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PONE-D-26-10019

Chronic CBD treatment differentially modulates neurobehavioral outcomes and endocannabinoid signaling in an aged HIV-1 Tat transgenic mouse model

PLOS One

Dear Dr. Yadav-Samudrala,

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Reviewers' comments:

Reviewer’s Responses to Questions

-->Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #1: Partly

Reviewer #2: Yes

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-->2. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: Yes

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-->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

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-->4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: Yes

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-->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: The study addresses a clinically relevant and understudied question—the effects of prolonged CBD exposure on neuroHIV in an aging context, and represents a meaningful extension of the group’s prior work on acute CBD exposure in this model. The inclusion of sex as a biological variable across behavioral, lipidomic, and proteomic endpoints strengthens the translational relevance of the findings. However, several limitations should be addressed and/or discussed.

1. While all very important factors, the sample size is small to assess sex*genotype*treatment. It is impressive that the differences in readouts reached significance, but a post-hoc power analysis should be included with discussion of plans to affirm the findings in a larger cohort.

2. There is much discussion about the potential protective effects of CBD on the endocannabinoid system (ECS) and brain function, but there is little acknowledgement of the literature on state of the ECS in the brain (and systemic) in people with HIV. Considering the study is on the effects of CBD in a tat mouse, this seems pertinent to the intro and discussion.

3. The data availability section appears to be missing important information... XXX

4. Memory was tested in the mice and some lipids were measured in the hippocampus, but no western blots of the hippocampus. This should be added or discussed.

5. While not entirely necessary, it is surprising that no measures of neuroinflammation were included, ie GFAP, IBA1, TNFa.

Reviewer #2: PONE-D-26-10019: Peer-Review Comments

This study examines the effects of chronic cannabidiol (CBD) treatment on aged HIV-1 Tat transgenic mice, investigating behavioral outcomes and changes in endocannabinoid system. The authors employed a 12-week CBD treatment protocol (3 mg/kg, s.c.) in 15-18 month-old mice, assessing cognition, anxiety-like behavior, nociception, and endocannabinoid signaling across multiple CNS regions. Key findings include gender-specific improvements in recognition memory (particularly in females), significantly altered thermal nociception, and complex region-dependent restructuring of the endocannabinoid system.

The work is supported by rigorous experimental design, clinically relevant aged model, multi-modal assessments across multiple brain regions, and inclusion of sex as a biological variable. The study’s longitudinal approach with multi-modal assessments (behavioral, biochemical) across four CNS regions (prefrontal cortex, amygdala, brainstem, spinal cord) provides valuable temporal and mechanistic insights. The inclusion of both sexes as a biological variable is particularly commendable given known sex differences in cannabinoid responses. Using aged mice addresses a critical gap in the literature, as most prior studies focus on young adults. This is highly relevant given the aging population of people living with HIV. The endocannabinoid system mapping is thorough. The combination of mass spectrometry for endocannabinoid ligand quantification and Western blotting for protein expression provides robust molecular characterization. The authors acknowledge and report three-way interactions (sex × genotype × treatment), demonstrating statistical sophistication and avoiding oversimplification of complex biological phenomena. Authors also acknowledge important limitations, for example, authors acknowledge that bulk tissue analysis cannot distinguish neuronal vs. glial contributions, which is critical given that Tat expression is astrocyte-driven in this model.

Specific Comments:

1. The authors report CBD-induced hyperthermia as a "critical physiological finding" (Page 31) yet continue to interpret/ discuss hot plate results as hyperalgesia. If baseline body temperature is elevated, isn’t the thermal gradient between body and hot plate is reduced? mechanically decreasing latency regardless of true nociceptive sensitivity? While the authors acknowledge this confounding issue ("explaining the paradoxical reduction in hot-plate latencies"), authors may need to adjust their conclusions accordingly, and revise the entire supra-spinal nociception narrative or, at minimum, explicitly state the limitations in discussion with regards to the extent of reliability of thermal nociception assessed in this context. Focusing solely on the thermoregulatory mechanisms and removing interpretive claims about hyperalgesia should be considered.

2. The "psychostimulant-like effect" (Page 32) attributed to Tat-mediated dopaminergic dysregulation appears speculative without dopamine measurements. This aspect can be clearly stated. Clearly mentioning these as hypotheses based on preliminary observations requiring future testing is recommended.

3. All assessments occurred at week 16-17, at least 72 hours after the final injection, if I am correct. No information is provided about CBD half-life or acute/chronic pharmaco-physiological levels, making it unclear whether observed effects reflect drug presence or long-term neuroplastic changes (or both). I recommend discussing this briefly.

4. Tables 1-3 containing Tukey post-hoc comparisons should be moved to supplemental material. This improves readability while maintaining statistical rigor. Readers can grasp key patterns from annotated figures without navigating lengthy tables, while those requiring detailed comparisons can consult the supplement.

5. The discussion is lengthy and somewhat repetitive. Integration of findings across brain regions occurs sporadically rather than systematically. A structured synthesis organized by behavioral domain (cognition, affect, pain) rather than region-by-region analysis would make it more succinct while improving clarity. Extensive discussion of CBD’s antioxidant properties, cerebral blood flow effects, and 5-HT1A/TRPV1 interactions is provided; while referencing supporting literature is appropriate, the narrative sometimes implies these mechanisms were demonstrated in the current study when they were not.

This manuscript addresses a clinically relevant question using an appropriately aged animal model and comprehensive methodology. The sex-specific cognitive benefits of chronic CBD in the context of HIV-associated neurological complications represent a meaningful contribution.

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Reviewer #1: Yes: Jerel Adam Fields

Reviewer #2: No

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Chronic CBD treatment differentially modulates neurobehavioral outcomes and endocannabinoid signaling in an aged HIV-1 Tat transgenic mouse model

PONE-D-26-10019R1

Dear Dr. Yadav-Samudrala,

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.

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Kind regards,

Vinod K. Yaragudri, M.Sc., Ph.D

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer’s Responses to Questions

-->Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.-->

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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-->2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #1: Yes

Reviewer #2: Yes

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-->3. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: Yes

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-->4. 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: Yes

Reviewer #2: Yes

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-->5. 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

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-->6. 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: The authors did a nice job responding to critiques. The major and minor concerns were addressed and the manuscript is much improved.

Reviewer #2: Revision is adequate. Authors have made a sincere effort and have satisfactorily addressed the reviewers' comments.

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-->7. 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: Yes: Jerel Adam Fields

Reviewer #2: No

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