PONE-D-21-30899Adjusting the neuron to astrocyte ratio with cytostatics in hippocampal cell cultures from postnatal rats: A comparison of cytarabino furanoside (AraC) and 5-fluoro-2’-deoxyuridine (FUdR)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: Partly

Reviewer #2: Yes

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

Reviewer #1: No

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: Yes

Reviewer #2: Yes

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Reviewer #1: No

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 authors of this paper investigated the use of AraC and FUdR at varying concentrations on reducing numbers of glia in cultures from P0-2 and P3-4 rats. They claim that FUdR is favorable at reducing glial cell numbers at low concentrations while increasing the number of neurons as compared to AraC. They also demonstrate that using FUdR at high concentrations doesn’t impact neuronal cell function through Na+ current measurements.

This study focuses on an interesting and relevant area that is useful for in vitro studies looking at neuron-glia interactions. The study aims to address an issue in the field, where reductions in glia in cell cultures are necessary to prevent their overgrowth and to prevent reductions in neuronal cells. The methods used in the study are scientifically sound, however, the claims made from the findings are not supported by the data and statistical analysis. The background information relating to what is known about FUdR, specifically the caveats, is lacking. The discussion also fails to expand upon some of their assumptions and how other literature supports these conclusions. Despite my disagreement with their major findings, I believe the addition of key experiments may help to support some of these claims. However, the likely major finding of this manuscript is on reporting that AraC is not cytotoxic at higher concentrations when using antioxidants but this would need to be further developed.

Major points:

Fig. 2:

1. Authors mention that astrocyte and neuron morphology are not affected and then show representative images but give no further information on how that was determined. It would be helpful to describe how this was decided (i.e. specific morphological features, size, etc.). Quantifying morphology would increase the rigor for this claim.

2. The authors frequently mention that neuronal numbers are increased in Fig 2B, however, no significant differences were shown. There may be a trend for increase in the P3-4 age cultures at low concentrations for FUdR, but this distinction is not made throughout the text.

3. The authors claim that 4 μM FUdR reduces GFAP+ cells more significantly than AraC, however, they fail to distinguish that this is only the case in the P3-4 cultures but not for the P0-2 cultures.

4. Overall, FUdR and AraC treatment appear to be equally effective at all concentrations for P0-2 except when comparing the neuron to astrocyte ratio (this difference isn’t seen on individual numbers of neurons in 2B and astrocytes in 2C). The only notable difference in cell numbers appears to be the effect on reducing astrocytes at low concentrations of AraC at P3-4. What could explain this discrepancy in no change in neurons and astrocytes in 2B and 2C, yet a significantly higher ratio of neurons vs. astrocytes in 2D?

5. The text states “In P0-2 cultures with increasing concentration of antimitotic agent the neuron to glia ratio increased to a maximum of about 6:1” suggesting that this is a dose-dependent mechanism but the data does not support this claim (significant differences at 10 μM and again at 75 μM).

6. Perhaps the most important finding of the paper lies within these graphs, and it’s that AraC is not effective at reducing astrocytes at low concentrations only in older P3-4 cultures, but at high concentrations at all ages, it is effective and doesn’t appear to be cytotoxic to neurons. This information is valuable and needs to be further explored.

Fig. 3:

1. Authors state that “unlabelled cells almost disappear after treatment” which is not followed up by any quantification or p-value.

2. Quantification of microglia was done using TMEM119, however, in the figure it is stated that both BIII-tubulin and TMEM119 are both in green. How were they separated for analysis if under the same channel?

3. The text discusses reductions in microglia and GFAP negative glial cells but doesn’t have any graph or data supporting this. Please include this figure.

4. The text states “The preceding experiments showed that concentrations of 10 μM or lower of FUdR were more efficient than AraC in reducing glia cell numbers” this is only true in the case of P3-4 cultures, but not for P0-2 cultures.

5. The discussion states “Judged by morphology, the further unlabeled cells in our cultures might be GFAP-negative astrocytes [28], activated microglia, less well stained with TMEM119, or oligodendrocytes and their precursors.” This can be determined using other markers for these cell types, including NG2 for OPCs, A2B5 for pre-oligos, and Iba1 or CXCL4 for microglia. Also, TMEM119 is developmentally regulated and doesn’t arise until later postnatal time points up to P14 so some negative cells can exist, whereas Iba1 or CXCL4 will identify all microglia (Bennett, ML et al., 2016, PNAS).

Fig. 4:

1. The text states “There was, however, an insignificant tendency for the highest absorbance per cell in the cultures treated with 50 μM FUdR, which contain the highest number of neurons (see Fig 2D).” this isn’t supported by the data in 2B which quantifies numbers of neurons.

2. The authors report that 50 μM FUdR treatment condition causes increased formazan production which does not reach statistical significance. They claim that this may be due to increased mitochondrial activity in neurons due to Na+ load from higher neuronal cell numbers. As stated previously, the data does not support that there is an increase in neuronal cells, however there is indeed a reduction in glia. Including an additional analysis of the higher FUdR concentrations will be helpful to see if any changes to overall cell viability are occurring. Furthermore, investigating the effects on astrocytes as a whole is essential, including the possibility of excitotoxicity due to reduced astrocyte coverage.

