Neuroproteomic Profiling of the Anxiolytic Potential of Stypopodium zonale in Drosophila

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Introduction
The authors correctly define neuroproteomics, highlight its advantages over genomics, and appropriately situate it in the context of biomarker discovery and drug development. However, this section has a distinctly textbook-like character and should be shortened in favor of stronger linkage to the specific research problem. The authors ascribe excessive inferential power to proteomics, failing to acknowledge the method’s limitations, particularly the lack of information on protein activity, post-translational modifications, and subcellular localization. Consequently, the observed changes could just as well reflect a nonspecific stress or metabolic response rather than processes directly related to anxiety regulation. Additionally, the formulated research hypotheses are very general and poorly falsifiable: the expectation of “broad proteomic changes” following extract exposure is trivial, and the concept of “anxiety-related proteins” is not precisely defined in the context of Drosophila, creating a risk of post hoc interpretation based on bioinformatic analyses rather than clearly defined a priori criteria.

Results
The presented results clearly show that the Stypopodium zonale extract induces significant and reproducible changes in the Drosophila melanogaster proteome. However, they do not provide sufficient grounds to infer a specific anxiolytic mechanism. The data rather indicate broad, non-selective modulation of the neuronal proteome, whose functional significance remains unclear without behavioral, physiological, and biochemical evidence.

Discussion
The most serious limitation of the study is the extensive mechanistic model, which relies on a cascade of assumptions not supported by direct functional data. Reduced abundance of G-proteins, components of the cAMP–PKA–CREB pathway, SNARE proteins, SAP47, or GAD1 does not allow inferences about receptor activity, kinase signaling, or changes in synaptic transmission. The observed global decrease in protein levels could equally reflect a nonspecific stress response, starvation effects, or extract toxicity rather than selective modulation of “anxiety” circuits. Attributing these changes to anxiolytic action is particularly problematic given the lack of behavioral data and the absence of an established anxiety model in Drosophila. The discussion of MP1–MVP2 circuits and translational analogies to human anxiety disorders is speculative and gives the impression of post hoc interpretation. Additionally, inconsistencies appear in the discussion, e.g., simultaneously suggesting reduced synaptic plasticity while accepting IPA predictions indicating LTP activation.

Materials and Methods

This section should be moved before the Results and Discussion sections, as its current placement disrupts the logical flow of the paper—the methodology appears after the interpretation of results, making it difficult to assess the rigor of the analyses, the choice of tools, and the validity of the conclusions. Justification is lacking for key experimental parameters, such as the extract dose, the 6-hour exposure period, and the number of biological replicates. It is unclear whether males and females were analyzed together or separately, and whether potential sex differences were statistically controlled. The use of 95% ethanol as a vehicle is not adequately discussed regarding its possible impact on Drosophila physiology and proteome, and the control conditions do not fully match the experimental conditions (e.g., no clear information on the presence/amount of ethanol in controls). The exposure procedure based on consumption of a colored solution was not quantitatively verified, and no data were provided to confirm the actual level of extract ingestion by the flies or individual variability. In the proteomics section, there is insufficient distinction between biological and technical replicates, and randomization and blinding of samples are not clearly described. Statistical analysis methods are described only generally, without justification for significance thresholds, and the use of MetaboAnalyst for proteomic data is not critically discussed.

Conclusions
The conclusions focus mainly on the potential social and economic impact of the project, giving them an appealing narrative, but they do not meet a key scientific requirement—namely, the summary and critical interpretation of the actual study results and the explanation of their mechanistic significance.

Author Response

Comment: The authors correctly define neuroproteomics, highlight its advantages over genomics, and appropriately situate it in the context of biomarker discovery and drug development. However, this section has a distinctly textbook-like character and should be shortened in favor of stronger linkage to the specific research problem. The authors ascribe excessive inferential power to proteomics, failing to acknowledge the method’s limitations, particularly the lack of information on protein activity, post-translational modifications, and subcellular localization.

