ࡱ> ]_\[ RUbjbj The sentence was shortened as The PCER (50 mg/kg, p.o.) recovered the scopolamine-induced reduction in step-through latency in the passive avoidance test; however, PSO did not modulate the memory function on this task. Page 1, Line 28-30: The authors should revised the Abstract. These sentences contain repetitive information: The effects of phytoceramide on spatial learning were evaluated by the Morris water maze test. The memory enhancing effect of Pcer was also acertained in Morris water maze test. => The sentence was revised as The effects of PCER on spatial learning were confirmed by the Morris water maze test. Page 3, Line 1: The authors should not start a sentence with And. And then Pso is acylated by various fatty acids to form phytoceramides. => Phytosphingosine is acylated by various fatty acids to form phytoceramides. Page 3, Line 10-17. Is there an interaction between cholinergic system and sphingolipids? It should be mentioned in the Introduction section. => The explanation was added in Introduction as Profound losses in the cholinergic system of brain are associated closely with cognitive deficits observed in Alzheimer disease [Bartus et al.,] Page 4, 1-5: Legend of Figure 2 should indicate the statistical difference between the groups. For instance, the asterisk (*) indicated in the Figure 2C represented statistical difference when compared to the L-glutamate group? Why did not Pc 0.1 present the asterisk (*)? => We present the asterist(*) in the column of Pc0.1 of Figure 2C. How were the doses of Pso and Pcer chosen for behavioral experiments? The authors performed a dose-response curve? => We choose the dose of 50mg/kg phytoceramide as therapeutic dose based on the pretest since 10, 20mg/kg of phytoceramide did not affect the memory recover. A two-way analysis of variance should be performed considering that it is necessary to evaluate that there is the effect of scopolamine and Pso or Pc and their interaction. => We reanalyzed the two-way ANOVA and represent the significance on the Figures. The manuscript is poorly discussed. The discussion should analyze the different effects observed by Pcer and Pso and the involvement of neurochemical mechanisms in the effects observed. => We expand the discussion as Ceramide has been known as a sphingolipid with potent proinflammatory and proapoptotic properties [14-16] and its active metabolite, ceramide 1-phosphate, stimulates macrophage proliferation through activation of the PI3-kinase, JNK and ERK pathways [17]. However, studies using primary cultures of neurons demonstrated that ceramide has multiple functions, depending on the cell type and the developmental stage. In immature hippocampal neurons, ceramide plays bipotential roles in cell survival and dendrite outgrowth in a dose-dependent manner [18]. It was also found that ceramide prevent cell death of motoneurons cultures through inhibition of oxidative signal [19]. These results suggest that the cellular level of ceramide is critical for regulation of neuronal survival and differentiation. In our result, PCER inhibited the glutamate-induced neurotoxicity in cultured neuronal cells. However, PSO did not show the neuroprotective effect with same dose of PCER in the cultured neuronal cells. Phytosphingosine is structurally similar to sphingosine, except that PSO has a hydroxyl group at C-4 of the sphingoid long-chain base instead of trans double bond between C-4 and C-5. Scopolamine, which is a muscarinic receptor antagonist, can cause amnesia in animals by blocking cholinergic neurotransmission and impairs learning and memory in rodents and humans. It was determined whether Pcer attenuates scopolamine-induced learning and memory impairments through the passive avoidance, Y-maze test, and water-maze tests. Pcer reversed the memory deficits induced by scopolamine. The passive avoidance test is a useful tool for the estimation of standard learning and memory. Then, this test has been used as an indicator of short-term and long-term memory [20]. Reviewer 2: 1. Overall the paper uses acceptable Standard English; however, several areas of the paper are more difficult to understand due to nonstandard uses of English words. I would suggest a through edit paying close attention to word choice. => We need English editing. 2. Phytoceramide and phytosphingosine should be abbreviated (Pcer) and (Pso) once and then only the abbreviation should appear throughout the rest of the manuscript including figures and figure captions. => We have checked abbreviation and used PCER for phytoceramide and PSO for phytosphingosine. 