-->PONE-D-26-07602-->-->Y-maze and Vertical Pole Performance Correlate with Fine Motor Learning in Mice-->-->PLOS One

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Section Editor

PLOS One

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Reviewers' comments:

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-->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: Yes

Reviewer #2: No

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

Reviewer #1: N/A

Reviewer #2: No

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-->3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: No

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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: To strengthen the rigor and impact of your findings, I have several critical points that require clarification and further consideration.

1. On the Core Conclusion and the Claim of "Prediction"

The central conclusion posits that the Y-maze and Vertical Pole tasks are "suitable predictors" of pellet-reaching performance. However, the study design is inherently cross-sectional and correlational, not longitudinal or predictive. All tasks were assessed within a similar timeframe. Can you provide stronger justification or refine the language to clarify how a correlation measured concurrently translates into a reliable predictionof future performance on a distinct, more complex task? Is the implied predictive power based on shared neural substrates (e.g., hippocampal-striatal circuits) or a more general "learning readiness" trait? The term "predictor" may be too strong without evidence from a true pre-screening followed by a later assessment of motor learning.

2. On Experimental Design and Confounding Factors

Fixed Order of Testing: The Methods indicate that all behavioral assays (Open Field, NOR, Y-maze, Vertical Pole) were conducted prior to the Pellet Reaching task. This fixed order is a significant confound. Could the testing sequence itself introduce practice effects, fatigue, or cumulative stress that systematically influences performance in the final, most complex task? For instance, are mice that are more active/less anxious in the initial tasks also the ones that show higher engagement in the final motor learning task? While you briefly acknowledge this in the discussion (lines 378-386), a deeper analysis is needed to disentangle whether the observed correlations reflect intrinsic ability links or are artifacts of the testing schedule.

Conflating "Working Memory" with "Activity/Exploration" in the Y-maze: You interpret the Correct Alternation Ratio (CAR) as an index of "working memory" and link it to pellet-reaching success. However, a high CAR in the Y-maze is also intrinsically linked to a higher number of arm entries (r=0.96), which is a direct measure of general locomotor activity and exploratory drive. How can you rule out the possibility that the improved pellet-reaching success is not directly due to superior spatial working memory, but rather a correlate of a more active, trial-and-error prone behavioral phenotype? The neural mechanisms underlying these two interpretations could be different.

3. On Data Analysis and Statistics

Correlation vs. Causation/Directionality: You report several significant correlations but the direction of interpretation sometimes requires more nuance. For example, the negative correlation between Vertical Pole time-to-turnand the total number of reachesis interpreted as better motor coordination leading to more attempts. Could the reverse, or a third variable, be plausible? For instance, could a higher general motivation level (interest in sugar) or lower baseline anxiety simultaneously cause faster turning on the pole andmore attempts in the reaching task?

Clarity in Statistical Reporting:

P-values in Table 1: In the provided table, many p-values are listed as "p=0.0". This is statistically improbable and likely a display artifact. Please verify and report all p-values accurately (e.g., as p < 0.001).

Multiple Comparisons: You state that no multiplicity correction was applied due to the exploratory nature of the full correlation matrix, which is acceptable. However, you highlight five specific significant correlations in the results. Did you consider calculating the False Discovery Rate (FDR) to assess the reliability of these "significant" findings within an exploratory framework? This is particularly important given the modest sample size.

4. On Sample and Generalizability

Sample Size and Statistical Power: The final sample size for key analyses is 11-12 mice, and only 9 for NOR. While some strong correlations were found (e.g., r=0.79 between Y-maze CAR and success ratio), the small sample may be underpowered to detect moderate or weak correlations and may render the estimates of the found correlations unstable. Did you perform a priori or post hoc power analysis? For the many task pairs where no correlation was found (most of Table 1), is the lack of association genuine, or a potential Type II error due to low power?

Single-Sex Limitation: The study uses only female mice, justified by avoiding male aggression and citing literature showing no sex differences in the baseline metrics used. However, in motor learning, especially involving reward and fine motor skills, the estrous cycle can modulate motivation, activity, and learning rate. While the cited literature supports a lack of difference in the individual tasks, the cross-task correlations themselves remain unvalidated across sexes. This limits the generalizability of the conclusions. This limitation should be more prominently discussed.

5. Other Specific Points

Evidence of "Learning" in the Pellet Reaching Task: The task is described as assessing "fine motor learning," but the results primarily report aggregate measures (total successes, attempts) over three test days. Is there data showing a within-subject improvement in success ratio across the three days? Presenting learning curves (even if non-significant due to few days) would better support that "learning" occurred, not just differences in "performance."