Author, s Response: We acknowledge that the observed differences in protein levels between control and experimental groups reported in the manuscript and the inferences made from those differences do not consider post-translational modifications, however, subcellular localization is addressed in Figure 1B. The criteria for the selection of our proteins of interest, which are emphasized in the paper, are related to function and/or signaling canonical pathway. Refer to a new table (Table 3) in the manuscript.

Comment: Consequently, the observed changes could just as well reflect a nonspecific stress or metabolic response rather than processes directly related to anxiety Additionally, the formulated research hypotheses are very general and poorly falsifiable: the expectation of “broad proteomic changes” following extract exposure is trivial, and the concept of “anxiety-related proteins” is not precisely defined in the context of Drosophila, creating a risk of post hoc interpretation based on bioinformatic analyses rather than clearly defined a priori criteria,

Author's Response: We hypothesize that the administration of the Stypopodium zonale extract could alter expression in the experimental flies, and that the observed changes could be related to the decrease in fear related behavior we reported previously. We agree completely with your observation that the expression “broad proteomic changes” might be misleading, and that these changes might also reflect metabolic responses and general stress response not directly linked to anxiety regulation. We took notice and addressed that in the revised manuscript.

 

Comment: The presented results clearly show that the Stypopodium zonale extract induces significant and reproducible changes in the Drosophila melanogaster proteome. However, they do not provide sufficient grounds to infer a specific anxiolytic mechanism. The data rather indicate broad, non-selective modulation of the neuronal proteome, whose functional significance remains unclear without behavioral

Author’s Response: We are proposing a general mechanism of action based mainly on the dysregulated proteins in the experimental group with respect to the control group. We are aware that the proposed model of the mechanism of action of Stypopodium zonale related to anxiolysis needs substantial future validation and probably important modifications. However, we have previous published behavioral results that clearly demonstrate an anxiolytic effect of the extract in anxiety-related behavior in Drosophila, measured using the Open Field Test and the Dark/light Box Test. Thus, the behavioral findings already reported by our laboratory can at least be partially rooted on the findings of this proteomic analysis.

Comment: The most serious limitation of the study is the extensive mechanistic model, which relies on a cascade of assumptions not supported by direct functional data. Reduced abundance of G-proteins, components of the cAMP–PKA–CREB pathway, SNARE proteins, SAP47, or GAD1 does not allow inferences about receptor activity, kinase signaling, or changes in synaptic transmission.  The observed global decrease in protein levels could equally reflect a nonspecific stress response

Author’s Response: As stated above in this reply, the use of extracts of Stypopodium zonale for the experiments and results presented in this manuscript are based on our publication reporting anxiolysis in Drosophila after the administration of the extract, measured using the Open Field Test. The extract is not toxic to the flies since it does not alter sexual and reproductive behavior, development of the flies since oviposition, feeding behavior nor locomotor activity (measured using the rapid iterative negative geotropism assay). The fasting period is inside the normal time range used in these types of experiments with Drosophila. We agree with your comment hat the observed global decrease in protein levels can partially reflect a nonspecific stress response and we addressed that in the revised manuscript.

Reduced markers of synaptic plasticity in proteomic data from whole Drosophila heads can coexist with IPA predictions of long-term potentiation (LTP) pathway activation because these findings reflect different biological scales and directions of regulation. First, “reduced synaptic plasticity” in this context likely refers to lower abundance of proteins involved in structural remodelling, synapse turnover, or activity-dependent destabilization, which are often elevated during stress and anxiety-like states. An anxiolytic algal extract could normalize neural circuits by stabilizing synapses and decreasing excessive or maladaptive plasticity, especially in stress-responsive circuits such as the mushroom bodies and central complex. At the same time, IPA infers “LTP activation” based on the pattern of protein changes mapping onto conserved vertebrate learning and memory pathways; many of these proteins (e.g., CaMKII, PKA pathway components, actin regulators) also support efficient synaptic transmission and signal amplification, not just structural growth. Thus, the extract may promote a state of functional potentiation with structural stabilization — enhancing signal fidelity and adaptive circuit strength while reducing noisy, stress-induced remodelling. Additionally, because the proteome was derived from entire fly heads, opposing changes in different neuron populations (e.g., decreased plasticity in stress circuits but increased potentiation in modulatory or inhibitory circuits) can produce a global signature that IPA interprets as LTP-like activation even when some plasticity-associated proteins decline.

