Rats remember: Lack of drug-induced post-retrieval amnesia for auditory fear memories

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1. 

   eLife

   Sep 22, 2020

   ##Author Response

   ###Reviewer #1:

   This manuscript provides evidence that drug administration during a reconsolidation window does not necessarily prevent memory recall, as has been shown by many groups. The authors attempted to replicate several published experiments and despite demonstrating that the drugs had other effects on the animals' behavior and physiology (e.g. weight gain), no effects on memory were observed.

   The paper is nicely prepared.

   We sincerely thank the reviewer for these kind words and the support to publish our replication efforts.

   ###Reviewer #2:

   General assessment:

   In this study, Luyten et al. aimed to replicate post-retrieval amnesia of auditory fear memories reported numerous times in the literature. They used a variety of behavioural approaches combined with systemic pharmacological treatments …

   ##Author Response

   ###Reviewer #1:

   This manuscript provides evidence that drug administration during a reconsolidation window does not necessarily prevent memory recall, as has been shown by many groups. The authors attempted to replicate several published experiments and despite demonstrating that the drugs had other effects on the animals' behavior and physiology (e.g. weight gain), no effects on memory were observed.

   The paper is nicely prepared.

   We sincerely thank the reviewer for these kind words and the support to publish our replication efforts.

   ###Reviewer #2:

   General assessment:

   In this study, Luyten et al. aimed to replicate post-retrieval amnesia of auditory fear memories reported numerous times in the literature. They used a variety of behavioural approaches combined with systemic pharmacological treatments (propranolol, rapamycin, anisomycin, cycloheximide) after reactivation of fear memories. Interestingly, none of the treatments induced a significant decrease of freezing responses during subsequent retrieval tests. Authors strengthened their null results by using Bayesian statistics, confirming the absence of drug-induced amnesia.

   Overall, the study is really interesting. Experiments and analyses are very well designed and bring some important findings to the debated topic of post-retrieval amnesia and its clinical relevance.

   We are grateful that the reviewer appreciates our work and recognizes the general importance of our null findings. We genuinely thank them for the time that they took to evaluate our paper in detail and hope to provide some clarifications in our responses below.

   I have nevertheless several comments for the authors to consider.

   -Despite being very detailed, the authors should clarify and uniformize their Methods section and Supplemental information (e.g. number of CS, contexts used...) to improve the understanding of the different approaches. Similarly, methods for the reinstatement protocol (Exp 2) are missing.

   We understand that the information in the main text is quite dense, but we explicitly chose to focus on the central message here, i.e., that we applied standard procedures that should have allowed us to detect amnestic effects in consideration of most of the published literature. In addition, the crucial overview of the number of training and test trials, as well as the context that was used for each session is depicted in Fig. 1-3, immediately above the results of the respective experiments.

   In the Supplement, we provide a more extensive (and repetitive) report of the experimental procedures. The idea is that the reader can find the most important information in the main text, and all additional details in the Supplement (or in our preregistrations on the Open Science Framework: https://osf.io/j5dgx ). For example, in the main text, it is mentioned that reinstatement in Experiment 2 consisted of two US presentations in context A, one day before the final test (see p. 6 and Fig. 1C). The Supplement (p. 1) adds that the reinstatement session started with 300 s of acclimation, followed by the first US and 180 s later by the second US, and that the rat was removed from the context 120 s after last US onset. For all phases of Experiment 2, the US was a 0.7-mA, 1-s shock.

   • In exp 5, tests 1 and 2 are supposed to have 12 CS each. However, only 8 dots are represented on the graph. Did the authors average some freezing values after the initial 4 first CS presentations?

   Thank you for noticing this. We did not average freezing values, but just did not measure freezing on all trials, as we were not specifically interested in the concrete freezing levels on each trial, but rather in the overall extinction curve. As mentioned in the legend of Fig. 2, freezing during CS5-7-9-11 was not measured (and hence also not shown). In other words, the 8 dots on the graph represent CS1-2-3-4-6-8-10-12.

   -There is an obvious difference in baseline freezing response before the test in Exp 7 (Figure 5A-B). Discussion of these differences is an important point and was thoroughly discussed by the authors in the Supplement.

   Thank you for pointing this out.