3. The use of the serial comma needs to be added throughout the manuscript (see Strunk and White, 2000). => We have checked the serial comma and corrected. 4. Minor point: I would change the phytosphingosine and phytoceramide abbreviations from Pso and Pcer to all uppercase PSO and PCER throughout the manuscript (see American Psychological Association., 2009). => We have checked abbreviation and used PCER for phytoceramide and PSO for phytosphingosine. 5. All ANOVAs should be written with the degrees of freedom numerator and denominator and the F value. For example, F(3,36) = 5.17, p = 0.0123. Also, the authors need to state the p-value that accompanies the pairwise post-hoc test. For example, The Ctl and PSO groups were significantly higher than the PCER group (post-hoc ps < 0.0123). Please alter the text throughout the results and discussion section accordingly. => We have checked the statistics and denote the significance between groups in the figures 6. Minor point: I would take all descriptions of the statistical tests and post-hoc procedures out of the figure captions and move them to the statistical analysis section. => We have moved all description of the statistical tests procedures out of the figures to the statistical section. 7. What the error bars represent in each figure needs to be explained in each of the separate figure captions (e.g., Data represent the mean 1 standard deviation or mean SEM). => We have corrected the expression of data represent as meanstandard errors (SE) 8. If possible, the authors should provide references to support their PSO and PERC dosing procedures. => We have explained why we selected 50mg/kg PCER in the text. Specific Comments Abstract: 1. On line 20, define CNS before the abbreviation is used. => We have corrected as suggested. 2. On line 22, I would change animal models to mice since only mice were used in this study. => We have corrected as suggested. 3. Minor point: on line 27, change the , after latency to a ; (see Strunk and White, 2000). => We have corrected as suggested. 4. On line 27 and 28, do the authors mean, did not modulate the memory function on this task? => We have corrected as The PCER (50 mg/kg, p.o.) recovered the scopolamine-induced reduction in step-through latency in the passive avoidance test; however, PSO did not modulate the memory function on this task. 5. On line 28, phytoceramide should be changed to PCER since it had been previous abbreviated. => We have corrected as suggested. 6. On line 28, do the authors mean, spatial learning and memory? The Morris maze results are not presented in such a manner to dissociate learning from memory. => We have corrected learning into memory. 7. The two sentences in lines 28 - 30 essentially say the same thing. One sentence should be removed. => We have corrected as suggested. One sentence was removed. 8. A short summary of the effects of PCER on Morris maze performance should be added since it was included for passive avoidance. => We have corrected as suggested. 9. On line 32, Pcer can be applied as therapeutic agent for the treatment of neurodegenerative disease, should be changed to something akin to, Pcer is a potential new therapeutic agent for the treatment of neurodegenerative disease. => We have corrected as suggested. 10. On line 34, is ischemia considered a neurodegenerative disease? => We have removed the ischemia as suggested. Introduction: 1. A reference/citation is necessary in order to substantiate the claims made on page 2 lines 3 5. => We have cited the reference as suggested. 2. On page 2 line 11, certain compound should be changed to certain compounds or a certain compound. => We have corrected as compounds. 3. Lines 2-13 on page 2 present information without tying the three points together into a cohesive story. I suggest that this section be rewritten so that it can set up the rationale for the study. Additionally, the rationale for using scopolamine needs to be further explained in the context of how it affects the cholinergic system and mimics some AD symptomology. => We have rewritten the suggested section; More focused on the cholinergic system is involved in AD disease. 4. Minor point: On page 2 line 14, the major findings should be changed to major findings. => We have corrected as suggested. 5. The serial comma was used previously used on line 3 and needs to be added on page 2 line 16 and throughout the manuscript (see Strunk and White, 2000). => We have corrected as suggested. 6. Especially should be removed from line 16 on page 2. => We have corrected as suggested. 7. A reference/citation is necessary in order to substantiate the claims made on page 2 lines 21 23. => We have cited the reference as suggested. 