Comment 5: This section should be moved before the Results and Discussion sections, as its current placement disrupts the logical flow of the paper - the methodology appears after the interpretation of results, making it difficult to assess the rigor of the analyses, the choice of tools, and the validity of the conclusions. Justification is lacking for key experimental parameters, such as the extract dose, the 6-hour exposure period, and the number of biological replicates. It is unclear whether males and females were analyzed together or separately, and whether potential sex differences were statistically controlled. The use of 95% ethanol as a vehicle is not adequately discussed regarding its possible impact on Drosophila physiology and proteome, and the control conditions do not fully match the experimental conditions (e.g., no clear information on the presence/amount of ethanol in controls). The exposure procedure based on consumption of a colored solution was not quantitatively verified, and no data were provided to confirm the actual level of extract ingestion by the flies or individual variability.

Author’s Response: We strictly followed the journal template and instructions for authors. The extract dose that was administered for these experiments was the same used for the behavioral assays that we previously published. Both for the control and experimental groups, four replicas were used. Each replica consisted of 120 Dropsophila heads. The six-hour exposure time is inside the regular time range for acute administration of drugs to Drosophila. Males and females were used for the experiments, since sex differences were out of our scope for this study. Ethanol was used as a transient vehicle for the organic extract, since it was already evaporated when the flies were exposed to the extract on the paper discs. Ethanol is an adequate solvent and vehicle for extracts in which polar chemical entities are abundant. We agree on your comment about the extent of extract consumption by each fly. However, the volume of extract in each paper disc exceeds the potential food ingestion of a fly during a 6-hour period.

Comment: The conclusions focus mainly on the potential social and economic impact of the project, giving them an appealing narrative, but they do not meet a key scientific requirement—namely, the summary and critical interpretation of the actual study results and the explanation of their mechanistic significance.

Author’s Response: Refer to revised manuscript.

 

 

 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

A study that identifies 951 proteins, across four biological replicates per condition, of adult Drosophila melanogaster flies, used as the animal model for new directions in drug discovery using marine-derived natural products and invertebrate models for the treatment of anxiety disorders.

 

The strengths are (1) the study is novel, (2) the Introduction presents the context of the problem and defines the terms, (3) the tables and figures—appropriately examined—are helpful in their presentation of the Results, (4) the Discussion is comprehensive, (5) methodological limitations are considered in the Discussion, (6) future research directions are suggested, (7) the Materials and Methods are explained sufficiently for study replication.

 

The weaknesses are (1) several of the citations in the Introduction and the Discussion are to outdated research lacking the additional support of citations to research published since 2022, (2) the hypotheses are not highlighted sufficiently in the Introduction nor in the Conclusion, (3) some of the citations are out of order, (4) some references on the list are not cited in the text, and (5) the Conclusion does not include information regarding drug development for anxiety—the reason for undertaking this study.

 

Line-by-line suggested edits

Several of the citations in the Introduction are outdated. Please add supporting citations of research published since 2022. These are the line numbers where they appear: 67, 70, 86, 90, 95, 113, 121. If there is no more recent research regarding any of these citations, please state the date of publication in the text of the citations.

According to the Discussion, there are two hypotheses for this study. Please add a paragraph after line 140 that states these two hypotheses directly.

142–144 Please delete this information. It is from the template.

