Neonatal Seizures and Purinergic Signalling

Round 1

Reviewer 1 Report

The current work of Menendez Mendez et al, is a very interesting and original review in the field of purinergic signaling and neonatal seizures. It is a well-written and organized work that represents an added value for the study of these topics.

In the first part of the paper they described first neonatal seizures and then purinergic systems. The authors comprehensively explain the state-of-the-art in regard to neonatal seizures etiology, therapeutic treatments and animal models used.

Paragraphs 4 and 5 get to the heart of the topic by describing the physiological and pathological role of purinergic signaling in CNS. In these sections, the review adequately reports studies that aim to target specific purinergic receptors as possible therapeutic strategies. Very interesting and innovative, section 5.3. dedicated to the suggestion of possible alternative therapeutic strategies by the authors. 

For these reasons I have only one strong suggestion for the authors: review the organization of Table 1. Some bibliographic references are missing and the division of spaces is unclear and confusing. I therefore suggest that this should be improved for a better understanding.

Minor suggestion regards Figure 1A: I would make a single Figure 1 without division in 1A and 1B. While the mechanisms represented in 1B are interesting, Figure 1A adds nothing new to what is already explained in the text.

Others minor suggestions follow:

• Section 3. The Purinergic System: the text should be left align; I would further divide the sub-paragraph 3.2 in two sub-sections P1 receptor family and P2 receptor family
• Line 207: "annion" should be changed in "anion"
• Line 258: check the brackets
• Figure 2, line 516: dot mark before "Cell debris" is missing
• References 9, 12, 43: removing link from the doi (http)

Author Response

We would like to thank the reviewer for the overall positive comments and constructive suggestions to improve our manuscript.

1. For these reasons I have only one strong suggestion for the authors: review the organization of Table 1. Some bibliographic references are missing and the division of spaces is unclear and confusing. I therefore suggest that this should be improved for a better understanding.

According to the reviewer’s suggestions, the format of the Table has now been changed to landscape orientation. This avoids information from a singular study being spread over two pages and in turn avoids creating confusion.

2. Minor suggestion regards Figure 1A: I would make a single Figure 1 without division in 1A and 1B. While the mechanisms represented in 1B are interesting, Figure 1A adds nothing new to what is already explained in the text.

According to the reviewers suggestions we have removed Figure 1A from our manuscript and have changed Figure 1B into Figure 1. This has also been changed in the Figure legend.

Others minor suggestions follow:

1. Section 3. The Purinergic System: the text should be left align; I would further divide the sub-paragraph 3.2 in two sub-sections P1 receptor family and P2 receptor family.

Accordingly, these subheadings have now been included for these paragraphs in Line 267 and Line 284.

2. Line 207: "annion" should be changed in "anion"

This has been corrected (Line 233): “Non-exocytotic mechanisms include the release of nucleotides by different types of channels, such as anion channels, pannexins and connexins.”

3. Line 258: check the brackets

This has been corrected (Lines 277): ” (e.g., epilepsy, neuropathy, neurodegenerative disorders or psychiatric conditions [118-121])”.

4. Figure 2, line 516: dot mark before "Cell debris" is missing

This has been added (Line 559) “Increases in intracellular calcium and cell death can trigger the release of glio/neurotransmitters (e.g., glutamate) into the extracellular space, that increases neurotransmission. Cell debris can trigger microgliosis, astrogliosis and release of pro-convulsive cytokines.”

5. References 9, 12, 43: removing link from the doi (http)

This has been removed accordingly.

Reviewer 2 Report

The paper is a useful review of the contribution of purinergic signaling to neonatal seizures. To the best of my knowledge, this topic has been largely ignored by other authors. Thus, the review is timely and of great value.

Minor comments:

Line 79. Should “compressive” be “comprehensive”?

Lines 88-89 compare to lines 104-105. There is an inconsistent usage of parentheses within parentheses.  [()] as compared to (()).

Lines 156-158. “Rates of developing cerebral palsy and developmental delay

157 were reduced following therapeutic hypothermia when examined at 18 - 22 months [60], however, no significant distinction on preventing disability could be made in later life [61].” This sentence suggests that the distinction cannot be made in later life rather than no conclusion can be drawn regarding the use of hypothermia to prevent disability later in life. I think the authors mean to convey the second sentiment.

Lines 188-200. Formatting problem. Not fully justified.

Lines 251-252. A3 receptors are also implicated in chronic pain (neuropathy). And, there is good evidence for A3 gene transcripts and receptors in microglia (Lines 273-274).

Line 328. “increasing” should be “increasingly”.

Lines 423-425. Reference 238 did not use electrophysiology.

