioral tagging, it is possible that NITESGON on the first task might help reduce the overall interference effect on the second task.”

    Additionally, we explained in further detail that comparing the percentage of correctly recalled word pairs on the second task 7-days after learning from the percentage of correctly recalled word pairs on the first task 7-days after learning was done to measure for an interference effect.

    Please see the adapted text from the Experiment 4 subsection on page 11 below:

    “Upon assessment for a potential interference effect, the active group displayed no significant difference in how many words participants were able to recall between the first and the second task (difference: .76 4.93) (F = .29, p = .60), whereas the sham group demonstrated the first task rendered an interference effect on the second task (difference: 5.16 5.99) (F = 14.11, p = .001).”

    Lastly, in the methods section describing how the interference effect was calculated was changed. The newly edited text better explains that the percentage of words pairs learned were subtracted from one another to measure the significance of interference one may have potentially had on the other.

    Please see the amended text in the Methods section on page 38 below:

    “In addition, an interference effect was calculated by subtracting the percentage of correctly recalled word pairs on the second task 7-days after learning from the percentage of correctly recalled word pairs on the first task 7-days after learning. This number gave a proxy of interference.”

    1. In Experiment 5 the learning and test phases for the two sleep groups were conducted at different times of day (sleep group: training at 8pm and testing the next morning at 8am, sleep deprivation group: training at 8am and testing at 8pm) which introduces the possibility of circadian effects between the two groups. Additionally, the memory test occurred at the 12h point for this experiment instead of the 7-day point. Therefore, the authors' conclusions are not addressed by this experiment, and it remains unclear whether the 7-day long-term memory effects of NITESGON are sleep-dependent.

    Upon reading your comment and reviewing the paper, we have decided to add a limitations paragraph to the paper which highlights the two sleep groups being conducted at different times of day and the memory test occurring at the 12-hour point as opposed to 7-days after initial learning. In addition to acknowledging these limitations, we have also provided explanations regarding what potential effects are introduced by having the sleep groups learn and test at different times of day, such as circadian effects between the two groups, and the memory tests occurring at 12-hours rather than 7-days after initial learning.

    Please see the new addition from the Discussion section on page 21 below:

    “Additionally, in Experiment 5, the learning and test phases for the two groups were conducted at different times of day (i.e., sleep group: training at 8 p.m. and testing at 8 a.m., sleep deprivation group: training at 8 a.m. and testing at 8 p.m.), thus introducing the potential for circadian effects between the two groups. Furthermore, the recall-only memory testing occurred at the 12-hour point rather than 7-days later, allowing us to conclude that the observed effect seen 12-hours later was not affected by sleep; however, it remains unclear whether the 7-day long-term memory effects of NITESGON are sleep-dependent.”

    Weaknesses (minor)

    1. Salivary amylase is being used as a proxy of noradrenergic activity; however, salivary amylase levels increase with stress as well, which impacts memory performance. It would be helpful if the authors addressed this and whether they measured other physiological indicators of stress/sympathetic nervous system activation.

    Upon review of your comment, we have edited the paper so that it includes text in the Discussion section that brings attention to the fact that stress can enhance salivary amylase and advises readers that this should be considered when interpreting results. We also add an additional measure which measure pupil size, a measure well-know for sympathetic measure. In addition we add also a VAS score to ask people about their stress levels.

    Please see the added new addition from page 22 below.

    “Although the use of indirect proxy measures, such as sAA for NA activity and sEBR for DA activity, enabled the tracking of LC-NA activity changes from baseline measurements and demonstrated the potential of an LC-DA relationship, caution must be advised when interpreting results considering these proxy measures are affiliated with limitations, such as being substantially variable, as well as the potential of other brain regions and monoamine neurotransmitters being associated with changes seen in sAA concentration levels(80), an enzyme that is provoked by both central parasympathetic and sympathetic nervous system activation, including acute stress responses(81). Additionally, although sEBR has been increasingly linked to DA, it has been defined as a more viable measure of striatal DA activity(52, 82). At the same time, some evidence suggests that sEBR and DA levels may be unrelated(83, 84), thus requiring further validation as a behavioral proxy measure.”

    1. Insufficient details of how the blinding experiment was conducted make it difficult to determine whether participants had awareness or subjective responses during the NITESGON stimulation. Adding physiological indicators of heart rate, skin conductance, and respiration would provide a better indicator of a sympathetic nervous system response. Additionally, a series of randomized stimulation and sham trials delivered to the participant would provide a more objective measure of the detectability of the stimulation.