Figures and tables must follow the paragraph in which they are first mentioned. Please move Table 1 and Table 2 after line 171. Then, start a new paragraph and move Figure 1B after line 182. Please also join lines 174 and 175.

The following citations in the Discussion are outdated. Please add supporting citations of research published since 2022. These are the line numbers where they appear: 281, 308, 317, 328, 336, 343, 351354, 355, 357, 368, 378, 383, 403, 426, 437, 489, 491, 498, and 524. If there is no more recent research regarding any of these citations, please state the date of publication in the text of the citations.

The citations in the Materials and Methods and the Discussion are numbered inappropriately. Although the Materials and Methods is the fourth section, the citations are numbered as if this section were the second. Additionally, the Discussion citations are numbered as if they follow the Materials and Methods—which they do not. Please renumber all the citations appropriately.

524 Citation [47] is the last citation number in the text. There is no mention of either reference 48 or 49. As they are outdated, they can be eliminated. If they are relevant, please include them where appropriate, along with a citation to research published since 2022 as support.

In line 343, to confirm this claim, please examine the following publication that I located in a search of the topic on Google Scholar: Mejías, K., Robles, G., Martínez, Z., Torres, A., Algarín, L., López, G., & Chiesa, R. (2016). Effects similar to anxiolysis in an organic extract of Stypopodium zonale on an anxiety related behavior in Drosophila melanogaster. American Journal of Undergraduate Research, 13(4). https://www.researchgate.net/profile/Ricardo-Chiesa/publication/311985357_Effects_similar_to_anxiolysis_in_an_organic_extract_of_Stypopodium_zonale_on_an_anxiety_related_behavior_in_Drosophila_melanogaster/links/586717ba08ae8fce490f22a8/Effects-similar-to-anxiolysis-in-an-organic-extract-of-Stypopodium-zonale-on-an-anxiety-related-behavior-in-Drosophila-melanogaster.pdf.

728–745 There is no mention of the hypotheses tested or the purpose of the study regarding the development of drugs for anxiety, laying the foundation for future behavioral validation. Please rewrite these Conclusions to focus on these points. Please also delete the information on stimulating Puerto Rico’s economy and the environmental impact, as neither is a feature of the study.

Author Response

 

Reviewer Comment:
Several of the citations in the Introduction are outdated. Please add supporting citations of research published since 2022. These are the line numbers where they appear: 67, 70, 86, 90, 95, 113, 121. If there is no more recent research regarding any of these citations, please state the date of publication in the text of the citations.

Response:
We thank the reviewer for this valuable observation. To address this comment, we carefully revised the Introduction and updated the cited literature at the indicated locations to include recent studies published since 2022. Specifically, we added contemporary references to support advances in neuroproteomics applied to neuropsychiatric disorders, proteomics-driven drug discovery, the neuroprotective and pharmacological potential of marine macroalgae, and the use of Drosophila melanogaster as a translational model for anxiety- and stress-related research.

Where recent literature was available, new citations were incorporated to complement or replace older references (lines 73, 77, 107, 128, and 139). In cases where foundational or seminal studies were retained due to their continued relevance, the publication year has been explicitly stated in the text to clarify their historical context. These revisions ensure that the Introduction reflects the current state of the field while maintaining appropriate acknowledgment of foundational work.

Reviewer Comment: According to the Discussion, there are two hypotheses for this study. Please add a paragraph after line 140 that states these two hypotheses directly.

Response: We thank the reviewer for this suggestion. To improve clarity, we have added a dedicated paragraph after line 140 explicitly stating the two central hypotheses of the study. This paragraph clearly outlines (1) the expected proteomic changes following Stypopodium zonale exposure and (2) the proposed association of these molecular changes with anxiety-related signaling pathways. This addition ensures that the study hypotheses are clearly defined and easily identifiable for the reader.

Reviewer Comment:
142-144 Please delete this information. It is from the template.