   -Ln 384-387: "... additional Bayesian analyses were carried out that collectively suggested substantial evidence for the absence of an amnestic effect". Despite the "substantial effect" given by the meta-analysis, I am a bit confused by the meaning of an "anecdotal evidence against drug < control" reported in half of the experiments. How do the authors interpret these results?

   In short, Bayesian analyses provide evidence that is categorized starting from ‘no evidence’, to ‘anecdotal’, ‘substantial’, ‘strong’, etc. depending on the obtained Bayes factor. Grouping studies with anecdotal and substantial evidence in a meta-analysis can result in overall substantial evidence, which is what we observed here.

   Addressing this remark in more detail, we want to point out that the use of frequentist analyses (ANOVAs and t-tests) allowed us to conclude that we could not replicate the amnestic effects of previously published studies – we did not obtain a statistically significant amnestic effect although we had sufficient power to detect the effect sizes that had been previously reported. However, those analyses do not permit us to make inferences about the evidence against an amnestic effect. Bayesian analyses, on the other hand, do allow us to quantify the obtained evidence against an amnestic effect (i.e., the null hypothesis) for each single experiment or by combining the results of several studies. When a single study suggests only anecdotal evidence against an amnestic effect, this implies that we cannot conclude based on that study alone that we have proper evidence for the absence of an effect. Rather, we can only conclude that we have no evidence for the presence of an amnestic effect and weak (‘anecdotal’) evidence for its absence. However, a collective analysis of our studies does lead to the conclusion of substantial evidence for the absence of an amnestic effect overall.

   -The effect of cycloheximide on memory consolidation is indeed unexpected. Even if beyond the scope of the current study, what is the authors' hypothesis to explain that cycloheximide in their conditions induced a pro-mnesic effects on the consolidation of fear memories but altered the consolidation of extinction?

   As indicated by the reviewer, this is beyond the scope of the current study. We have no additional data on this effect and can only guess at its meaning. Also note that the effect was rather small and disappeared quickly during the test under extinction.

   One purely speculative hypothesis is that the injection with cycloheximide was more arousing than the vehicle injection, either due to sensations caused by the substance during injection or due to the rapidly emerging malaise it induced (or a combination of both), which we have documented in the Supplement (p. 5).

   In line with work by McGaugh, Roozendaal and colleagues, such arousal around the time of training could, in theory, enhance consolidation of a fearful memory, and thus explain greater fear memory during test (see e.g., Roozendaal & McGaugh (2011), https://doi.org/10.1037/a0026187 ). Then again, a similar argument could be made for improved consolidation of the extinction memory (de Quervain et al. (2019), https://doi.org/10.1007/s00213-018-5116-0 ), which we did not observe. One could suggest that – assuming that we have observed ‘true’ effects here – the arousal component had the upper hand during the consolidation of the fear memory, while the protein synthesis inhibition overruled such effects during consolidation of the extinction memory. As this is all highly speculative, we prefer to not add this to the Discussion.

   -Cycloheximide seemed to induced post reconsolidation amnesia of fear memory after extinction training (Exp 8, Fig 3G) but not after single CS reactivation. Can the authors please develop this point? Is it possible that several presentations of the CS is required to destabilise the initial memory trace?

   First of all, it is important to emphasize that cycloheximide-treated rats in Experiment 8 (Fig. 3G) froze more during the CSs of Test 2 than control animals, arguing against a drug-induced reconsolidation blockade of the initial fear memory. Furthermore, the obvious within-session extinction during Test 1 in Experiment 8 suggests that it did not function as a typical reactivation-without-extinction session (Merlo et al. (2014), https://doi.org/10.1523/JNEUROSCI.4001-13.2014 ).

   In light of the current literature, reactivation with a single CS is by far the most common way to destabilize a memory trace that was formed with one or three CS-US pairings. As mentioned in our paper, this should provide an appropriate degree of prediction error for the memory to become malleable (p. 12).

   Theoretically, it is indeed possible that more than one (e.g., two) CS presentations could allow for destabilization of the memory trace, although others who have used reactivation sessions with more than one CS presentation did not find the amnestic effects that they did observe with a single CS (Merlo et al. (2014); Sevenster et al. (2014), https://doi.org/10.1101/lm.035493.114 ).