Dissociation of "Turn" vs. "Descent" in the Vertical Pole: The interesting finding that only time-to-turn, not time-to-descend, correlates with pellet reaching is interpreted as the turn requiring finer motor control. Is there any kinematic analysis or behavioral observation to support this mechanistic interpretation? Otherwise, a "fear of heights" could plausibly affect both turning and descending latency.

Reviewer #2: I think this exploration of cross -assay performance in mice is interesting, and well-presented but ultimately this study is heavily limited, and requires substantial revision. The sample size is very small, which is understandable, but combined with the analysis methods used reduce confidence in the results. There is a big issue with correlation across independent run sub correlations with different sample sizes. There are also results of a slew of variables that do not directly address the question, think could be removed to focus.

Conceptually as well, this study is unconvincing. I think many of the results could be attributed to behavioural trait linked to speed/ activity rather than memory/ motor skills. Many of the methods used to describe the assays and procedures are solid, but then the measures used are not well justified and do not clearly separate activity from actual cognitive performance.

Line 174: why 4 days? Is that sufficient for all to achieve habituation, how do we know?

Line 185: a minimum of ? So there was not a standardised rest interval for all individuals? What was the range of variation in rest days?

Line 190: including events when they reach ‘for the pellet when no pellet was available’ is not really a failure that can be attributed to fine motor skill. I would argue that the authors re-run the analysis with these ‘failures’ removed.

Line 194: …Anova’s as multiple ones were used. But see below.

Methods: There are two important general things, as this analysis section is both overly complex and also not detailed enough to be reproducible. First, as it was the same individuals, this data would be better analysed in a single model not as separate one-way Anova’s it would allow variation introduced by individual differences to be better accounted for within and between tests, and to better account for the different sample sizes across tests. As to the second point, the level of detail varied widely. The text on lines197 – 200 about basic regression is redundant for an academic audience and is extremely basic. But then there is not detail about how the authors ‘… computed the multiple correlation coefficient based on their pairwise correlation coefficients, representing the shared linear variance.’

Line 202: why use a triple correlation coefficient computed from the pre-existing pairwise correlation coefficients rather than from the raw data ? It is not standard practice, and readers need to know what formula were used. Far more importantly, Deriving these pairwise correlations assumes those correlations were computed on the same sample sizes with no missing data, and any pairwise correlation using different subsets of individuals as is the case with this study the result would be hard to interpret at best and potentially invalid.

Line 214: So no videos were manually scored? If not how do you ( and thus we) know that Ethovision was working correctly and effectively distinguishing between different behaviours? I would ask the authors what aspect of research they actually wanted to engage in.

Line 240: This is not an outlier, unless you measurements were off for this one individual. It is very likely that this reflects actual relevant among-individual variation within the group. Treating it and or naming it an outlier is terminologically incorrect and conceptually odd. I am glad the measurements from this individual (I assume – the wording is slightly ambiguous) was kept in the analysis as the sample size is already very low for this study, but the authors must be careful with what they term a statistical outlier. Especially when comparing a data point to a group average – which is not a method that can be used to determine a statistical outlier.

Line 253: ‘escape responses – what are these ? Also this this makes sense from the perspective of ‘declarative’ memory - more successful maze entries = good memory and less time spent exploring novel object. But this depends on a more refined measure of novel object exploration time (i.e. relative duration of time spent exploring a novel object does not say much – if some individuals just take longer to approach. Also, how much time do we expect a mouse to take to satisfy their curiosity about a novel object. Were the ones that spent longer in the investigation area actually inefficient? I think interpreting this test as measured is prone to ambiguity.

Line 254: higher *number of* arm entries.

I note that the data are to be provided upon acceptance.

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Reviewer #1: Yes: Yixuan Ku

Reviewer #2: No

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Y-maze performance predicts refined motor learning in mice

PONE-D-26-07602R1

Dear Dr. Ciralli,

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.

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Kind regards,

Stephen D. Ginsberg, Ph.D.

Section Editor

PLOS One

-->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 #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 #2: Yes

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

Reviewer #2: Yes

**********

-->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 #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 #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 #2: I think the authors have addressed all the issues raised, and the re framing around the new results is good. Also, I note that two of my earlier comments were not worded very well at all, and might have come across as more critical than intended, my apologies to the authors.

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Reviewer #2: No