Response:
We thank the reviewer for pointing this out. The template text located at lines 142-144 has been removed from the manuscript as suggested.

 

The following citations in the Discussion are outdated. Please add supporting citations of research published since 2022. These are the line numbers where they appear: 281, 308, 317, 328, 336, 343, 351,354, 355, 357, 368, 378, 383, 403, 426, 437, 489, 491, 498, and 524. If there is no more recent research regarding any of these citations, please state the date of publication in the text of the citations.

Line 390, 437 include new citations,

 

The citations in the Materials and Methods and the Discussion are numbered inappropriately. Although the Materials and Methods is the fourth section, the citations are numbered as if this section were the second. Additionally, the Discussion citations are numbered as if they follow the Materials and Methods-which they do not. Please renumber all the citations appropriately.

The citations have fixed .

Reviewer Comment:

524 Citation [47] is the last citation number in the text. There is no mention of either reference 48 or 49. As they are outdated, they can be eliminated. If they are relevant, please include them where appropriate, along with a citation to research published since 2022 as support.

Reviewer comment (Line 343):
In line 343, to confirm this claim, please examine the following publication…

Response:
We thank the reviewer for highlighting this relevant study. The work by Mejías et al. (2016) demonstrates anxiolytic-like behavioral effects of a crude organic extract of Stypopodium zonale in Drosophila melanogaster, and we agree that it provides important prior evidence supporting the relevance of S. zonale as a candidate for anxiolytic research.We have therefore incorporated this reference into the Introduction, where it appropriately supports the rationale for selecting Stypopodium zonale and justifies its relevance for anxiolytic drug discovery.

The statement in line 343, however, specifically addresses intracellular signaling mechanisms involving Gβγ-mediated regulation of PLCβ and calcium mobilization. As the study by Mejías et al. (2016) does not investigate molecular or signaling mechanisms related to Gαq, Gβγ, PLCβ, or Ca²⁺ dynamics, we did not cite it in this mechanistic context to avoid misattribution. We have clarified the text to emphasize that, while prior behavioral evidence exists, the specific molecular mechanisms discussed here have not yet been documented in Drosophila, thereby strengthening the distinction between established behavioral findings and the novel mechanistic insights proposed in this study.

Reviewer comment (Lines 728–745):
There is no mention of the hypotheses tested or the purpose of the study regarding the development of drugs for anxiety…

Response:
We thank the reviewer for this insightful comment. We have fully rewritten the Conclusions section to explicitly restate the two central hypotheses guiding the study and to clarify the purpose of the work within the context of anxiolytic drug discovery. The revised Conclusions now emphasize (i) the hypothesis-driven identification of differential protein abundance following Stypopodium zonale exposure, (ii) the translation of these proteomic changes into a mechanistic framework relevant to anxiety-related signaling pathways, and (iii) the role of these findings as a molecular foundation for future behavioral validation in Drosophila melanogaster. In accordance with the reviewer’s suggestion, we have removed references to stimulating Puerto Rico’s economy and environmental impact, as these elements are outside the scope of the present study. The revised section is now fully aligned with the scientific objectives and translational focus of the manuscript.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I thank the Authors for their detailed responses to the raised comments. While these responses address some of the concerns, they remain insufficient in several key aspects from a reviewer’s perspective. In particular, the issue of excessive inferential power attributed to proteomic analyses has not been fully addressed. The Authors acknowledge that post-translational modifications were not analyzed and refer to the discussion of subcellular protein localization; however, they do not engage with the fundamental limitation of this approach, namely the lack of information on protein activity and signaling dynamics. As a result, the response appears largely defensive and does not lead to a meaningful attenuation of the mechanistic interpretations whose validity was the subject of the original critique.

The Authors respond more convincingly to the concern regarding the generality of the research hypotheses and the risk of post hoc interpretation. Their explicit acknowledgment that the term “broad proteomic changes” may be misleading, and that the observed differences may also reflect nonspecific metabolic or stress-related responses, is appreciated. The declaration that appropriate revisions have been introduced into the manuscript indicates that this comment has been genuinely taken into account.