   ###Reviewer #3:

   Luyten et al's study examines the phenomenon of drug-induced post-retrieval amnesia for auditory fear memories in rats, and report that after several experiments using Propranolol, Rapamycin, Anisomycin or Cycloheximide that they essentially observe no disruption of reconsolidation, (i.e., no amnesia). This is a well-executed, written and meticulous study examining an important phenomenon. The author's lack of observing amnesia using these "reconsolidation blockers" highlights an important fact that systemic administration of these drugs at the time of memory retrieval may not robustly influence reconsolidation processes despite what the existing literature may collectively indicate. The author's data clearly indicate this point and it is important the scientific community be made aware of these difficulties in blocking reconsolidation using systemic administration of these drugs.

   We are thankful for these generous comments and value the reviewer’s thorough and thoughtful assessment of our work. We also appreciate the reviewer’s position that it is important to get this message across to the scientific community.

   This group has previously published similar studies disputing similar phenomena. First highlighting a lack of amnesia following the reconsolidation-extinction paradigm and then more recently demonstrating a lack of amnesia attempting to block the reconsolidation of context fear memories. This is now their third installment focusing on Cued fear memories. Certainly, these findings are important, but arguably the novelty of such findings may be diminished a bit.

   We appreciate that the reviewer is well aware of some of our other work in this domain that supports a more general and widespread reproducibility crisis in this field.

   Regarding the novelty, one key point to stress here, which is also articulated in the paper (p. 3, 13), is that the current rodent findings (which we could not replicate) are the ones that provide the most direct basis for the clinical translations that have been proposed (e.g., by giving patients a propranolol pill after retrieval of a traumatic or phobic memory, see e.g., https://kindtclinics.com/en/ or Kindt & van Emmerik (2016), https://doi.org/10.1177/2045125316644541 ), and are therefore critical in their own right, not only because of their fundamental scientific relevance, but certainly also in light of their clinical reach.

   In one of the "control" experiments where the experimenters administer anisomycin immediately post training, they observe a paradoxical result - they observe memory strengthening instead of the expected blockade of consolidation and amnesia. This result highlights a number of things to consider when we interpret these overall results. For one protein synthesis inhibitors(PSIs) are toxic and when administered systemically usually result in inducing the animals to have diarrhea and generally just makes them sick. This of course will make the animals stressed and agitated and result in increasing their stress and likely amygdala activity. All of this could likely be the reason why the animals exhibited memory strengthening or no impairment in consolidation even with a PSI on board. See PMCID: PMC7147976. Figure 6. In this study, they could rescue the impairment of PSI on consolidation by increasing BLA principal neuron firing. Thus an important take away is something like this could easily be happening in the reconsolidation experiments - that there is no blockade because the animals are stressed either due to PSI on board or because some issues with experimenter/animal interactions, etc lead to higher BLA neural activity and rescue of the reconsolidation process.

   We agree that (systemic) protein synthesis inhibitors can induce signs of sickness in the animals (particularly in the first hours after injection) and have provided a detailed description of our relevant observations in the Supplement (p. 4-5). The reviewer is completely correct in stating that this may cause some amygdala activation which could interfere with the amnestic effects that we expected to see, as described in the paper by Shrestha, Ayata et al. (2020), and in line with our reply to Reviewer #2’s first comment regarding our cycloheximide experiment. Yet, effective induction of amnesia with these drugs has repeatedly been reported in the literature.

   Nevertheless, although relevant, the current remark has relatively little implications for our findings. In the large majority of our experiments, we did not use these toxic protein synthesis inhibitors (PSIs) (such as cycloheximide and anisomycin), but drugs that have generally been administered systemically throughout the literature (with successful amnestic effects). Furthermore, in the experiments where we did administer systemic cycloheximide or anisomycin, we observed no differences compared to vehicle-treated rats in contextual freezing (e.g., 9% on average in Experiment 7) immediately prior to the crucial test tones (Test 1, 24h after injection) – which argues against high levels of stress or agitation. Moreover, a blinded experimenter could not tell the difference between PSI-treated versus vehicle-treated animals while handling the animals for the test session, and observed no behavioral abnormalities, nor signs of pain or distress, as mentioned in the Supplement. We acknowledge that these experimenter observations may not entirely reflect what is happening in the animals’ amygdala, but they at least go against the notion that PSI-treated animals would be too sick to be tested properly.

