Ethanolamine and Vinyl–Ether Moieties in Brain Phospholipids Modulate Behavior in Rats

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

 

 

The manuscript describes the effects of hydrophilic moiety and the bond present at n-1 position in the glycerophospholipid on the behavioural changes in mice. The manuscript is beneficial in understanding how molecular changes in the glycolipid would modulate their functions and how the same is then manifested in form of behavioural effects in mice. Authors have claimed after battery of tests that replacing ethanolamine with choline helps in memory restoration and substitution of ester bond with vinyl-ether bond at sn-1 in phosphatidyl ethanolamine increases recall in NOR. Authors also claimed that in exploratory behaviour tests, vinyl-ether bonds in PE 18:0/22:6 restore exploratory behavior nature.  Although, the manuscript at this stage is written in a good understandable form but it still needs some minor revision as suggested below.

 

 

Q1. Line 68: Please provide abbreviation for OA, AA.

 

Material and Method:

 

Q2. OF Test: For this behaviour test, has author done any mice adaptation or was it not needed?

 

Q3.NOR test: Authors needs to mention here are the mice used for NOR test are the same as used for OF test or are they different sets of mice? If they are same set of mice, then how long after OF test these animals were used for NOR test.. Please explain if the authors have done any mice adaptation in this test or not?

 

 

Q4. EPM Test: If any adaptation was done for thie mice in EPM as EPM measures anxiety then even control mice may have some anxiety for first time in EPM test so how do we know if the effect if due to liposome injection?

 

Result:

 

Q5 Incorporation of dope into mice brain: Can authors quantify the fluorescence signals in both abdominal and brain regions and put in figure 1?

 

 

Author Response

The manuscript describes the effects of hydrophilic moiety and the bond present at n-1 position in the glycerophospholipid on the behavioural changes in mice. The manuscript is beneficial in understanding how molecular changes in the glycolipid would modulate their functions and how the same is then manifested in form of behavioural effects in mice. Authors have claimed after battery of tests that replacing ethanolamine with choline helps in memory restoration and substitution of ester bond with vinyl-ether bond at sn-1 in phosphatidyl ethanolamine increases recall in NOR. Authors also claimed that in exploratory behaviour tests, vinyl-ether bonds in PE 18:0/22:6 restore exploratory behavior nature.  Although, the manuscript at this stage is written in a good understandable form but it still needs some minor revision as suggested below.

 

Response: Thank you very much for your important comments. We are thankful for the time and energy you expended.

 

 Q1. Line 68: Please provide abbreviation for OA, AA.

 

Response 1: Thank you for your suggestion. As the reviewer suggested, the reader may be confused about the meaning of OA because it is difficult to correspond oleic acid to OA in phosphatidylcholine-plasmalogen-oleic acid (PlsCho-OA). To recognize that OA is the abbreviation of oleic acid at a glance, I revised the name of the phosphatidylcholine-plasmalogen-oleic acid to choline plasmalogen-oleic acid (PlsCho-OA) because of the following reasons: (1) choline plasmalogen is a synonym for phosphatidylcholine-plasmalogen, and (2) in the case of PE, we have used the term, ethanolamine plasmalogen (PlsEtn), but not phosphatidylethanolamine-plasmalogen.

We have spelled out the abbreviation, AA, in full at line 64.

 

Material and Method:

 

Q2. OF Test: For this behaviour test, has author done any mice adaptation or was it not needed?

 

Response 2: Thank you for your important question. In our study, we have examined the exploratory behavior under a novel environment as well as locomotor activity, therefore, adaptation of rats for the arena before OF test was not needed.

 

Q3.NOR test: Authors needs to mention here are the mice used for NOR test are the same as used for OF test or are they different sets of mice? If they are same set of mice, then how long after OF test these animals were used for NOR test.. Please explain if the authors have done any mice adaptation in this test or not?

 

Response 3: We used the same rats for all behavioral tests. We have added the schedule of the behavioral tests in figure 1. The rats were acclimated to the arena by OF test before conducting the NOR test because the same arena was used for OF and NOR tests. The NOR test was conducted 3 hours after the OF test on Day 1. We have corrected the sentences from lines 147-150 as described follows: Briefly, rats explored two identical objects (two 500 mL cylindrical water bottles measuring 10 cm in diameter) placed in the arena for 10 min on Day 1, three hours after the acclimation of the rats to the arena during the OF test, and were then returned to their home cages.

Further, we have explained the reason why we completed all behavioral tests in three days after injection with liposomes (lines 127-130) as described below.

The level of PE P-18:0/22:6 was still elevated in the prefrontal cortex at four days after injection with liposomes incorporating PE P-18:0/22:6 in our previous study, and therefore, all the behavioral tests were conducted within three days after liposome injection in this study (Figure 1).

 

Q4. EPM Test: If any adaptation was done for thie mice in EPM as EPM measures anxiety then even control mice may have some anxiety for first time in EPM test so how do we know if the effect if due to liposome injection?

 

Response 4: Thank you for your important query. We agree with your comments.

All behavioral tests were conducted under the same conditions among all groups as described in the figure 1. Furthermore, we used the saline-injected rats and the control liposome-injected rats as the control groups. However, we did not find significant differences in the results of the EPM test among all groups. I believe that these results suggest no effect of the phospholipids examined in this study on the anxiety-like behaviors.

 

Result:

 

Q5 Incorporation of dope into mice brain: Can authors quantify the fluorescence signals in both abdominal and brain regions and put in figure 1?

 

Response 5: Thank you for your suggestion. We have data about fluorescent signal intensities in the frontoparietal region of the brain although fluorescence intensity was not quantified unfortunately. We have added mean fluorescent signal intensity/pixel of the frontoparietal regions in Figure 2.