With respect to the lack of sufficient grounds for inferring a specific anxiolytic mechanism, the Authors’ response is only partially satisfactory. Although they concede that the proposed mechanism is hypothetical and requires further validation, they rely on previously published behavioral results to support their interpretation. This does not resolve the core issue raised in the review, namely that proteomic analysis alone—particularly when performed on homogenates of whole Drosophila heads—does not allow for inference about specific neuronal mechanisms underlying the observed anxiolytic effect.

The most serious concerns remain in relation to the expanded mechanistic model proposed in the manuscript. Rather than restricting interpretation to a descriptive level, the Authors develop a speculative narrative involving synaptic plasticity and activation of LTP-related pathways, based primarily on bioinformatic analyses. While internally coherent, this argumentation is not supported by direct functional data and, in practice, exacerbates the very issue highlighted in the review: the construction of a cascade of assumptions that exceeds the inferential capacity of the applied methodology.

Equally unconvincing is the response addressing methodological issues. Referring to the journal template does not constitute sufficient justification for a suboptimal manuscript structure in which the methodology is presented after the interpretation of results. The lack of control for potential sex-specific differences cannot be justified by stating that this issue was beyond the scope of the study. Similarly, claims regarding the absence of effects of ethanol as a vehicle and the adequacy of extract exposure are not supported by quantitative data, which hinders proper assessment of the reliability and comparability of the control and experimental conditions.

Author Response

 

REVIEWER 1 COMMENTS AND REPLIES – 2^ND REVISION – FEBRURARY 2026

COMMENT 1:

I thank the Authors for their detailed responses to the raised comments. While these responses address some of the concerns, they remain insufficient in several key aspects from a reviewer’s perspective. In particular, the issue of excessive inferential power attributed to proteomic analyses has not been fully addressed. The Authors acknowledge that post-translational modifications were not analyzed and refer to the discussion of subcellular protein localization; however, they do not engage with the fundamental limitation of this approach, namely the lack of information on protein activity and signaling dynamics. As a result, the response appears largely defensive and does not lead to a meaningful attenuation of the mechanistic interpretations whose validity was the subject of the original critique.

RESPONSE:

We thank the reviewer for this important clarification and agree that proteomic profiling of total protein abundance does not provide direct information on protein activity, post-translational modifications, or signaling dynamics. In response, we have further revised the Discussion to explicitly state these methodological constraints and to avoid any implication that pathway activation or functional synaptic plasticity changes were directly measured. The sections describing synaptic and LTP-related processes have been rewritten to emphasize that these associations arise from enrichment analyses and known functional annotations of the differentially expressed proteins, rather than from direct assessment of signaling state or protein function. We have also added a dedicated paragraph outlining the specific limitations of whole-head proteomics, including the inability to resolve cell-type specificity, and dynamic regulation of signaling pathways. These revisions substantially temper the mechanistic narrative and more clearly position the proposed model as a hypothesis-generating framework intended to guide future targeted functional studies.

 

COMMENT 2:

With respect to the lack of sufficient grounds for inferring a specific anxiolytic mechanism, the Authors’ response is only partially satisfactory. Although they concede that the proposed mechanism is hypothetical and requires further validation, they rely on previously published behavioral results to support their interpretation. This does not resolve the core issue raised in the review, namely that proteomic analysis alone -particularly when performed on homogenates of whole Drosophila heads-does not allow for inference about specific neuronal mechanisms underlying the observed anxiolytic effect.