    Thank you for your comment regarding the portion of the experiments that were included to determine the efficacy of the measures taken to ensure the experiments were well blinded. After reviewing the comment and reading over the paper, we were concerned that it was not clear enough to the reader that the efficacy of blinding was determined by having each participant of every experiment complete the same single-answer questionnaire after all NITESGON and testing had been experienced. Therefore, we edited the wording below to elucidate that there was not an individual blinding experiment but that there was a questionnaire for every participant in every experiment to help determine the efficacy of blinding for each experiment and the research.

    Please see the text from the Blinding section on pages 17-18 below:

    “Blinding. To determine if the stimulation was well blinded, all participants in Experiments 1-7 were asked to guess if they thought they were placed in the active or control group (i.e., what stimulation participants received compared to what participants expected). Our findings demonstrated that participants could not accurately determine if they were assigned to the active or sham NITESGON group in each experiment, suggesting that our sham protocol is reliable and well-blinded (see fig. 8).”

    Additionally, please see the text in the Methods section that has been reworded to clarify how the questionnaire of blinding was conducted on page 47 below:

    “Blinding: To determine if the stimulation for all experiments was well blinded, all participants who participated in Experiments 1-7 were asked to complete a single-response questionnaire after the conclusion of the NITESGON procedure. Here, participants were asked to guess if they thought they were placed in the active or control group. A χ2 analysis was used to determine if there was a difference between what stimulation participants received compared to what participants expected.”

    1. It would be appreciated if the authors could speak to the possible role of the amygdala in the memory-enhancing effects of NITESGON, as this region is a well-known modulator of many types of memory consolidation and is implicated in noradrenergic-related memory enhancement.

    Upon consideration of your comment, we added text providing the reader with insight into how NITESGON has activated the amygdala in previous research, similar to the VTA in the current study, and how the LC and amygdala were shown to be activated during emotionally arousing stimuli in another study. Furthermore, we have acknowledged that the amygdala is understood to have modulatory implications in long term memory and how future investigations are needed to establish the amygdala’s role with NITESGON.

    Please see the text from the Discussion section on page 20 below:

    “Additionally, it is well-known that the amygdala is not the final place of memory storage, but rather has major modulatory influences on the strength of a memory(74). Similar to the VTA in the current study, prior research has shown that the amygdala is activated during NITESGON but ceased post-stimulation; however, NITESGON was not accompanied by a task during the experiment(14). Moreover, a recent fMRI study spotlights the dynamic behavior of the LC during arousal-related memory processing stages whereby emotionally arousing stimuli triggered engagement from the LC and the amygdala during encoding; however, during consolidation and recollection stages, activity shifted to more hippocampal involvement(75). Considering the impact the VTA and amygdala can have on memory, future experimental investigations are needed to establish their role in the memory-enhancing effects of NITESGON.”

  3. eLife

    eLife assessment

    This paper will be of fundamental interest to many sub-disciplines of neuroscience, ranging from cognitive neuroscientists to cellular neuroscience. It provides compelling and substantial brain and behavioral evidence of a novel intervention that can boost long-term memory. The key claims of the manuscript are generally well supported by the data, though the correlational nature of the data in different types of experiments raises some issues about interpretation.

  4. eLife

    Reviewer #1 (Public Review):

    Luckey et al. used a sophisticated, multimodal approach to test the hypothesis that engaging LC-hippocampal pathways promote behavioral tagging processes in humans. To activate this mechanism in a causal manner, they apply transcutaneous electrical stimulation of the greater occipital nerve (NITESGON), a relatively novel and non-invasive technique for stimulating brainstem pathways linked to arousal-related neuromodulation. To test the behavioral tagging hypothesis, they use a variety of indirect methods, including pharmacology, EEG, fMRI, saliva assays, and eye-tracking to measure LC-related activity, hippocampal activity/connectivity, and potential dopamine states/release. At the behavioral level, they demonstrate that NITESGON stimulation during or after learning benefits long-term but not immediate associative memory. These long-term memory improvements were related to increased gamma power in the MTL. In another set of experiments, they show that NITESGON during associative learning promotes associative learning on a subsequent unrelated (object-location) or highly overlapping (paired word associates) task. Consistent with prior VNS and other NITESGON studies, they show robust evidence that this intervention leads to significant increases in salivary alpha-amylase, a putative marker of central noradrenergic activity. This increase in sAA was also correlated with long-term associative memory across several experiments using paired word associates. Using fMRI, they demonstrate resting-state increases in local hippocampal, LC, and VTA low-frequency fluctuations as well as increased rs-FC between the LC and hippocampus during and after stimulation. Finally, they show that NISTESGON does not enhance long-term associative memory in individuals taking a dopamine antagonist medication, implicating a potential dopamine mechanism in these stimulation-induced memory effects.