8. On page 3 line 1, sentences should not start with And. => We have corrected as suggested. 9. On page 3 lines 10-16, these sentences contain several nonstandard uses of English words making their meaning harder to comprehend. I suggest that the sentences be rewritten. => We have rewritten as Profound losses in the cholinergic system of brain are associated closely with cognitive deficits observed in Alzheimer disease [Bartus et al.,]. Therefore, the current study focused on the effects of the PCER and PSO on the memory impairment by suppressing cholinergic activity in the central nervous system of mice. The memory performance was examined by using passive avoidance and Morris water maze tasks in mice which were impaired in memory by scopolamine treatment. Results and Discussion: 1. A reference/citation is necessary in order to substantiate the claims made on page 3 line 19. => We have cited the reference as suggested. 2. LDH needs to be designated as an abbreviation for lactate dehydrogenase. => We have corrected as suggested. 3. In text reports of the statistical test that evaluated LDH release need to be added (e.g., F(3,36) = 5.17, p = 0.0123). => We have calculated the significance. 4. On page 3 lines 19 27, phytoceramide and phytosphingosine should be changed to PCER and PSO throughout since they had both been previous abbreviated. => We have corrected as suggested. 5. On page 3 line 28, passive test should be changed to passive avoidance test. => We have corrected as suggested. 6. A reference/citation is necessary in order to substantiate the claims made in the sentence on page 3 lines 30 31. => We have cited the reference as suggested. 7. On page 3 line 32, define p.o. before the abbreviation is used. => We have corrected as suggested. 8. On page 3 line 33, please report the other statistical test information aside from the p-value (e.g., F(3,36) = 5.17, p < 0.01) and change the , after (p < 0.01) to a ;(see Strunk and White, 2000). => We have corrected as suggested. 9. At the end of page 3, the authors should summarize what the passive avoidance data suggest about the effects of PSO and PCER on memory. => We have corrected as suggested. 10. If possible, representative photomicrographs displaying the neuronal morphology of each treatment in culture (i.e., Vehicle, PCER, PSO, and L-glutamate) should be added to figure 2 (see Diaz Brinton et al., 2000). This will allow the reader to form their own conclusions regarding the neuroprotective effects of PCER. => We have represented the photoimage of neuroprotection of PCER in Fig 2 as suggested. 11. On figure 2, phytoceramide and phytosphingosine should be changed to PCER and PSO to keep consistent with the text. => We have corrected as suggested. 12. For figure 3, the authors need to add a reference/citation to validate their timeline of behavioral testing 30 minutes after Sco treatment. This should be added to the experimental section. => We have cited the reference as suggested. 13. Minor point: On figure 3, I would change the scopolamine abbreviation from Sco to all uppercase SCO (see American Psychological Association., 2009). => We have corrected as suggested. 14. On page 5 line 2, if the authors wish to only test spatial learning and not learning and memory, then a clear operational definition for learning on the Morris mazes needs to be added here or in the experimental section that is substantiated by previous literature and supported by their data. => We have corrected as spatial learning and memory. 15. On page 5 line 5, On the day following the final day of training trial sessions should be changed to On the probe trial (see de Hoz et al., 2005). => We have corrected as On the probe trial 16. On page 5 lines 2 10, phytoceramide and phytosphingosine should be changed to PCER and PSO throughout since they had both been previous abbreviated. => We have corrected as suggested. 17. On page 5 lines 2 - 10, please report the other statistical test information aside from the p-values (e.g., F(3,36) = 5.17, p < 0.01). 