Intense training prevents the amnestic effect of inactivation of dorsomedial striatum and induces high resistance to extinction

Martha Martínez-Degollado; Andrea C. Medina; Paola C. Bello-Medina; Gina L. Quirarte; Roberto A. Prado-Alcalá

doi:10.1371/journal.pone.0305066

Peer Review History

Original SubmissionFebruary 14, 2024
4 Apr 2024 Decision Letter - Yukiori Goto, Editor PONE-D-24-06106Intense training prevents the amnestic effect of inactivation of dorsomedial striatum and induces high resistance to extinctionPLOS ONE Dear Dr. Prado-Alcalá, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by May 19 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: No Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Roberto et al. investigated the effect of intense training on memory and extinction. They reported that intense training induces resistance to the extinction. I believed this manuscript is good but there are several concerned that should be attention by authors. The main point is that this finding should be support by histological parameters. I think neurological index such as Neru-D. Why did the researchers not use other learning tests such as the Morris Water Maze? How did the researchers divide the intensity into low, medium and high? Is there a standard? Point 1: PTSD abbreviation should be explaining previously. Point 2: It is surprising that a 9-week-old animal has a 350 g weight. Point 3: number of rats in each group and study design should be mention in materials and methods. Point 4: The quality of figure 1 is not clear. Reviewer #2: This study examined the effects of TTX inactivation of dorsomedial striatum in memory consolidation of foot shocks. TTX impaired consolidation with low intensities of foot shocks , but not with the high intensity, which are highly interesting. The major shortcoming of the study is obviously a lack of explanation for the underlying mechanisms, although the authors have proposed a highly speculative idea that spinogensis may be involved in the observations. Alongside this issue, I have only a few minor concerns. The word "intense training" has been used repeatedly in the title and over the manuscript, which misleads the study, as the study examined different intensities of foot shock, but not intensive training itself. Thus, it is better to replace the word with a more appropriately describing the experimental condition. It would be helpful to include a diagram describing the experimental design and timeline of drug treatments and behavioral training/testing. For instance, data in Figure 5 and 7 have reported the data alike, but the experiment was conducted differently for each of them are obscure. Please indicate whether the error bars in the figures are SD or SEM. Line 47-48 The sentence "Hindrance of memory consolidation is produced by ... tetrodotoxin" can read as if taking TTX causes amnesia, which is actually not. This should state more accurately that infusion of TTX to inactivate a specific brain region could cause amnesia. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: Plos one review.docx https://doi.org/10.1371/journal.pone.0305066.r001
Revision 1
17 May 2024 Author Response Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? Reviewer #1: Yes Reviewer #2: Yes ________________________________________ 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ________________________________________ 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: No Reviewer #2: Yes Reply: All the data underlying our findings are fully available in the file we uploaded in our original submission (Supporting Information; File Name: S1 DATA.pdf). ________________________________________ 4. Is the manuscript presented in an intelligible fashion and written in standard English? Reviewer #1: Yes Reviewer #2: Yes ________________________________________ 5. Review Comments to the Author Reviewer #1: Roberto et al. investigated the effect of intense training on memory and extinction. They reported that intense training induces resistance to the extinction. I believed this manuscript is good but there are several concerned that should be attention by authors. The main point is that this finding should be support by histological parameters. I think neurological index such as Neru-D. Reply. We tried hard to find the suggested neurological index Neru-D with no success; PubMed, Scopus, and other sources yielded 0 positive results. If this reviewer is asking about the injection sites, Figure 1 shows the location of the injector tips, all of which were within the dorsomedial striatum. Why did the researchers not use other learning tests such as the Morris Water Maze? Reply This study aimed to investigate the effects of the inactivation of the dorsomedial striatum on memory consolidation and subsequent extinction of learning acquired with different degrees of training. To this end, we decided to use a behavioral task, the inhibitory avoidance task, that has been used in my laboratory for many years. One of the advantages of this procedure is that it entails only one trial and one aversive stimulus, thus allowing to determine with precision the duration of the consolidation process; furthermore, we have defined the parameters of aversive stimulation that yield those different degrees of training and the characteristics of extinction learning. Hence, this task is quite appropriate to answer our experimental questions. In the case of the Morris Water Maze task, on the other hand, rats require several trials and sessions to learn, so it is difficult to determine when consolidation takes place; the other reason we did not use this task is that it would take us a significant amount of time to establish the correct parameters to produce distinct degrees of training and extinction curves for each training condition. How did the researchers divide the intensity into low, medium, and high? Is there a standard? Reply In lines 263-270 of the revised manuscript, we have now defined low, moderate, and intense training as follows: “We have studied the phenomenon of extinction using the same strain of rats, inhibitory avoidance conditioning chambers, and footshock parameters. We have found that 3.0 mA induces stronger learning (and memory) than 2.0, 1.0, 0.5, and 0 mA; rats trained with 1.0 or 2.0 mA learn better than those trained with 0.5 mA; 0 mA does not produce IA learning. We measured the strength of learning through resistance to extinction (resistance to extinction is stronger when the learning experience is also stronger). Based on these results we have termed low, moderate, and intense training as that mediated by 0.5 mA, 1.0 or 2.0 mA, and 3.0 mA, respectively (see references 12, 29, 30, 42, and 45).” In the revised manuscript we have also shown the comparison of resistance to extinction among the control groups; resistance to extinction was statistically higher in the 3.0 mA group, followed by the 1.0 mA