    This paper is impressive in scope and takes advantage of both causal and indirect methods to cross-validate their results. Behavioral tagging is a relatively nascent area of research in humans, and this paper provides compelling evidence for the role of noradrenergic activity (whether related to behavioral tagging or more general arousal-related consolidation processes) in facilitating memory encoding and consolidation. Beyond basic science research, these findings also have important clinical implications. In recent years, there has been intense interest in studying the LC's role in promoting healthy cognitive function and its involvement in AD-related neuropathology. The LC is one of the earliest sites of tau pathology and thereby represents an important target for clinical intervention in early AD. The current study advances our understanding of a non-invasive technique that may be used to bolster learning in both healthy populations and potentially in older individuals with AD.

    The key claims of the manuscript are generally well supported by the data. However, while the large number of studies is a significant virtue of this paper, it is also - at times - a potential weakness. There are many measures and pieces to this puzzle to assemble. While the multimodal approach is admirable and rigorous, the fit between some of these pieces is sometimes overstated. The correlational nature of the data helps cross-validate some of the predictions about the LC mechanisms involved in behavioral tagging. But the most compelling test of this hypothesis would be to link the LC/hipp/VTA fMRI data - arguably the most direct outcome measure in this study - to long-term memory performance and the other neurophysiological measures (e.g., sAA, blink rate, etc.). Many of the results are compelling but they are often observed in parallel studies. Thus, interpreting them as engaging a common mechanism is tenuous. This important shortcoming notwithstanding, there is still a strong replication in other findings (e.g., sAA-memory correlations) across experiments that lend support to some of the hypotheses.

    A related issue is that the reliability of these indirect measures of noradrenergic signaling and dopaminergic receptors, including salivary alpha-amylase and spontaneous eyeblink rate, is oversold. While this stimulation technique elicits parallel increases in many of the neurophysiological and behavioral measures, these patterns might not reflect the engagement of a shared underlying mechanism. It's an especially big stretch to interpret the eyeblink effects as relating to LC-DA, which cannot be verified using the current methods. In addition, the spatial resolution of the neuroimaging data is poorly suited for testing predictions about such a small brain structure. This represents a potential weakness of the paper, as the large smoothing kernel in the fMRI data may capture the contributions of other brainstem nuclei and regions activated by NITESGON. It is also worth noting that many of the individual differences findings are confounded by group clustering effects. That is, the between-group effects belie whether the same linear relationships exist in the sham and stimulation groups individually. This necessitates additional correlation analyses within groups to verify that stimulation doesn't decorrelate the relationship between physiological measures and performance.

    While the behavioral tagging predictions are intriguing and supported by some findings in the literature, they may not be entirely appropriate for this study. In short, I'm not fully convinced these data satisfy all assumptions of BT (see Dunsmoor et al., 2022 for an overview). Behavioral tagging is thought to be a process that stabilizes weak learning. While it's very difficult to operationalize the "strength" of a memory representation, I'm not sure if the current paired-associates paradigm yields weak learning. Participants have multiple opportunities to learn the memoranda, which casts some doubt as to whether these are weak memory representations. This possibility is supported by the generally high memory performance (~80% on average) during the immediate test and even accurate recall after 7 days.

    Behavioral tagging also does not make any explicit predictions about interference effects. Much of this theory centers upon the idea that arousing learning events lead to memory enhancements/benefits; but it does not speak directly as to whether these events confer protection from memory interference (and there was no baseline condition in Dunsmoor et al., 2015 to test any predictions regarding reduced retroactive interference for CS+ stimuli, for example). I find the protective effects of stimulation in Experiment 4 very interesting, and they speak to the importance of this technique as a memory intervention. However, I think this is an example of the authors relying too heavily on a behavioral tagging framework when these could simply reflect arousal-related (Nielson et al., 1996; 2014) and/or noradrenergic-related (e.g., McGaugh, 2013) consolidation benefits more broadly. In summary, I think it would strengthen the paper to walk back claims related to behavioral tagging specifically and address the possibility of alternative (but related) mechanisms.