18. On page 5 lines 2 - 10, since a tracking system using EthoVision was employed, a more appropriate Morris maze dependent variable to analyze other than escape latency would be swim distance or path length. Please reanalyze the Morris maze data using swim distance. I would report swim distance as the only Morris maze dependent variable or as the dependent variable that is supported by escape latency and swim speed. => Unfortunately, our EthoVision is not working now (something wrong), we could not reanalyze the old data. 19. On page 5 lines 2 - 10, an omnibus repeated measures or mixed model ANOVA should be used to evaluate a Treatment main effect across all days and trials or in specific day and trial blocks followed by the appropriate post-hoc test to evaluate pairwise differences. With a repeated measures ANOVA, Treatment is the between-subjects factor and Days and Trials are the nested within-subjects factors. Please reanalyze the Morris maze data in this manner. This will allow interpretation of Treatment main effects across the Morris maze task instead pairwise effects within each day, post-hoc tests can then be used to evaluate pairwise comparisons. => We have evaluated the pairwise comparison and marked the figures 20. For figure 4 line 15, define i.p. before the abbreviation is used and what was the rationale for using 1mg/kg (i.e., can the authors provide a citation for this dose?). => We have corrected and cited reference as suggested 21. The authors interpretation of Morris (1984) paper is incorrect on page 6 lines 4 - 5. An evaluation of Trial 1 to trial 2 within a day is only a measure of working or short-term memory when one is employing a matching to sample procedure where the platform is moved to a new location each day. The authors did not move their platform each day according to their description and thus could not evaluate working/short-term memory. This sentence needs to be removed or revised to account for this fact. => We have corrected the sentence as suggested. 22. On page 6 lines 9 11, I am not sure why the authors conclude their discussion with an explanation of structural differences between PSO and PCER. This sentence should be revised or another sentence included explaining to the reader why this information is important at this point in the manuscript. Furthermore, phytosphingosine should be changed to PSO since it had been previous abbreviated in the manuscript. => We have expanded and reorganized the discussion section. Experimental section: 1. The sentence starting line 12 on page 7 is unneeded in the experimental section. This sentence should be moved to the introduction or the appropriate results and discussion section with an accompanying citation describing why LDH is a great marker of cellular toxicity. => We have removed unnecessary sentence as suggested. 2. On page 8 line 17, I suggest that the sentence, All values were expressed as mean standard error (SE) should be removed and added to each figure caption. This statement is also in opposition to the statement in the caption for figure 2 that stated data represent mean standard deviation. This inconsistency needs to be rectified. => We have added thesentence Data represent the mean ( standard error (SE) to each figure caption. Conclusion: 1. Phytoceramide and phytosphingosine should be changed to PCER and PSO and not be redefined as abbreviations in brackets since they had both been previous abbreviated. => We have corrected as suggested. 2. On page 8 line 24, For, The ameliorating effect of Pcer, what is the ameliorating effect of Pcer on? Do the authors mean the ameliorating effect of Pcer on learning and memory? Please revise this sentence. => We have corrected as suggested. 3. A sentence or two should be added that describes what the implications of this studys findings mean in the grand scheme of things (i.e., a take home point for the reader), similar to the one that concludes the abstract. => We have added more explanation in conclusion section as suggested.  Reviewer 3: This study reports the data of two molecules and their neuroprotective memory enhancing effects of Pcer and Pso. The methods used are suitable to investigate this hypothesis. Although the data of the present study seem interesting, I have several comments the authors need to address. Only one dose of Pcer and Pso were used. The authors should explain where these dosages were based on. Could the different effects of Pcer and Pso be explained by not using the optimal dose? => We have tested the phytoceramide and phytosphingosine with variable dose in preliminary experiment. We found the lower dose of phytoceramide (<25 mg/kg) did not show the ameliorating effect on memory impairment.. Minor: - Although Im not a native English speaker, I think the English could be improved. => We need English editing service. - In the Introduction it is stated: In regard to brain functions, the major findings have emphasized the significance of sphingolipid as bioactive molecules. It is my opinion that this is a too strong statement. There are many other brain functions not related to these molecules. => We explain the function of sphingolipid as bioactive molecule in the brain function.       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