RESPONSE TO COMMENT 2:

We appreciate the reviewer’s emphasis on the interpretative limits of whole-head proteomic data. We agree that such analyses alone cannot establish specific neuronal or circuit-level mechanisms underlying the observed behavioral phenotype. To address this concern, we have revised the manuscript to decouple the behavioral findings from any direct mechanistic inference at the neuronal level. Prior behavioral data are now cited solely to support the reproducibility and robustness of the anxiolytic-like phenotype, not as evidence for a particular molecular or synaptic mechanism. The Discussion has been further modified to clarify that the proteomic results identify molecular correlates and enriched functional categories associated with the treatment condition, but do not permit conclusions regarding the specific neural substrates, cell types, or synaptic processes involved. This distinction is now stated explicitly to ensure that the scope of inference remains aligned with the resolution of the experimental approach.

 

COMMENT 3:

The most serious concerns remain in relation to the expanded mechanistic model proposed in the manuscript. Rather than restricting interpretation to a descriptive level, the Authors develop a speculative narrative involving synaptic plasticity and activation of LTP-related pathways, based primarily on bioinformatic analyses. While internally coherent, this argumentation is not supported by direct functional data and, in practice, exacerbates the very issue highlighted in the review: the construction of a cascade of assumptions that exceeds the inferential capacity of the applied methodology.

RESPONSE TO COMMENT 3:

We appreciate the reviewer’s careful evaluation of our proposed mechanistic model and agree that the proteomic and bioinformatic analyses do not constitute direct functional validation of synaptic plasticity or LTP-related processes. Our intention was not to present these pathways as experimentally demonstrated mechanisms, but rather as biologically plausible hypotheses generated from the observed protein expression patterns. To address this concern, we have revised the manuscript to clearly distinguish descriptive findings from mechanistic interpretation and have moderated the language throughout the Discussion to avoid causal implications. The proposed model is now explicitly framed as a working hypothesis intended to guide future functional studies, rather than as a confirmed signaling cascade. We believe this clarification preserves the value of the integrative analysis while remaining within the inferential limits of the applied methodology.

COMMENT 4:

Equally unconvincing is the response addressing methodological issues. Referring to the journal template does not constitute sufficient justification for a suboptimal manuscript structure in which the methodology is presented after the interpretation of results. The lack of control for potential sex-specific differences cannot be justified by stating that this issue was beyond the scope of the study. Similarly, claims regarding the absence of effects of ethanol as a vehicle and the adequacy of extract exposure are not supported by quantitative data, which hinders proper assessment of the reliability and comparability of the control and experimental conditions.

RESPONSE TO COMMENT 4:

We thank the reviewer for highlighting important methodological and reporting issues. In response, we have reorganized the manuscript so that the Methods section now precedes the Results, improving clarity and adherence to standard scientific structure. Regarding sex-specific effects, we acknowledge this as a limitation and have added a statement in the Discussion explicitly recognizing that sex was not analyzed as a biological variable and that future studies should address potential sex-dependent responses. Concerning ethanol vehicle exposure and extract dosing, we have expanded the Methods and Results sections to include quantitative details on vehicle concentration, exposure duration, and feeding conditions, and we now report the absence of detectable behavioral or proteomic effects in vehicle-only controls. These additions improve transparency and allow readers to better assess the comparability and reliability of experimental and control conditions.

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you to the authors for the changes to the manuscript. All have improved it. A few remain

 

Line-by-line suggested edits.

71–117 The Introduction has several outdated citations. Please add support to them from research published since 2022. If no such newer research exists, please state the publication year in the text. These are the line numbers and the older citations: 71 [4], 74 [5], 96 [10], 108 [11], 113 [12], and 117 [10].

128 Please cite research published since 2022 to support the claim “Benzodiazepines, although widely prescribed”.

128–129 Please cite research published since 2022 to support the claim “present well-documented limitations associated with long-term use”.

131 Please cite research published since 2022 to support the claim “many patients fail to achieve sustained remission with existing first-line treatments”.