   I don't think the authors go far enough articulating the important differences between systemic and intra-cranial administration of these drugs. Time is a potential factor. Immediate administration of the drug at high concentration in the target brain region (BLA) versus many minutes until the drug gets to the target region with uncertain concentration levels that may not mirror levels reached with intracranial administration. It's unfortunate the authors were not able to include intra-BLA administration of these drugs in this study. I do not necessarily expect them to do such experiments, since they have already done so much and it is not clear the laboratory has the appropriate expertise to conduct such experiments, but this comparison would be helpful.

   We fully agree that our results do not provide any information about the replicability of intracranial administration of drugs to induce post-retrieval amnesia of cued fear memories. We had already clearly acknowledged this in the first version of the paper (p. 11), but have now added an extra section to the Discussion (p. 13) to highlight this point in the new version posted on BioRxiv (Version 2). Notwithstanding the expertise of our laboratory to carry out intracranial infusions, we agree with the reviewer that such experiments are beyond the scope of this article.

   It is, however, noteworthy that the drugs that we used in 6 experiments did not necessarily rely on intracranial administration in prior successful studies. Rapamycin, for example, has generally been used systemically (not intracranially). Propranolol has been used either systemically or intracranially in rodents and always systemically in human subjects (healthy and patients). Bearing in mind the timing issue that was raised by the reviewer, we moreover included an experiment with pre-reactivation administration of propranolol (Experiment 4), where the drug was injected 5-8 minutes before the rats heard the reactivation tone.

   I think it is important that the authors make some statement of training conditions on cannulated versus cannulated rats. For example, every animal in Nader's 2000 study was bilaterally cannulated targeting the BLA. In contrast every animal in this study underwent no such surgery. I think this is relevant. In my experience non cannulated animals are a bit smarter than cannulated animals and the training conditions across these two differing groups may not equate to the same level of learning. And of course, differences in learning levels can lead to differences in the ability of the retrieved memory to destabilize.

   Thank you for pointing this out. We are aware that there may be differences between operated and non-operated animals and already briefly discussed this matter in the Supplement (p. 4). We have now also added this issue to the Discussion in the new section (p. 13) where we emphasize the differences between systemic and intracranial drug administration in relation to the previous comment.

   That being said, the comment regarding (non-)cannulated rats only really applies to Experiment 7 where we tested the effects of systemic anisomycin or cycloheximide. Prior cued fear conditioning studies indeed used intracranial administration of these drugs. The argument does not hold for Experiments 1-6, as systemic propranolol and rapamycin have repeatedly been reported to have amnestic effects in non-operated rats, with procedures identical to or closely resembling ours.

   The authors mention possibly examining markers of memory destabilization. GluR1 phosphorylation, Glur2 surface levels, protein degradation/ubiquitination have all been used to assess if destabilization has occurred. I do not fully agree with their reasons for not performing such experiments. They could examine some or one of these phenomena across differing training conditions between retrieval, no-retrieval animals. This likely could be informative. However, the authors may not possess the necessary expertise to conduct such experiments, so I'm not stating these experiments need to be completed, but certainly the study could be strengthened with such data.

   We agree that including yet more control experiments, using different experimental approaches could further strengthen the study. Nevertheless, the main conclusion of our paper – i.e., reconsolidation blockade using systemic administration of several drugs is considerably more difficult to reproduce than what the literature collectively indicates – is strongly and sufficiently supported by the data that we already report here. Overall, we believe that our conclusion does not require such additional controls. Moreover, even though the comparisons suggested by the reviewer could indeed be scientifically interesting, it is still unclear whether such experiments would provide sufficiently clear cut-offs as to which experimental condition would then allow for adequate memory destabilization and interference.

   Experiment 3E - Propranolol without reactivation. I don't see any data for this on the graphs. Am I missing something?

   Our apologies for the confusion. The legend shown next to Fig. 1F applies to all panels of Fig. 1, but only Experiment 1 (shown in Fig. 1A-B) contained a no-reactivation group as an additional control. Experiment 3 (shown in Fig. 1E-F) did not. We have moved the legend to the bottom of Fig. 1 to clarify this.

   The authors should probably cite this paper too, PMID: 21688892. The authors in this study find no evidence that propranolol inhibits cued fear memory reconsolidation.