Fig. 5:

1. The authors only investigated the effects of FUdR on the Na+ currents in these cultures but did not look at the effect of AraC. The text is lacking in rationale for why they left this out. It is especially relevant given that the field believes AraC to be cytotoxic at higher doses, where this paper proves this not to be the case.

Minor points:

6. Fig. 1: The text says that you look at 6 μM for AraC and 25 μM for FUdR but then figure 1 shows all concentrations and throughout the rest of the text, 6 μM for AraC is never mentioned again.

7. Fig 2 : The text says that AraC is in orange and FUdR is in purple but in 2A, the text shows AraC in purple and FUdR in orange.

8. Fig 3: Fig 3E, pink arrow on bottom right is pointing to a GFAP positive cell.

9. Fig 4: Figure legend for 4A says “shown as circles” but there are no circles.

Reviewer #2: In this manuscript the authors proposed FUdR as an alternative cytostatic that can be used instead of AraC to increase the ratio of neurons to astrocytes. They compared different doses of both treatments and found that at high doses both cytostatic have similar effects, but at low doses FUdR is more effective in killing astrocytes and therefore enriching neuronal cultures.

The research is important in the field as in vitro tools are still required to evaluate molecular mechanisms. There are, however, some weakness in the analysis of the data and some changes are suggested to improve quality of the manuscript.

1. In financial disclosure: Is stated that the author(s) received no specific funding for this work, however in acknowledge section some funding is mentioned

2. The cells were manually counted, and the cell numbers is key to the conclusions of the paper, the reviewer would suggest either an automatic cell counting (several plugins published for Image J allow automatic cell counting), a clear description of how the analyzer was blind to the culture condition, or ideally both

3. Add was found at the end of first paragraph of results… however, no significant differences of total cell count between any of the treated cultures was found (p>0.05).

4. Figure legend 1. Should indicate how many independent cultures were analyzed instead of numbers of dishes

5. Pooling data together from different drug concentrations is not a valid comparison and should be removed

6. I recommend changing the color of the ICC-IF since red, green colour blindness is the most common form of colour vision deficiency

7. Page 15, line 274 Treatment with 4 uM FUdR, however, inhibited astrocyte proliferation more efficiently than AraC in cultures prepared from P3-4 rats, resulting in a reduction of astrocytes to about 20% of the control level.

It needs to specify P3-4 since the difference is not significant for P0-2 culture, also eliminate already at a very low concentration of the cytostatic as the concentration is already mentioned.

8. Page 15, lines 284-286. There was “almost” no difference between the two age groups… or there was a significant age-dependent effect??

9. Page 16, line 287. In P0-2 cultures with increasing concentration of antimitotic agent the neuron to glia… be specific, replace antimitotic agent by FUdR

10. Using the same color for two different markers (in this case, TMEM119 and beta3-tubulin) is not acceptable. The authors need to repeat this staining if the same secondary was used or image the ICC-IFs again using a microscope with more cubes or lasers to be able to separate the 4 markers used.

11. Re-write page 17 lines 318-320 to improve readability. The sentence “To assess cell…” is repetitive and unclear

12. Remove sentence “There was, however, an insignificant tendency…” in page 17 lines 328. Insignificant tendency = no difference

13. Figure legends 4 and 5 states “shown as circles/single data point plotted as circles” no circles are shown in the figures

14. Page 23 lines 452-455, and 456-457. The authors make conclusions based on no significative effects. Slightly higher (p>0.1856), slightly improved, tendentially preserved… These need to be removed from the text as are not statistical significative effects and only distract the reader of the main conclusion of the paper that is clearly stated in Page 21 lines 416-418.

15. In conclusion, page 25, line 473 replace “minimize interaction with glia” for “minimize glia content” and “highly purified neuronal cultures” for “highly enriched neuronal cultures”

16. Page 25, line 489 replace “experimental conditions require more astrocytes to be present in the neuronal vicinity, low…” for “experimental conditions require mix cultures, low…”

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Reviewer #1: No

Reviewer #2: No

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Adjusting the neuron to astrocyte ratio with cytostatics in hippocampal cell cultures from postnatal rats: A comparison of cytarabino furanoside (AraC) and 5-fluoro-2’-deoxyuridine (FUdR)

PONE-D-21-30899R1

Dear Dr. Leßlich,

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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Alexander A. Mongin, 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: (No Response)

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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

**********

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: (No Response)

Reviewer #2: The authors did a good job addressing my comments on the first round of revision. Now I only suggest minor changes:

- Last two sentences of methods immunocytochemistry are repetitive, erase one.

- For consistency use the same format for the p values through the paper. Currently sometimes p<0.001 is used, others p=exact value, others p<0.006, p<0.004 ….

- Page 17, line 330 … however, the effect was not significant (p=0.262), page 17, line 334-336 Moreover, there was a significant difference of the … (p=0.24). Why p=0.262 is not significant and p=0.24 is it?

- Page 25, line 487, erase including microglia since TMEM119 staining was removed

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Reviewer #1: No

Reviewer #2: No