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closeReferee comments: Referee 2
Posted by PLOS_ONE_Group on 25 Feb 2008 at 11:47 GMT
Referee 2's review:
Review of the original version of the manuscript
The mouse and the cell data are tremendously exciting. This is a highly significant body of work. The manuscript would benefit from an expansion of the conclusions.
I have only relatively minor concerns:
1. Scale bars are required on all of the photomicrographs.
2. “Dopaminergic neurons constitute only a minor portion (~ 5%) of cells making up the SN.” Do you mean cells that can be dissected cleanly from the midbrain?
3. “20% of the striatal nerve terminals originate from SN dopaminergic neurons” statement needs a reference. What about interneurons, cortical and thalamic inputs?
4. “Synaptosomes in the flow-through fractions were primarily GABAergic” What about interneuron (ACHE) and glutamatergic cortical and thalamic inputs? Strictly speaking the synaptosomes that are left shouldn’t be called GABAergic. Only a proportion of the synaptosomes are GABAergic.
5. Results: “H2O2 levels were measured in isolated ST DAergic synaptosomes following injection of dichlorofluoramine (DCF)” But in the Methods (p 22): Mice were injected in the tail vein with ~200 µ l of dihydroethidium (DHE, Calbiochem). Is one a mistake.? H2O2 can be measured by DCF; DHE measures only superoxide. Also, DCF reacts with several other ROS. To determine whether the signal reflects H2O2, it needs to be extinguished by treatment with catalase: the abolished signal is then attributable to H2O2. EUK189 will abolish both H2O2 and O2- DCF signals.
6. Page 7, there are references listed “[46,47][6, 48-52]” which should be [6, 46-52].
7. Page 12, “…the striatum (ST) easier is larger, easier to dissect…” Please correct.
8. Page 15 and Figure 8: How old were the mice tested? Were there any differences between M and F?
9. Page 16, second last line: “affective” should be “effective”.
Discussion.
I would like to see the discussion expanded to include the following points:
1.After the initial cell death (3 weeks MAO elevation) cell loss does not seem to proceed. Why wasn’t the cell loss grater in animals that had elevated MAO for 12 + months? Why doesn’t the cell death continue?
2.Explain why should elevation of MAO increase cell death? Is DA processed to hydrogen peroxide produced by the glia? What happens to the DA normally?
Review of the first revised version of the manuscript
The authors have successfully addressed the reviewers comments and in particular the re-writing of the introduction and discussion has made it significantly easier to read the manuscript. This is a significant body of carefully controlled work that will have an important impact on the field.
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N.B. These are the comments made by the referee when reviewing an earlier version of this paper. Prior to publication the manuscript has been revised in light of these comments and to address other editorial requirements.
RE: Referee comments: Referee 2
julieandersen replied to PLOS_ONE_Group on 29 Feb 2008 at 21:54 GMT
Responses to reviewer #2:
Q1. Scale bars are required for the micrographs.
A1. Scale bars have been added to all micrographs as requested.
Q2/3. Please provide references for 5% SN neurons and 20% striatal nerve terminals being dopaminergic.
A2/3. This has been done; please see response #A7 to reviewer #1 above.
Q4. The flow-through striatal synaptosomes should not be referred to as GABAergic.
A4. This has been changed in the text to ‘non-dopaminergic’ rather than ‘GABAergic’.
Q5. Which was injected to measure intracellular H2O2, DCF or DHE? To show that it is truly H2O2, catalase treatment should be used.
A5. DCF was injected; catalase addition to ST DA synaptosomes resulted in prevention of the MAO-B-mediated ROS increase suggesting that it is specifically H2O2. This makes sense since the ROS species produced by the enzymatic reaction of MAO-B with substrate is H2O2.
Q6. Pg 7, change references listed to 6, 46-52.
A6. This correction has been made.
Q7. Pg 12, please correct sentence “…the striatum easier is larger, easier to dissect…”
A7. This sentence has been corrected.
Q8. Pg. 15/Fig. 8, How old were the mice tested in the behavioral tests? Where there male/female differences?
A8. Animals for locomoter analyses were 2-3 months of age; only male mice were used for all of the analyses described in the manuscript so we did not investigate in this current study any male:female differences.
Q9. Pg 16, second to the last line: “affective” should be changed to “effective”.
A9. This correction has been made.
Q10. Expand the Discussion to include: (a) why cell loss isn’t greater with age and (b) why MAO-B elevation increases cell death.
A10. It is not clear why there was not a more dramatic increase in SN DA cell loss with age in the MAO-B transgenics. Normally, cell loss coincides with an age-related increase in MAO-B enzyme activity. Our results suggest that perhaps the elevation of MAO-B to aging levels in the young animals was sufficient on its own to produce significant cell loss and that the additional stress of an aging brain contributed only marginally (yet significantly) to this effect. In terms of MAO-B-mediated cell death, we assume this is in part a consequence of selective complex I inhibition in the dopaminergic neurons which has been observed following both rotenone and MPTP treatment to result in a similar cell death pattern. Secondary microglial activation also appears to contribute. These points have been added to the Discussion.