   Thank you for bringing this to our attention. We were aware of this paper, but it had slipped through the cracks. We have cited it in the new version of the paper (p. 11).

   Read the original source
2. 

   eLife

   Sep 22, 2020

   ###Reviewer #3:

   Luyten et al's study examines the phenomenon of drug-induced post-retrieval amnesia for auditory fear memories in rats, and report that after several experiments using Propranolol, Rapamycin, Anisomycin or Cycloheximide that they essentially observe no disruption of reconsolidation, (i.e., no amnesia). This is a well-executed, written and meticulous study examining an important phenomenon. The author's lack of observing amnesia using these "reconsolidation blockers" highlights an important fact that systemic administration of these drugs at the time of memory retrieval may not robustly influence reconsolidation processes despite what the existing literature may collectively indicate. The author's data clearly indicate this point and it is important the scientific community be made aware of these difficulties in blocking …

   ###Reviewer #3:

   Luyten et al's study examines the phenomenon of drug-induced post-retrieval amnesia for auditory fear memories in rats, and report that after several experiments using Propranolol, Rapamycin, Anisomycin or Cycloheximide that they essentially observe no disruption of reconsolidation, (i.e., no amnesia). This is a well-executed, written and meticulous study examining an important phenomenon. The author's lack of observing amnesia using these "reconsolidation blockers" highlights an important fact that systemic administration of these drugs at the time of memory retrieval may not robustly influence reconsolidation processes despite what the existing literature may collectively indicate. The author's data clearly indicate this point and it is important the scientific community be made aware of these difficulties in blocking reconsolidation using systemic administration of these drugs.

   This group has previously published similar studies disputing similar phenomena. First highlighting a lack of amnesia following the reconsolidation-extinction paradigm and then more recently demonstrating a lack of amnesia attempting to block the reconsolidation of context fear memories. This is now their third installment focusing on Cued fear memories. Certainly, these findings are important, but arguably the novelty of such findings may be diminished a bit. In one of the "control" experiments where the experimenters administer anisomycin immediately post training, they observe a paradoxical result - they observe memory strengthening instead of the expected blockade of consolidation and amnesia. This result highlights a number of things to consider when we interpret these overall results. For one protein synthesis inhibitors(PSIs) are toxic and when administered systemically usually result in inducing the animals to have diarrhea and generally just makes them sick. This of course will make the animals stressed and agitated and result in increasing their stress and likely amygdala activity. All of this could likely be the reason why the animals exhibited memory strengthening or no impairment in consolidation even with a PSI on board. See PMCID: PMC7147976. Figure 6. In this study, they could rescue the impairment of PSI on consolidation by increasing BLA principal neuron firing. Thus an important take away is something like this could easily be happening in the reconsolidation experiments - that there is no blockade because the animals are stressed either due to PSI on board or because some issues with experimenter/animal interactions, etc lead to higher BLA neural activity and rescue of the reconsolidation process.

   I don't think the authors go far enough articulating the important differences between systemic and intra-cranial administration of these drugs. Time is a potential factor. Immediate administration of the drug at high concentration in the target brain region (BLA) versus many minutes until the drug gets to the target region with uncertain concentration levels that may not mirror levels reached with intracranial administration. It's unfortunate the authors were not able to include intra-BLA administration of these drugs in this study. I do not necessarily expect them to do such experiments, since they have already done so much and it is not clear the laboratory has the appropriate expertise to conduct such experiments, but this comparison would be helpful.

   I think it is important that the authors make some statement of training conditions on cannulated versus cannulated rats. For example, every animal in Nader's 2000 study was bilaterally cannulated targeting the BLA. In contrast every animal in this study underwent no such surgery. I think this is relevant. In my experience non cannulated animals are a bit smarter than cannulated animals and the training conditions across these two differing groups may not equate to the same level of learning. And of course, differences in learning levels can lead to differences in the ability of the retrieved memory to destabilize. The authors mention possibly examining markers of memory destabilization. GluR1 phosphorylation, Glur2 surface levels, protein degradation/ubiquitination have all been used to assess if destabilization has occurred. I do not fully agree with their reasons for not performing such experiments. They could examine some or one of these phenomena across differing training conditions between retrieval, no-retrieval animals. This likely could be informative. However, the authors may not possess the necessary expertise to conduct such experiments, so I'm not stating these experiments need to be completed, but certainly the study could be strengthened with such data.

