Paper title: Anxiolytic effects of fluoxetine and nicotine exposure on exploratory behavior in zebrafish Aim(s): 1. To determine the anxiolytic effects of fluoxetine and nicotine in zebrafish and comparing the two drugs 2. To determine sex differences in anxiety parameters in zebrafish

Abstract, title and references The authors describe a study to investigate sex differences in the response of zebrafish to two anxiolytic drugs. The aim is evident, and the abstract adequately explains the methods and results. Most references are recent (within the past 5-10 years). All of them are relevant and in the correct reference format.

Q.1 The title does not directly correlate with the aim. Although the authors focus on sex differences, the current title only mentions what is already known--that fluoxetine and nicotine have anxiolytic effects. The title needs to be reworded to include "sex differences." A good title for this paper would be “Anxiolytic effects of fluoxetine and nicotine exposure and sex differences in anxiety parameters in zebrafish.” Introduction/background Q.2 Starting from a general background on zebrafish as a model organism to study stress and anxiety, the authors delve deeper into the sex-based differences in response to anxiolytic drugs. However, extensive citation of references could be avoided as many of the facts are relatively well-known and therefore, need not be cited.

Q.3 (Introduction, paragraph 1) The authors state that zebrafish are similar to humans, in that, they use cortisol as the stress hormone and not corticosteroids. This is a logic issue as cortisol is a type of glucocorticoid, and glucocorticoids are also corticosteroids. Moreover, cortisol is not a neurotransmitter. The authors should therefore carefully review the facts stated in the Introduction.

Q.4 (Introduction, paragraph 3) While the authors state that the lack of adequate reports on sex-dependent stress responses of zebrafish stimulated them to conduct this research, a compelling rationale is not evident. Lack of previous reports cannot be the sole motivation for a study; therefore, the possible applications of the results that the authors foresaw before conducting the study should also be highlighted in the Introduction.

Methods Some major clarifications are required for the Methods section.

Q.5 (Methods, “Subjects”) The authors state the use of four-month-old adult zebrafish in their experiments. However, the grouping is not clear. The number of fish used in each group is mentioned in the results section, rather than the methods section. A justification for the number of fish used in each group seems to be lacking.

Q.6 (Methods, “Dosing”) While the authors state that the high dose of fluoxetine may cause non-specific effects, no attempt to compensate for this in the experiment was made. Only a single dose was studied.

Q.7 (Methods, “Dosing”) For drug exposure, the fish were individually placed in a 100-mL beaker; however, this volume seems to be small enough to induce restraint stress on the fish, and the authors should factor this in their interpretation of the results.

Q.8 (Methods, “Dosing”) While the dosing conditions (dose and exposure duration) are mentioned clearly, the authors have not verified the amount of drug absorbed by each fish. The drug concentration remaining in the beaker after the 3-min exposure duration should have been measured to determine the amount of drug absorbed by each fish.

Q.9 (Methods, “Behavior assay & video tracking”) The video tracking protocol needs to be clarified further. Throughout the paper, the angle of video capture (top or side view) has not been mentioned.

Q.10 (Methods, “Analysis”, “Freezing”) The authors describe the use of a binary (yes/no) system to determine freezing. This protocol is not validated and may not be very useful. Hence, the authors should consider only freezing duration.

Q.11 (Methods, “Analysis”, “Speed”) While the authors describe slow swimming as an indicator of anxiety, erratic behavior, which involves sudden bursts of swimming, would also be tracked and recorded to affect the average values. In this analysis, the authors should also consider measuring ‘number of erratic movements’ and exclude speed during such erratic movements in the calculation of average swimming speed.

Q.12 (Methods, “Analysis”, “Depth”) The authors state variability in depth as a factor indicating anxiety. Fish that stay close to the surface, considered active (less anxious), would also show less variance in depth compared to fish that are anxious and stay on the bottom. Typically, the novel tank diving test is performed to study anxiety and depth. It would be a good idea to mention this in the paper.

