Exploring the Pathological Effect of Aβ42 Oligomers on Neural Networks in Primary Cortical Neuron Culture

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This paper discussed the functional aberrations of cortical neuronal networks induced by the Aβ42 oligomer Aβ42. Overall, the paper is interesting. The authors are advised to consider the following suggestions to further improve the paper’s quality.

(1) It is recommended to avoid lumped references in the Introduction section, such as [15-17].

(2) The quality of the figures needs to be further improved. Among them, the display of many details of the diagram was not clear. For example, the words in Figure 3B were not clear. The authors are advised to enlarge all the figures.

(3) The authors are suggested to add a Summary/Conclusions section, which should include the contribution of this paper and the major findings.

(4) The whole paper needs to further polish. The current version still has grammar mistakes and typo errors.

 

Author Response

• It is recommended to avoid lumped references in the Introduction section, such as [15-17].

 

The number of references is trimmed to 24 from 27 including 16.

 

• The quality of the figures needs to be further improved. Among them, the display of many details of the diagram was not clear. For example, the words in Figure 3B were not clear. The authors are advised to enlarge all the figures.

All figures are modified for a better visual recognition.

• The authors are suggested to add a Summary/Conclusions section, which should include the contribution of this paper and the major findings.

Conclusions are inserted as below.

The study demonstrates the irregular activation of neurons and the disruption of neural network organization by Aβ42 oligomer treatment using HD MEA. Aβ42 neuronal culture displays significant impairments in basic topological properties over time, including spike frequency and duration. Network analysis based on graph theory also indicates a disruption in the neural network as a result of changes in network parameters, such as CC, PL, and ND. Furthermore, CAT analysis reveals asynchronous communication be-tween neurons and inhomogeneous network bursts. Through this study, we will gain a better understanding of how neural networks change as AD progresses.

• The whole paper needs to further polish. The current version still has grammar mistakes and typo errors.

All grammatical and typo errors are corrected.

Reviewer 2 Report

Comments and Suggestions for Authors

 

The authors of « Exploring the Pathological Effect of A_42 Oligomers on Neural Networks in Primary Cortical Neuron Culture” explore the electrical activity and network formation using MicroElectrode Arrays (MEA) of primary neuronal cultures over time, in a normal and pathological development, treating the cultures Ab42 oligomers to mimic Alzheimer’s disease conditions.

Even if the experiments and results seems to be interesting, the experiments (Materials and Methods) are not properly explained in the text of the article, making it difficult to judge the soundness of the discoveries or even the interpretation of the results.

Together, there are several formal omissions (not presenting the terms or hypothesis, acronyms not defined) and probably misrepresentations on the figures or other mistakes. By the way, all the figures are too small; I had to look at them zooming in my computer.

Finally, all the statistical analysis is wrong. A t-test allows comparing two conditions, in a more complex analysis as the one showed here, it either should be a 2-way ANOVA, repeated measures or mixed model, depending on the experimental design. Since many of the details are lacking I do not know which analysis would be the appropriate…

 

Having talked in general terms, now I am going to point some specific issues. As I said, the M&M section is really short, and there is missing information. How many MEA have they used? How many cultures? Just one? Did they prepare several replicates? Are the different time points (DIV) the same MEA reanalysed over time? Alternatively, did they dedicate a MEA to each registration? When does the oligomers treatment start? From the seeding? Just one application or it is renewed every 3 days? Did they apply the oligomers 1h-24 h before the registration? What kind of oligomers are predominant? Dimers, trimers, tetramers or higher density oligomers? As the authors said in the article 10 mM Ab42 is neurotoxic. Quite high for nowadays standards, but without discussing that, what kind of impact does it have in the long term (if there is a long term) of the culture? Have the cultures similar neuronal densities?