Line 460. I’m not sure it’s correct to say that P2X7R activation leads to phagocytosis of apoptotic cells. P2X7Rs are scavenger receptors that promote phagocytosis in the absence of activation (that is, in the absence of agonist). Activation of P2X7Rs leads to a decrease in phagocytosis because agonist binding leads to a conformational change that dissociates the cytoskeleton from the C-terminus of X7.

Line 466. Dependend.

Line 598. (5% O₂ for 15 min) or (5% O₂, 15 min).

Line 618. Regarding P2X4R, has anyone shown an effect mediated by this receptor subtype in HUMAN microglia?

Author Response

We would like to thank the reviewer for the positive feedback and constructive comments.

Minor comments:

1. Line 79. Should “compressive” be “comprehensive”?

 This has been corrected (Line 83): ‘In fact, a comprehensive review of studies which evaluated an overall population of 4538 newborns with neonatal seizures observed 17.9% developed post-neonatal epilepsy [26].’

2. Lines 88-89 compare to lines 104-105. There is an inconsistent usage of parentheses within parentheses.  [()] as compared to (()).

 This has been changed accordingly (Line 92): “Moreover, rare cases of an inborn genetic component of neonatal seizures exist, with the majority altering metabolic pathways, including KCNQ2 mutations, infantile hypophosphatasia (mutations in the tissue nonspecific alkaline phosphatase (TNAP)), and propionic acidemia (deficiency of propionyl-CoA carboxylase).”

3. Lines 156-158. “Rates of developing cerebral palsy and developmental delay

157 were reduced following therapeutic hypothermia when examined at 18 - 22 months [60], however, no significant distinction on preventing disability could be made in later life [61].” This sentence suggests that the distinction cannot be made in later life rather than no conclusion can be drawn regarding the use of hypothermia to prevent disability later in life. I think the authors mean to convey the second sentiment.

We would like to thanks the reviewer for pointing this out. We have amended the sentence accordingly (Line 164): “Rates of developing cerebral palsy and neurodevelopmental delay were reduced following therapeutic hypothermia when examined at 18 - 22 months [60], however, no significant conclusion regarding therapeutic hypothermia’s ability to prevent disability could be made when followed up in later life”

4. Lines 188-200. Formatting problem. Not fully justified.

This has been aligned properly in our revised manuscript.

5. Lines 251-252. A3 receptors are also implicated in chronic pain (neuropathy). And, there is good evidence for A3 gene transcripts and receptors in microglia (Lines 273-274)

According to the reviewer’s suggestions we have included two citations to address this.

Line 278: Salvemini D, Jacobson KA. Highly selective A3 adenosine receptor agonists relieve chronic neuropathic pain. Expert Opin Ther Pat. 2017;27(8):967. doi:10.1080/13543776.2017.1341018.

Line 307: Evidence for functional adenosine A3 receptors in microglia cells. Christian Hammarberg, Gunnar Schulte and Bertil B. Fredholm. Journal of Neurochemistry, 2003, 86, 1051–1054

6. Line 328. “increasing” should be “increasingly”.

We have decided to leave increasing as stated in our original submission (Line 340): “ The P2Y₁₁ receptor is an exception as it is also able to couple to Gs which stimulates adenylyl cyclase thereby increasing cAMP production [149,166]”

7. Lines 423-425. Reference 238 did not use electrophysiology.

Thank you for pointing this out. We have removed sentence, ‘using electrophysiology techniques’ and has the sentence has been changed to (Line 463): “Multiple groups have observed neuronal P2X7 localised to presynaptic terminals [237,238].”

8. Line 460. I’m not sure it’s correct to say that P2X7R activation leads to phagocytosis of apoptotic cells. P2X7Rs are scavenger receptors that promote phagocytosis in the absence of activation (that is, in the absence of agonist). Activation of P2X7Rs leads to a decrease in phagocytosis because agonist binding leads to a conformational change that dissociates the cytoskeleton from the C-terminus of X7.

We would like to thank the reviewer for spotting the error. The paragraph has now been changed to (Line 497): “Additionally, P2X7 might regulate the population of NPCs though innate phagocytosis of dead cells throughout development. In this regard, neuroblasts isolated from human fetal telencephalons are able to phagocytose apoptotic cells in the absence of P2X7 receptor activation [253].”

9. Line 466. Dependend.

This has been corrected

10. Line 598. (5% O₂ for 15 min) or (5% O₂, 15 min).

This has been corrected accordingly.

11. Line 618. Regarding P2X4R, has anyone shown an effect mediated by this receptor subtype in HUMAN microglia?

To our knowledge P2X4R in human microglia under hypoxic conditions has not been investigated. However, P2X4 has been observed in human microglia and is upregulated in microglia in multiple sclerosis patients, where it is hypothesized to control the fate and survival of activated microglia.

Vázquez-Villoldo, N., et al. (2014). "P2X4 receptors control the fate and survival of activated microglia." Glia 62(2): 171-184.