Q.13 (Methods, “Behavior assay & video tracking”) Because body weight did not affect any parameter, it should be justified as to why body weight was measured, and the fish were subjected to anaesthesia.

Q.14 (Methods, “Analysis”, “Statistical analysis”) Especially because the focus of the study was to determine the effect of sex on anxiety in zebrafish, a correlation analysis should have been performed with sex and the various parameters as variables. This is not clearly indicated in the Statistical Analysis section.

Q.15 (Methods, “Analysis”, “Horizontal place preference”) Instead of the horizontal place preference test, the authors should have studied social interaction, using a physically divided 3-chamber tank with the test fish in the middle portion and conspecifics in one side. Depth analysis would anyway provide the same information as the horizontal place preference test, but social interaction is known to be affected in anxiety.

Q.16 (Methods, general) Some minute, but important details have not been mentioned. These include water changes between testing each fish (an anxious fish releases stress hormones in the water that could affect the behavior of the subsequently tested fish), intensity of backlighting, transfer (net handling) stress, temperature and pH of the water, method of tracking (color-based or light distortion-based), etc.

Overall, the authors clearly state that all experiments were approved by the appropriate ethics committee and the protocol number is provided. The methods section might seem to be complete to a lay person, but reproducibility testing will not be possible with the current level of methodological details. Results Q.17 (Results, Paragraph 1) The number of male and female fish in the three groups is different. Because the study is mainly on sex effects, the authors would need to justify how the results of this paper are valid even when the sex distribution of the fish was not even. This can be done in the Discussion.

Q.18 (Results, “Individual components of behavior”) The authors have indicated significant differences in Table 1 using bold font. This is not a conventional method. Moreover, it is not very comprehensible. Therefore, I suggest that the authors use symbols or letters to indicate significant differences.

Q.19 (Results, general) All numerical values should be presented in a consistent manner with only 2 decimals. For p-values significantly below 0.05, it would be sufficient to state it as p < 0.05 or 0.01. Q.20 (Results, “Individual components of behavior”, “Freezing behavior”) Figure 1A–Instead of calculating freezing probability, would it make better sense if the number of freezing bouts was shown? Q.21 (Results, “Individual components of behavior”, “Freezing behavior”) Figure 1B–The Y-axis shows freezing time with the units as the fourth root of seconds. Would it be possible to show the time simply in seconds? Q.22 (Results, general) All figures–Instead of using light and dark bars for females and males, I suggest that the authors use patterns and present all figures in black and white. All labels need to be consistent in letter casing. Some are written in complete lowercase, while some start with a capital letter. Q.23 Since MANOVA and ANOVA are practically the same (barring minor differences), either term should be used consistently throughout the paper. Q.24 I am not sure why the authors have shown values for a particular parameter as well as for its variance. While the authors have commented on variance in depth and speed, it is unclear as to how these values affect the interpretation of the results.

Q.25 (Results, “Individual components of behavior”, “Horizontal place preference”) In the result for horizontal place preference, the contradictory values obtained should have been justified better. The authors state that they are unsure about the data. This is a loose end and further experiments to allow an accurate interpretation are needed. Q.26 (Results, general) Terms used for measurements (variance–consistency; depth usage–vertical usage; average–mean; middle third horizontally–center) need to be written consistently. This could lead to confusion among the readers.

Overall, it appears that the authors have quantified several parameters to interpret the data, but most of them are derived parameters and, therefore, are not necessary. Discussion and Conclusions The Discussion section is typically not structured, but in this case, I appreciate that the authors have clearly grouped their results to address two important research questions. However, the Discussion should start with a high-level summary of whether the research aims were met and the major findings.

Q.27 Figures need not be cited again in this section.