group, and then by the 0.5 mA group.” Lines 345-353. Point 1: PTSD abbreviation should be explaining previously. Reply We thank the reviewer´s comment. We have now defined PTSD as post-traumatic stress disorder (line 43). Point 2: It is surprising that a 9-week-old animal has a 350 g weight. Reply We checked the age of the rats used in this study. They were, actually, 12-14 weeks old; we have made this correction in the new manuscript (line 100). Point 3: number of rats in each group and study design should be mention in materials and methods. Reply The number of rats varies in each experiment, so, in the original manuscript the sample size of each group was specified in the Figure legend of each experiment. Point 4: The quality of figure 1 is not clear. Reply The PDF version of the original Figure 1, made by the Journal, looks like this: However, the original figure is the following one, clearly showing the principal details. In case it is accepted, this is the figure that would be presented in our article. Reviewer #2: This study examined the effects of TTX inactivation of dorsomedial striatum in memory consolidation of foot shocks. TTX impaired consolidation with low intensities of foot shocks, but not with the high intensity, which are highly interesting. The major shortcoming of the study is obviously a lack of explanation for the underlying mechanisms, although the authors have proposed a highly speculative idea that spinogensis may be involved in the observations. Reply In the Discussion of the original manuscript, based on experimental results from other laboratories as well as our laboratory, we suggested several possible mechanisms that may underly the protective effect of high-intensity aversive stimulation training on memory consolidation; these suggested mechanisms are: A. Intense training induces the transfer of information to a wider neuronal network, and the affected structures are no longer critical sites for the encoding of overtraining [4,9,29]. In agreement with this interpretation, recent findings from our laboratory have provided compelling evidence that intense training induces the recruitment of a higher number of neurons in the amygdala [42] and ventral striatum [45] which may contribute to the formation of a stronger memory trace (lines 406-412). B. There is sound evidence showing that aversive training in rodents induces the release of the glucocorticoid hormone corticosterone (CORT), which is dependent upon the intensity of the aversive stimulation during learning [45, 48, 49]. Moreover, infusion of CORT or CORT-receptor agonists into the striatum, amygdala, and hippocampus facilitates memory consolidation of IA [50-54] (lines 443-447). It has also been shown that CORT administration increases the density of hippocampal dendritic spines in vitro in a dose-dependent manner. C. Preliminary data from our laboratory indicates that as a result of intense training new spines can be produced in DMS despite its inactivation by TTX (lines 439-441). Thus, based on these data, we have proposed that the protective effect of intense training is mediated by activation of plasmatic and/or membrane-bound corticosterone receptors. We are currently exploring these possibilities. Alongside this issue, I have only a few minor concerns. The word "intense training" has been used repeatedly in the title and over the manuscript, which misleads the study, as the study examined different intensities of foot shock, but not intensive training itself. Thus, it is better to replace the word with a more appropriately describing the experimental condition. Reply We agree that “intense training” might lead to some confusion. To avoid this possibility, in the original manuscript we had given an operational definition of intense training, that we have used over the years, as seen in lines 254-260 in the original manuscript “We defined intense or strong IA training as that in which training with a relatively high foot shock intensity produces higher resistance to extinction than lower foot shock intensities. Resistance to extinction is a measure of the strength of learning because resistance to extinction is stronger when the learning experience is also stronger; we had previously found that, in our hands, a foot shock of 3.0 mA fulfills this criterion [29, 41, 42]”; we have added more references in support of this definition (4, 12, 43, 45) (lines 261-262). In the revised manuscript we have also defined, operationally, these three levels of training, and have compared resistance to extinction among the control groups; resistance to extinction was statistically higher in the 3.0 mA group, followed by the 1.0 mA group, and then by the 0.5 mA group (lines 345-353). It would be helpful to include a diagram describing the experimental design and timeline of drug treatments and behavioral training/testing. For instance, data in Figure 5 and 7 have reported the data alike, but the experiment was conducted differently for each of them are obscure. Reply We have now included a diagram describing the experimental design and timeline of drug treatments and behavioral training/testing in all the figures showing behavioral results. Please indicate whether the error bars in the figures are SD or SEM. Reply In all the figures where error bars were represented (Figures 2, 3, 4, 5, and 7), it was stated that the data represent median step-through latencies, in seconds, with interquartile ranges. Line 47-48. The sentence "Hindrance of memory consolidation is produced by ... tetrodotoxin" can read as if taking TTX causes amnesia, which is actually not. This should state more accurately that infusion of TTX to inactivate a specific brain region could cause amnesia. Reply We fully agree with this reviewer and have changed this sentence to “Amnesia is produced by treatments that induce temporary disruption of normal electrical activity of brain structures involved in memory consolidation, such as tetrodotoxin (TTX) [1-8]” (lines 47-49). ________________________________________ 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0305066.r002
23 May 2024 Decision Letter - Yukiori Goto, Editor Intense training prevents the amnestic effect of inactivation of dorsomedial striatum and induces high resistance to extinction PONE-D-24-06106R1 Dear Dr. Prado-Alcalá, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Yukiori Goto, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Thank you for a revision on the manuscript, which has adequatly addressed the issues ratised by the referees at the initinal submission. Reviewers' comments: https://doi.org/10.1371/journal.pone.0305066.r003
Formally Accepted
28 May 2024 Acceptance Letter - Yukiori Goto, Editor PONE-D-24-06106R1 PLOS ONE Dear Dr. Prado-Alcalá, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Yukiori Goto Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0305066.r004

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