435–710 The Discussion has several outdated citations. Please add support to them from research published since 2022. If no such newer research exists, please state the publication year in the text. These are the line numbers and the older citations: 435 [20], 467 [22], 480 [23,24], 491 [22], 499 [25], 506 [26], 514 [27], 517 [28,29], 519 [50], 520 [31], 542 [30], 552 [32], 557 [33], 577 [34], 604 [35], 615 [36], 669 [40], 671 [41], 678 [42], and 710 [45].

Author Response

Comment: 71–117 The Introduction has several outdated citations. Please add support to them from research published since 2022. If no such newer research exists, please state the publication year in the text. These are the line numbers and the older citations: 71 [4], 74 [5], 96 [10], 108 [11], 113 [12], and 117 [10]. 

Response: We thank the reviewer for this important suggestion. We systematically revised both the Introduction and the Discussion to address outdated citations by prioritizing recent literature published since 2022 wherever possible. Specifically, we replaced several older references with updated reviews and primary research articles (2022–2025) that more accurately reflect the current state of the field. In cases where foundational or historical references were retained because no substantially newer mechanistic studies were available, we explicitly stated the publication year in the text to clarify the temporal context of the evidence.These revisions were implemented at the specific line numbers indicated by the reviewer in both the Introduction (lines 71–131) and the Discussion (lines 435–710). As a result, the reference list and in-text citations were updated to improve the timeliness, relevance, and accuracy of the manuscript.

Comment: 128 Please cite research published since 2022 to support the claim “Benzodiazepines, although widely prescribed”.

Response: We updated these citations by replacing older sources with recent reviews and primary studies published since 2022 that support the same statements. Where no newer mechanistic studies were available, the publication year was explicitly stated in the text.

Comment: "128–129 Please cite research published since 2022 to support the claim “present well-documented limitations associated with long-term use”.

Response: We replaced older citations with recent literature (2023–2025) addressing (i) current prescribing trends of benzodiazepines, (ii) well-documented limitations and adverse outcomes associated with long-term use, and (iii) incomplete remission rates with first-line anxiety treatments. These updates strengthen the clinical relevance and timeliness of the statements.

Comment: 435–710 The Discussion has several outdated citations. Please add support to them from research published since 2022. If no such newer research exists, please state the publication year in the text. These are the line numbers and the older citations: 435 [20], 467 [22], 480 [23,24], 491 [22], 499 [25], 506 [26], 514 [27], 517 [28,29], 519 [50], 520 [31], 542 [30], 552 [32], 557 [33], 577 [34], 604 [35], 615 [36], 669 [40], 671 [41], 678 [42], and 710 [45].

Response: We revised all instances of outdated references in the Discussion by incorporating recent reviews and primary research articles (2022–2025) on GPCR signaling, calcium-dependent neurotransmission, synaptic plasticity, anxiety-related mechanisms, and proteomic pathway interpretation. Foundational studies were retained only when mechanistic insights remain current, and their publication years are now stated explicitly in the text.

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for the changes to the manuscript. All have improved it. A few remain.

 

Line-by-line suggested edits

The following line numbers have outdated citations. If there is no research published since 2022 that can add support for them, please state the year of publication in the text: 104 [15], 145 [25], 514 [36], 542 [37], and 549 [38].

300–314 Please change the italicized text to normal text.

335 Please check the spelling of “rpiritized”.

Author Response

Comment: The following line numbers have outdated citations. If there is no research published since 2022 that can add support for them, please state the year of publication in the text: 104 [15], 145 [25], 514 [36], 542 [37], and 549 [38].

Response:
We thank the reviewer for highlighting this issue. For references [15], [25], [36], [37], and [38], we have now explicitly indicated the year of publication in the main text. No more recent studies directly addressing these specific points were identified; therefore, the year of publication was added to provide appropriate temporal context.

Comment: 300–314 Please change the italicized text to normal text.

Response: The text was located and fixed accordingly.

Comment: 335 Please check the spelling of “rpiritized”.

Response: The spelling error has been fixed.