   Experiment 3E - Propranolol without reactivation. I don't see any data for this on the graphs. Am I missing something?

   The authors should probably cite this paper too, PMID: 21688892. The authors in this study find no evidence that propranolol inhibits cued fear memory reconsolidation.

   Read the original source
3. 

   eLife

   Sep 22, 2020

   ###Reviewer #2:

   General assessment:

   In this study, Luyten et al. aimed to replicate post-retrieval amnesia of auditory fear memories reported numerous times in the literature. They used a variety of behavioural approaches combined with systemic pharmacological treatments (propranolol, rapamycin, anisomycin, cycloheximide) after reactivation of fear memories. Interestingly, none of the treatments induced a significant decrease of freezing responses during subsequent retrieval tests. Authors strengthened their null results by using Bayesian statistics, confirming the absence of drug-induced amnesia.

   Overall, the study is really interesting. Experiments and analyses are very well designed and bring some important findings to the debated topic of post-retrieval amnesia and its clinical relevance.

   I have nevertheless several comments for the …

   ###Reviewer #2:

   General assessment:

   In this study, Luyten et al. aimed to replicate post-retrieval amnesia of auditory fear memories reported numerous times in the literature. They used a variety of behavioural approaches combined with systemic pharmacological treatments (propranolol, rapamycin, anisomycin, cycloheximide) after reactivation of fear memories. Interestingly, none of the treatments induced a significant decrease of freezing responses during subsequent retrieval tests. Authors strengthened their null results by using Bayesian statistics, confirming the absence of drug-induced amnesia.

   Overall, the study is really interesting. Experiments and analyses are very well designed and bring some important findings to the debated topic of post-retrieval amnesia and its clinical relevance.

   I have nevertheless several comments for the authors to consider.

   -Despite being very detailed, the authors should clarify and uniformize their Methods section and Supplemental information (e.g. number of CS, contexts used...) to improve the understanding of the different approaches. Similarly, methods for the reinstatement protocol (Exp 2) are missing.

   -In exp 5, tests 1 and 2 are supposed to have 12 CS each. However, only 8 dots are represented on the graph. Did the authors average some freezing values after the initial 4 first CS presentations?

   -There is an obvious difference in baseline freezing response before the test in Exp 7 (Figure 5A-B). Discussion of these differences is an important point and was thoroughly discussed by the authors in the Supplement.

   -Ln 384-387: "... additional Bayesian analyses were carried out that collectively suggested substantial evidence for the absence of an amnestic effect". Despite the "substantial effect" given by the meta-analysis, I am a bit confused by the meaning of an "anecdotal evidence against drug < control" reported in half of the experiments. How do the authors interpret these results?

   -The effect of cycloheximide on memory consolidation is indeed unexpected. Even if beyond the scope of the current study, what is the authors' hypothesis to explain that cycloheximide in their conditions induced a pro-mnesic effects on the consolidation of fear memories but altered the consolidation of extinction?

   -Cycloheximide seemed to induced post reconsolidation amnesia of fear memory after extinction training (Exp 8, Fig 3G) but not after single CS reactivation. Can the authors please develop this point? Is it possible that several presentations of the CS is required to destabilise the initial memory trace?

   Read the original source
4. 

   eLife

   Sep 22, 2020

   ###Reviewer #1:

   This manuscript provides evidence that drug administration during a reconsolidation window does not necessarily prevent memory recall, as has been shown by many groups. The authors attempted to replicate several published experiments and despite demonstrating that the drugs had other effects on the animals' behavior and physiology (e.g. weight gain), no effects on memory were observed.

   The paper is nicely prepared.

   Read the original source
5. 

   eLife

   Sep 22, 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 reviewers all found this thorough report of the failure to replicate drug-induced post-retrieval amnesia to be interesting and the work was viewed as scientifically sound. But they were all concerned that the extent of the advance is not to the level that would be expected. They also raised substantive concerns regarding the reasons for the failures to replicate.

   Read the original source
6. 

   Version published to 10.1101/2020.07.08.193383 on bioRxiv

   Jul 8, 2020