    To summarize, the results of this study are very interesting and the project is very ambitious. There is much therapeutic potential for NITESGON to improve memory and this study represents an important advance towards achieving that goal. The work would primarily be improved by not relying on too many assumptions or inferences, and being more agnostic with respect to certain mechanisms (e.g., whether this is behavioral tagging or general consolidation mechanisms).

  5. eLife

    Reviewer #2 (Public Review):

    Luckey et al. investigated the mechanisms by which non-invasive transcutaneous electrical stimulation of the greater occipital nerve (NITESGON) enhances long-term memory. They find that NITESGON applied during or after a word-association task enhances memory recall at a retrieval test 7 days later but not at an immediate test, suggesting NITESGON's memory-enhancing effect involves the consolidation process. They show that NITESGON applied during a second spatial memory task not only enhances later recall for that task, but also for an initial word-association memory task unpaired with stimulation administered before the second task. This highlights NITESGON's ability to retroactively strengthen memories and provides further evidence for behavioral tagging. Furthermore, the authors perform a series of in-depth experiments to examine the mechanisms by which NITESGON enhances memory consolidation. They show that NITESGON increases salivary a-amylase levels, a marker of endogenous noradrenergic activity, and spontaneous eye blink levels, a proxy for dopamine levels, both in support of locus coeruleus involvement. Resting-state fMRI results further suggest NITESGON induces increased communication between the locus coeruleus and hippocampus, suggesting a circuit-based mechanism by which NITESGON enhances memory consolidation. Interestingly, the data also indicate that NITESGON's memory-enhancing effect is not sleep-dependent but is dopamine-receptor-dependent.

    The conclusions of this paper are mostly well supported by the data, however, some of the key mechanistic findings lack the appropriate controls required for the authors' claims.

    Strengths

    1. The manuscript is written in an easy-to-read manner with clarity for each of the individual experiments conducted.
    2. The authors provide convincing evidence that NITESGON targets the memory consolidation process and enhances long-term but not short-term memory. This provides a unique non-invasive method for enhancing memory and has an important potential impact on neurocognitive disorders.
    3. The manuscript provides convincing evidence that NITESGON increases LC-hippocampus connectivity as well as MTL gamma power, providing a circuit-based mechanism by which stimulation enhances memory.

    Weaknesses (major)

    1. Adding control groups (sham stimulation) to Experiment 5 and Experiment 8 would be needed to increase confidence that NITESGON's memory-enhancing effects do not depend on sleep but do depend on dopamine receptor activity.
    2. Task order in the interference study in Experiment 4 was randomized during the first visit for task training as well as during the memory test, however, the word-association and spatial navigation tasks used in Experiments 3 and 4 were not counterbalanced during training or memory testing. Thus, the authors cannot rule out the possibility of order effects.
    3. It is unclear how Experiment 3 and Experiment 4 differ. Percent of words recalled is the measure of memory performance, however, there is not a clear measure of interference in Experiment 4 (i.e. words recalled during Memory task II that were from Memory task I).
    4. In Experiment 5 the learning and test phases for the two sleep groups were conducted at different times of day (sleep group: training at 8pm and testing the next morning at 8am, sleep deprivation group: training at 8am and testing at 8pm) which introduces the possibility of circadian effects between the two groups. Additionally, the memory test occurred at the 12h point for this experiment instead of the 7-day point. Therefore, the authors' conclusions are not addressed by this experiment, and it remains unclear whether the 7-day long-term memory effects of NITESGON are sleep-dependent.

    Weaknesses (minor)

    1. Salivary amylase is being used as a proxy of noradrenergic activity, however, salivary amylase levels increase with stress as well, which impacts memory performance. It would be helpful if the authors addressed this and whether they measured other physiological indicators of stress/sympathetic nervous system activation.
    2. Insufficient details of how the blinding experiment was conducted make it difficult to determine whether participants had awareness or subjective responses during the NITESGON stimulation. Adding physiological indicators of heart rate, skin conductance, and respiration would provide a better indicator of a sympathetic nervous system response. Additionally, a series of randomized stimulation and sham trials delivered to the participant would provide a more objective measure of the detectability of the stimulation.
    3. It would be appreciated if the authors could speak to the possible role of the amygdala in the memory-enhancing effects of NITESGON, as this region is a well-known modulator of many types of memory consolidation and is implicated in noradrenergic-related memory enhancement.
  6. Version published to 10.1101/2022.07.06.498966v1 on bioRxiv