 

In addition to the corrections described above, I have corrected the order of the first, middle, and family names of the first author, MST ZENIKA NASRIN. I would like to apologize for changing the order of her name in the revised manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript from Zenika et al. details behavioral modulation induced by specific phospholipid moieties.  

- Including citations for the methods that are used in the paper would be helpful.

Experimental design although appropriate including some other methods would be beneficial. Such as : 

- In addition to the imaging studies done on the brain - running WB / IHC analysis on the hippocampus could provide more of a mechanistic insight. 

- Transcriptome analysis by RNA -seq on the hippocampus would have been another interesting approach to include in the scientific design of this project. 

 

 

Author Response

The manuscript from Zenika et al. details behavioral modulation induced by specific phospholipid moieties. 

 

- Including citations for the methods that are used in the paper would be helpful.

 

Response 1: Thank you for your important comments. We are thankful for the time and energy you expended. We have cited our published paper for the methods related to the organ imaging and behavioral tests (lines 107, 112, 129, 143, 146, 152, and 161).

 

Experimental design although appropriate including some other methods would be beneficial. Such as :

 

- In addition to the imaging studies done on the brain - running WB / IHC analysis on the hippocampus could provide more of a mechanistic insight.

 

- Transcriptome analysis by RNA -seq on the hippocampus would have been another interesting approach to include in the scientific design of this project.

 

Response: Thank you very much for your insight suggestions. We agree with you. WB/IHC analysis on the hippocampus was not conducted in our study, however, Hossain et al. reported that plasmalogen diet increased plasmalogen content in the mouse hippocampus, and increased p-ERK, p-Akt, p-CREB, synapsin-1, PSD-95, and SYT-1 proteins in the hippocampus (Hossain et al., 2022, cited in the manuscript). Hossain et al. also reported that plasmalogen diet upregulated Bdnf mRNA expression. Moreover, Li et al. suggest that plasmalogens by regulating hippocampal neurogenesis by activating the Wnt/b-catenin pathway (Li et al., 2023, cited in the manuscript). Similarly, PE P-18:0/22:6, PE 18:0/22:6, and PC 18:0/22:6 incorporated into liposomes in our study may act on the hippocampus through BDNF/TrkB/CREB signaling and/or Wnt/ b-catenin pathway. WB and IHC analyses are warranted in our future study.

We have corrected Discussion part from lines 293 to 308 as the following.

Conversely, a plasmalogen-enriched diet containing PlsEtn, PlsCho, sphingomyelin, and other phospholipids was found to increase Pl content in the hippocampus and to accelerate brain-derived neurotrophic factor (BDNF) signaling in mice by promoting tyrosine kinase receptor B (TrkB) expression in the lipid rafts of the hippocampus, and concomitantly to enhance learning and memory in the NOR test. Eicosapentaenoic acid (EPA)-enriched PE and the combination of Chlorella and ascidian PlsEtn activated BDNF/TrkB/cAMP response element-binding protein (CREB) signaling, which has an important role in neurogenesis, synaptic plasticity, and cognition, in the hippocampus. Additionally, a mixture of three ethanol amine plasmalogens, one choline plasmalogen, and ceramide alpha-hydroxy fatty-acid-sphingosine improved memory function by regulating hippocampal neurogenesis by activating the Wnt/ b-catenin pathway in a mouse model of Alzheimer’s disease. Collectively, these studies suggest that the enrichment of plasmalogens in the hippocampus can improve learning and memory through BDNF/TrkB/CREB signaling and/or the Wnt/ b-catenin pathway; however, the question of which intramolecular structures in the phospholipids are able to improve memory has not yet been definitively answered.

 

In addition to the corrections described above, I have corrected the order of the first, middle, and family names of the first author, MST ZENIKA NASRIN. I would like to apologize for changing the order of her name in the revised manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript by Zenika et.al. describing “Ethanolamine and vinyl–ether moieties in brain phospholipids 2

modulate behavior in rats” is a significant research work that will have multidisciplinary research applications. 
This reviewer would suggest that the authors to perform control experiments with the same phospholipid/liposome formulations, but using (1)  primary hippocampal neurons and (2) relevant BBB model cell lines in vitro. Such a study would give the readers a little more clarity of the intake and distribution process 

Author Response

The manuscript by Zenika et.al. describing “Ethanolamine and vinyl–ether moieties in brain phospholipids modulate behavior in rats” is a significant research work that will have multidisciplinary research applications.

This reviewer would suggest that the authors to perform control experiments with the same phospholipid/liposome formulations, but using (1)  primary hippocampal neurons and (2) relevant BBB model cell lines in vitro. Such a study would give the readers a little more clarity of the intake and distribution process.

 

Response: Thank you very much for your important suggestions. We are thankful for the time and energy you expended. We agree that control experiments with the same phospholipid/liposome formulations are needed to clarify the intake and distribution process by using primary hippocampal neurons and relevant BBB model cell lines in vitro: however, fluorescent labeled PE P-18:0/22:6, PE 18:0/22:6, and PC 18:0/22:6 are not yet available on the market and it is difficult to synthesize those phospholipids, especially plasmalogen, labeled with fluorescent materials or ¹³C. Development of the methods to trace those phospholipids are warranted in our future study.

We have corrected the limitation of this study (lines 372-374) as follows: Our present study lacks data involving the intake and distribution process of injected phospholipids and the activating or inhibiting pathway which led to the recognition memory and exploratory behaviors.

 

In addition to the corrections described above, I have corrected the order of the first, middle, and family names of the first author, MST ZENIKA NASRIN. I would like to apologize for changing the order of her name in the revised manuscript.