Dear Dr. Brodbeck,

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PLOS ONE

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[This research was supported by Natural Sciences and Engineering and Research Council of Canada (https://www.nserc-crsng.gc.ca/index_eng.asp) Discovery Grants awarded to SAM-S 21738, and by grant 2019-03620 from the Swedish Research Council (Vetenskapsrådet; https://www.vr.se/english.html) to RM. MIRB was supported by a Natural Sciences and Engineering Research Council of Canada Doctoral Scholarship.].

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Reviewer #1:

Reviewer #2:

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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?

Reviewer #1: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The manuscript titled “Brain activity in Cluster N and the hippocampus in non-migratory zebra finches completing a spatial orientation task using magnetic compass information” addresses an interesting and relevant topic. However, I believe that substantial revisions and re-analysis of the data are required before it can be considered for publication.

Major points

1) Behavioral procedure: The description of the behavioral procedure is incomplete. It is unclear whether the animals received adaptation trials before training or whether food deprivation was applied. Performance data during the training phase should be provided to clarify the baseline conditions and ensure the reliability of the results.

2) Individual-based analysis: Both behavioral data and Zenk expression results show considerable individual differences, indicating a heterogeneous sample. An individual-based analysis is therefore necessary. The authors should classify subjects according to performance (e.g., good vs. poor performers) and then analyze Zenk-positive cells within these subgroups. This would allow a more accurate interpretation of the data and may reveal patterns obscured by averaging across all individuals.

Reviewer #2: This manuscript explores an interesting and timely question: whether Cluster N is active in a non-migratory songbird during magnetic compass use. The study is well conducted, and combining behavioural testing with immediate early gene expression is a valuable approach. The results are largely negative, with no significant differences in orientation or Zenk expression observed between magnetic conditions. Such negative findings are entirely suitable for PLOS ONE, provided the conclusions are appropriately cautious and the limitations are clearly acknowledged. I suggest publication after major revision, provided the authors address the following points to better align their conclusions with the data and acknowledge the study’s key limitations.

Major comments:

In this study, zebra finches were trained to locate food in a plus maze using magnetic compass cues and then tested under static, sweeping, or vertical magnetic fields. While orientation was not significant, activity levels differed across conditions. These behavioural results are important but currently underemphasised. The differences in activity (Supplementary Figure 1) show that the birds perceived the different magnetic stimulations. This figure should be moved into the main Results section and incorporated into the Discussion. Emphasising these behavioural differences would strengthen the manuscript by highlighting that the birds responded to the magnetic manipulations, even though orientation itself was not statistically significant.

Another concern is that all three experimental conditions involved an active magnetic field. A true negative control (e.g., coil off, sham, or near-zero magnetic field) is absent. Such a condition would be essential to demonstrate whether Cluster N activation depends on magnetic input rather than dim light or task context. Please explicitly acknowledge this limitation in the Discussion.

Zenk expression did not differ between magnetic conditions, and without a dim-light or no-stimulus control it cannot be determined whether the observed signal reflects specific activation of Cluster N or merely baseline Zenk expression. It is valid to note that Cluster N is anatomically present in zebra finches, but functional activation under the test conditions cannot be concluded. Nevertheless, the manuscript sometimes states that Cluster N is “active” which overstates the findings. The conclusions should be tempered, and the Discussion should explicitly acknowledge the possibility that the observed Zenk levels reflect baseline activity rather than magnetically or light-induced activation.

Furthermore, the difference with Keary & Bischof (2012), which is used as a comparison, warrants a fuller discussion. In their study, zebra finches passively exposed to a strongly rotating magnetic field showed significant hippocampal activation (c-Fos) and a trend in the hyperpallium, whereas the present study found no differences between conditions (Zenk) during active navigation. Several factors could explain this discrepancy: (i) stimulus salience (continuous 180° oscillations versus static, sweeping, or vertical fields); (ii) passive perception versus active navigation; (iii) use of different immediate early gene markers (c-Fos versus Zenk); and (iv) the broader set of brain regions analysed by Keary & Bischof compared to the focus here on Cluster N and hippocampus. Notably, while Keary & Bischof still detected differences without a sham condition, the absence of a true negative control is a more serious limitation in this study and should be explicitly acknowledged.

Minor comments:

In the Introduction, please clarify whether Cluster N is primarily a visual area, a magnetic-processing area, or both. Currently, it seems that evidence for magnetic compass processing is limited, while light dependence is more firmly established. A clearer framing would help set up the rationale for your study.

Please use standard nomenclature throughout the manuscript: write zenk and c-fos in italics when referring to the genes, and use Zenk and c-Fos (not italicized, first letter capitalised) when referring to the proteins (for c-Fos you may also simply write Fos). Avoid all-caps "ZENK," which is uncommon in avian literature and mainly used for human gene symbols. For example, on page 17 the sentence “…Keary & Bischof found higher levels of c-fos…” should read “c-Fos.”

Please also clarify figure legends (e.g., explain orientation arrows for readers unfamiliar with orientation analyses).

Report exact p-values (and effect sizes where possible) rather than describing non-significant results as “trends.”

Ensure consistent terminology for brain regions (e.g., always “Cluster N”; specify whether “hippocampus” refers to the whole formation or a subdivision).

Light intensity (green LED, 27 mW/m²) is unusually high compared to prior Cluster N studies. Please elaborate on this in Methods/Discussion, as it may affect Cluster N activation.

I hope these comments are helpful in revising the manuscript.

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what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes: Shigeru WatanabeShigeru WatanabeShigeru WatanabeShigeru Watanabe

Reviewer #2: Yes: Uwe MayerUwe MayerUwe MayerUwe Mayer

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Brain activity in Cluster N and the hippocampus in non-migratory zebra finches completing a spatial orientation task using magnetic compass information

PONE-D-25-37676R1

Dear Dr. Brodbeck,

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,

Brenton G. Cooper, Ph.D.

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

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.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??>

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: I have read the revised MS and correspondence between the authors and reviewers. The MS has been well revised and I can recommend it for publication.

Reviewer #2: I am happy with the revised version of the manuscript.

PS: While reading, I noticed a small typo: on page 10, “DNH = dorsolaternal…” should be written without the “n”.

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what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). 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 For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our Privacy Policy..-->

Reviewer #1: No

Reviewer #2: Yes: Uwe MayerUwe MayerUwe MayerUwe Mayer

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