Inhibition of NKCC1 Ameliorates Anxiety and Autistic Behaviors Induced by Maternal Immune Activation in Mice
, Shufen Hu †, Shu-Ting Qu, Meng-Dan Lv, Jun-Jun Wang, Xin-Ting Liu, Jia-He Yao, Yi-Yan Ding and Guang-Yin Xu *
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe text lacks an explanation for the abbreviation 'PFC' throughout, including the abstract. Please include its full form.
In the introduction, mention the association between poly(I:C) and microglia activation.
Additionally, in the methods section, specify the number of samples used.
In the discussion, the caution regarding evidence linking maternal infection with coronaviruses to the birth of autistic children is noted. However, it remains questionable whether pregnancy infection caused by the RNA virus analogue poly(I:C) can adequately account for the aspect of pregnancy infection attributed to coronaviruses. It is advisable to delve into this issue more comprehensively in your discussion.
Furthermore, provide a causal explanation for the relationship between microglia activation by MIA and the sequential pathway of NKCC1 overexpression by microglia activation, as illustrated in Figure 6.
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Reviewer 2 Report
Comments and Suggestions for AuthorsThis animal study examined the role of maternal immune activation (MIA) induced by 15 poly(I:C) infection during pregnancy in leading to autistic behaviors in offspring; this study further examined whether the MIA offspring showed significant microglia activation, excessive dendritic spines and narrow postsynaptic density in prefrontal cortex (PFC); this study also examined the role of overexpression of NKCC1 in these abnormalities of PFC in MIA offspring PFC; and finally this study examined the ameliorating effects of PFC microinjection of NKCC1 inhibitor bumetanide on ameliorating the autistic behaviors by in MIA offspring.
This study has clear hypothetical frames. The study evidenced the role of inhibition of NKCC1 in ameliorating the autistic behaviors by in MIA offspring and provides ne knowledge to the etiology of autism spectrum disorder.
My only suggestion is that the abbreviations in the manuscript warrant being rechecked and revised. For example, the fulling spelling of BTN should be presented at its first appearance. Full spellings can be deleted whether the abbreviations have been introduced (for example, Line 202, Line 277).
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Reviewer 3 Report
Comments and Suggestions for Authors
In this study, the authors by using maternal immune activation (MIA) in pregnant mice for producing offspring with autistic behaviors aimed to show that the NKCC1 is critical for mediating the autistic behaviors in mouse MIA offspring. The authors stated that MIA induced by poly(I:C) injection (20 mg/kg, single administration ??) during pregnancy leads to autistic behaviors in offspring. They further stated that MIA offspring showed significant microglia activation, excessive dendritic spines, and narrow postsynaptic density in PFC. Furthermore, the authors stated that these abnormalities may be caused by overexpression of NKCC1 in MIA offspring PFC. Finally, they speculated that bumetanide injected into the brain (by two permanent cannulae into the two sides of PFC) could ameliorate autistic behaviors.
The paper is potentially interesting but is limited by an inadequate description of the methods and results.
- The MIA model aimed at changes in social behavior has been previously severely criticized (PMID: 32445939) (not cited in the paper).
- The authors administered poly(I:C) and bumetanide but no description of the n of the animals and the total number of groups. In the Figures, I did notice 2 groups only. This is quite odd.
- Two-way ANOVA should be used.
- F and d(F) should (must) be always included in the results.
- All results should be given according to both poly(I:C) and bumetanide administration.
- The idea of bumetanide is intriguing. Indeed, in the brain, bumetanide blocks the NKCC1 cation-chloride co-transporter and thus decreases internal chloride concentration in neurons. In turn, this concentration change makes the action of GABA more hyperpolarizing, which may be useful for the treatment of neonatal seizures, which quite often are not responsive to traditional GABA-targeted treatment, such as barbiturates. Bumetanide is therefore under evaluation as a prospective antiepileptic drug. However, it has gross side effects. Bumetanide administration by two permanent cannulae into the two sides of PFC administration is quite an invasive method. Did the animals react well?
- I did not understand the age of animal sacrifices. In the paper, as far as I read, the authors stated that all testing was performed at 4 weeks of age. Please explain.
- NKCC1 is also expressed in many regions of the brain during early development, but not in adulthood. Did the authors check for NKCC1 brain presence in naïve 28/30 days old animals by ELISA or immunocytochemistry?
- Is KCC2 the abbreviation of NKCC2? If yes, the authors by western blotting found it in the PFC. This is quite original and should be better discussed also by further presence demonstration by ELISA or immunocytochemistry.
- The plural of cannula should be cannulae or cannulas.
- The English editing should be improved.
Comments on the Quality of English LanguageMinor editing of the English language required
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Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsPlease make sure your references are in the correct format for this journal.
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Reviewer 3 Report
Comments and Suggestions for Authors
In this revised paper the authors replied to some of my concerns.
However, the description of the methods and results of the Microinjection of bumetanide into PFC is still poor.
-As far as I may understand the authors used twelve
offspring in each of the saline, poly(I:C), poly(I:C) + saline, and poly(I:C) + BTN groups. It is NOT clear to the reader whether or not the poly(I:C) + saline group was poly(I:C) + ip saline or poly(I:C) + PFC saline. This should be clearly stated by the authors since the poly(I:C) + PFC saline group is the proper control of the bumetanide.
- The results of the bumetanide testing should include the data of the 4 analyzed groups (saline, poly(I:C), poly(I:C) + ip/PFC saline, and poly(I:C) + BTN). Figure 5 is the core of the paper and should include the data of the 4 analyzed groups and not only the findings disclosed for the poly(I:C) + ip/PFC saline, and poly(I:C) + BTN groups. I'm wondering why the data of the other groups were not included in these results.
- Were the offspring brothers? How many litters were used by the authors? Since this paper deals with behavior and locomotion did the authors consider the litter effect? This should be clearly stated in the paper.
Comments on the Quality of English LanguageMinor editing of the English language required
Author Response
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Round 3
Reviewer 3 Report
Comments and Suggestions for AuthorsThe paper is ok