 

Usually on primary cultures, it is considered a mature neuron at 12-13 DIV, with well-formed synapses and dendritic spines. I understand the authors are interested in the formation and development of the network, so they study it when synapses are forming and still immature, but at DIV1, usually neurons have developed a few short neurites, and even if it is high-density cultures, I find surprising that they can measure electrical activity at this stage. Do they have some negative control registration? How do they explain this activity at this early stage?

 

As a said, there are many acronyms that have not been introduced in the text. I was wondering what CC stands for (cell cluster, connectivity…) until I read the figure 2B axis, correlation coefficient. Same thing happened to PL (Path Length), ND (Node degree), or DDW (double distilled Water?) and PDLO, that still I do not know what it means. It is normal to use acronyms, but they should be introduced somewhere in the text, so the readers, does not have to guess it, as they did for CAT, NBM or MFR (even if I would prefer that it will not defined in a figure legend).

 

The figures are too small to show all the data they want to show. In addition, I do not know if it is a problem of the software used to represent it, but there are some data that is not possible to be like represented. Figure 1A, is it the 64x64electrodes representation? In a given moment? The average of the 5 min?  In Figure 1B, the chart with the violin plot, most of the means are really close to 0 (I guess because most of the electrodes they had no spike), but it is not possible to have the tail below 0 as they have. Negative spikes it is not something possible, so I hope that it is just a misrepresentation. In addition, what depends on the violin size? Theoretically, all of them should have the same number of values 64x64 by 300 seconds. Is there a difference in the number of replicates?

In Figure2, A, still a representation of the 64x64electrodes? They classify the different types of nodes, but an explanation on the section 2.2 “An abnormality…” would be necessary to interpret it by the non-expert reader. What are the blue lines that appear in some of the images (Ex/DIV13 ctl right side?) ? Why there is no standard deviation or error on C & D ? Finally, I find really surprising that at DIV16 both conditions end up with the same values, around 3 for PL and 55 (maybe?) for ND. How do they explain it? What is their interpretation? Having different development pathways, at their maturity state they have reached similar values….

 

On Figure 3, I do not think that time scale is right. The figure seems to be more than 5 times bigger than the bar. They represent with two colours the difference between spike network bursts and spike bursts, but the authors do not define them neither in the text nor in the figure legend, nor explain their importance and differences nor the criteria to categorize them. In B,C and D, what does it mean the dot on each of the boxes? Supposing it is the median as in Fig1, then there would be several cases where the median would be on the 75^th percentile, but since they have not defined it, I cannot be sure.

 

On Figure 4, what the colour bar means? On the figure legend, they say that it represents initiation (gray) to termination (yellow). However, what does blue means then? I suppose that black means no trajectory, not? Why on B & C, there are some points without variation? Specially in DIV 1.

So, even I think there is an interesting experiment and probably interesting data, missing so much information makes it hard to interpret, with a lot of suppositions and doubts about how it was done. In addition, a new statistical analysis is necessary before trusting the conclusions achieved. Sorry to say, but in the present form I cannot recommend its publication.

 

Author Response

On top of the revised manuscript, an additional suplementary figure is prepared, too. (Somehow it allows only single file uploading.)

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I was going to read and review the paper " Exploring the Pathological Effect of A_42 Oligomers on Neural Networks in Primary Cortical Neuron Culture”, but I will restrain myself of doing it after reading the cover letter from the authors, that I will not call it cover letter since they just just answering my comments. By the way, the answers were extremely short, generally just showing that they had included the previous petitions, but sometimes the answer have no-scientifically arguments, as "The bundle software didn’t provide it." or "It could be coincidental".  

I remind the authors that the function of reviewing is a fundamental part of the publishing process, done with good will in exchange of nothing (or almost nothing, just be recognised as reviewer). Phrases as "Some non-professional scientists or graduate students believe that..." and "As you are not blind, there..." are aggressive and insulting.

Since insulting the reviewer has an impact on the review itself, I will not do it. Consider me out of the process and good luck finding some other reviewer.