Q.28 (Discussion, “Differences in fluoxetine and nicotine behavioral responses”, Paragraph 1) While reduction in freezing bouts and freezing time was not observed by the authors in their study, they offer a suitable explanation. However, freezing is an important behavioral trait that acute fluoxetine exposure cannot influence. Moreover, clinical courses of fluoxetine treatment are long-drawn, and therefore, the choice of acute exposure may not be appropriate in zebrafish. Could the authors explain why they chose acute dosing?

Q.29 (Discussion, “Differences in fluoxetine and nicotine behavioral responses”, Paragraph 1) The effect of cortisol on stress is known, and so, the authors need not speculate on the effect of fluoxetine.

Q.30 (Discussion, “Differences in fluoxetine and nicotine behavioral responses”, Paragraph 2) The fact that nicotine-exposed fish swam higher than control fish questions the nature of the control, which ideally should not show any sign of anxiety itself. The authors need to answer if nicotine induces excitation beyond its anti-anxiety effect. The authors acknowledge that the nicotine dose was too low but do not justify their selection of the dose and salt form.

Q.31 (Discussion, “Differences in fluoxetine and nicotine behavioral responses”, Paragraph 3) The differences between nicotine and fluoxetine that led the authors to describe the complexity of anxiety seem rather vague.

Q.32 (Discussion, “The effect of sex on behavior and drug efficacy”, Paragraph 1) In describing the effects of sex on anxiety, the authors claim that males were more anxious than females but the difference in freezing behavior was not significant. Thus, this claim seems to be overstated.

Q.33 (Discussion, “The effect of sex on behavior and drug efficacy”, Paragraph 2) The authors did not determine sex differences in baseline behavioral parameters; this is a fatal limitation.

Q.34 (Discussion, “The effect of sex on behavior and drug efficacy”, Paragraph 2) Again, the second limitation of using too low a dose of nicotine could have been completely avoided had the authors ran pilot tests on various doses or followed the dose most commonly reported in the literature.

Most of the results presented by the authors remain to be confirmed through further research. Therefore, the contribution of the present study to the literature stands questionable.

Overall comments The authors aimed to determine the anxiolytic effects of nicotine and fluoxetine as well as sex differences in anxiety parameters in zebrafish by measuring depth, speed, freezing, etc., which are well-known indicators of anxiety in zebrafish. The study design is, therefore, appropriate. While nicotine mainly affected swimming speed, fluoxetine affected depth and horizontal placement. However, novelty of this study is not clear. Previous studies have reported the effects of both fluoxetine and nicotine in zebrafish. Sex-based differences in anxiety behavior are also reported. The only thing that makes this paper stand out is that the sex differences reported in the current paper contrast those reported in the literature. Males were more frequently found to exhibit signs of anxiety than females–a result that contrasts previous reports that males are bolder than females. It would be great if the authors could support the contrasting results with conclusive results rather than dismissing this important contrast by stating limitations. Overall strengths: Zebrafish have been increasingly used to model anxiety. However, sex differences in the anxiety parameters are not studied extensively. This study reports that males are more anxious than females–a result that contrasts previous findings that females are more prone to show signs of anxiety. This study also shows the differences in the anxiolytic effects of nicotine and fluoxetine: nicotine mainly reduces swimming speed, whereas fluoxetine makes the zebrafish swim closer to the surface without significantly affecting speed. Points of improvement: Major points:

1. The main rationale of the study is not clear. Why did the authors decide to study the effect of nicotine and fluoxetine as well as sex differences? The effects are already known.
2. While there is not much novelty in the study described, the article would greatly benefit from a better discussion of the results and an explanation to the contrasting result on sex differences reported by the authors.
3. The Methods need to be thoroughly checked and any gaps need to be addressed to allow reproducibility.
4. The Discussion section needs to be rewritten because it does not clearly justify the results nor does it indicate how the results are placed in comparison with the existing body of literature.
5. Implications of the study findings need to be highlighted.

Minor points:

1. Presentation of values in figures needs to be rechecked. There is no need to present variance along with the actual values in separate graphs.
2. There are several language issues, and a thorough proofreading by a native English speaker would be required.