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GABALAGEN Facilitates Pentobarbital-Induced Sleep by Modulating the Serotonergic System in Rats

Curr. Issues Mol. Biol. 2024, 46(10), 11176-11189; https://doi.org/10.3390/cimb46100663

by Minsook Ye¹, Kyoung-min Rheu²

, Bae-jin Lee² and Insop Shim^1,*

Reviewer 1: Anonymous

Reviewer 2: Anonymous

Reviewer 3: Anonymous

Curr. Issues Mol. Biol. 2024, 46(10), 11176-11189; https://doi.org/10.3390/cimb46100663

Submission received: 14 August 2024 / Revised: 14 September 2024 / Accepted: 1 October 2024 / Published: 4 October 2024

(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

The manuscript appears to be lacking data in the results section. There is no mention of the chemical analysis of the GABALAGEN product, although there is information elsewhere of the amino acid content. In the letter accompanying the submission there are details of greatly increased levels of GABA, glycine, serine, glutamic acid etc. These results should be included in the actual manuscript. Also is there any 5HT or related chemicals, such as tryptamine, in GABALAGEN? Or was this not analysed for? If it was please provide the data.

The statement on page 2 "Although there are no current studies investigating the effect of GBL on sleep, its high GABA and glycine content could lead to a positive impact on sleep efficacy for those with sleep complaints." thus has no supporting data.

Also there is nothing in the manuscript to support the statement on page 8 that "GBL binds to both GABAA-BZD and 5-HT2C receptors, thereby synergistically stimulating serotonin and GABAergic systems in the brain." Where is the data that GBL binds to GABAA receptors?

Likewise on page 9 there is no data to support the statement "GBL binds to both GABAA-BZD and 5-HT2C receptors, thereby synergistically stimulating serotonin and GABAergic systems in the brain.

The case for a mechanism for the involvement of GABA in GBL influencing sleep appears to rest one (1) high GABA content in GBL, (2) known effects on GABA on sleep processes, (3) effects of pentobarbital and diazepam as known modulators of GABAA receptor activation.

Author Response

Dear Editor;

Attached please find a revised copy of our manuscript cimb-3182102 entitled “GABALAGEN Facilitates Pentobarbital-Induced Sleep by Modulating the Serotonergic System in Rats” which we have modified in response to the critiques of your referees. All changes in this revision are highlighted (yellow text).

We hope the manuscript is now in a form suitable for publication in CIMB.

Reviewer 1

Response: We have added details on the chemical analysis of the GABALAGEN product and its amino acid content to the results section of the revised manuscript as “To determine the GABA content in GBL, HPLC analysis was performed. The presence of GABA after fermentation was confirmed by comparing the retention times of the standard and GBL (Fig. 1). The HPLC analysis results indicated that the average GABA content in GBL was 11.39%. Additionally, the amino acid composition of GABALAGEN shows significantly increased levels of threonine, aspartic acid, serine, glutamic acid, glycine, alanine, valine, leucine, arginine, proline, carnosine, and ornithine compared to the initial collagen, as presented in Table 1." Also, it is shown that GABALAGEN does not contain 5-HT. GABALAGEN is produced through the fermentation of collagen using Lactobacillus brevis BJ20 and Lactobacillus plantarum BJ21, microorganisms that are renowned for their capability to produce high levels of GABA. These specific microorganisms were selected for their ability to enhance GABA production during the fermentation process.

Figure 1. GBL chemical analysis. HPLC chromatograph of GABA in GBL

Table 1. Amino acid composition in GBL

Response: We have revised the statement to better reflect the potential impact of GBL on sleep, with reference to the established roles of GABA and glycine. The updated sentence is as follows: "Although no studies have directly investigated the effect of GBL on sleep, the high concentrations of GABA and glycine in GBL indicate a potential positive impact on sleep efficacy for individuals with sleep complaints. This hypothesis is based on the well known roles of GABA and glycine in promoting relaxation and sleep, as demonstrated in previous studies [12, 23]."

Likewise on page 9 there is no data to support the statement "GBL binds to both GABAA-BZD and 5-HT2C receptors, thereby synergistically stimulating serotonin and GABAergic systems in the brain.

Response: Thank you for pointing out the lack of supporting data for the statements regarding GBL's binding to GABA_A-BZD and 5-HT_2C receptors. We acknowledge that the current manuscript does not provide direct evidence that GBL binds to GABA_A receptors. To address this, we added statement of the study limitations of the existing data, and plan to conduct further experiments to investigate this binding interaction.

The statement on page 8, "GBL binds to both GABAA-BZD and 5-HT2C receptors, thereby synergistically stimulating serotonin and GABAergic systems in the brain," has been revised to: "GBL has been demonstrated to bind to 5-HT2C receptors, potentially influencing the serotonergic system. However, there is currently no direct evidence of GBL binding to GABAA-BZD receptors, and further investigation is required to confirm this interaction." The statement on page 9, "GBL binds to both GABAA-BZD and 5-HT2C receptors, thereby synergistically stimulating serotonin and GABAergic systems in the brain" was revised to "Although the interaction of GBL with 5-HT2C receptors is supported by the data, its potential binding to GABAA-BZD receptors has not been confirmed and necessitates further research to elucidate this mechanism."

Response: We have revised the manuscript to better reflect your suggestions. Specifically, we have expanded on the role of GABA and its interaction with 5-HT_2C receptors, as well as reinforced the connection between GABA's established effects on sleep regulation and the high GABA content in GBL. We have included the following "In addition, GABA may can indirectly affect the activity of 5-HT_2C receptors. It is known that increased GABAergic inhibition via GABA_A receptors can enhance the activity of 5-HT_2C receptors in the hypothalamus, influencing functions such as sleep regulation, appetite suppression or temperature regulation. Therefore, the interaction between the GABAergic system and serotonin receptors like 5-HT_2C plays a crucial role in sleep control. The involvement of GABA in GBL's influence on sleep is supported by several key points: (1) the high GABA content in GBL, (2) the established effects of GABA on sleep regulation, and (3) the effects of known GABA_A receptor modulators such as pentobarbital and diazepam. GABA is widely recognized for its role in promoting sleep and reducing wakefulness, and its presence in GBL may contribute to the compound's sedative properties. This is consistent with the action of other sleep-promoting agents, such as pentobarbital and diazepam, which enhance GABAA receptor activation. Given these similarities, it is plausible that GBL exerts its effects through a related pathway, modulating GABAergic activity to influence sleep architecture."

Author Response File: Author Response.pdf

Reviewer 2 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

“GABALAGEN Facilitates Pentobarbital-Induced Sleep by Modulating the Serotonergic System in Rats”（cimb-3182102）

Thanks for the revisions and some concerns remain.

1. For the p value, please provide the specific data unless it is less than 0.001.

2. Because there are more than 2 conditions in the current investigation, corrections need to be done for multiple comparisons, such as Bonferroni correction.

3. Please provide the effect size where available.

Comments on the Quality of English Language

Minor editing of English language required.

Author Response

Dear Editor;

We hope the manuscript is now in a form suitable for publication in CIMB.

Reviewer 2

Thanks for the revisions and some concerns remain.

For the p value, please provide the specific data unless it is less than 0.001.

Response: We have revised the manuscript to include specific p values where they are 0.001 or above.

Because there are more than 2 conditions in the current investigation, corrections need to be done for multiple comparisons, such as Bonferroni correction.

Response: Corrections for multiple comparisons have been applied using the Bonferroni correction method, given that the current investigation involves more than two conditions in the revised manuscript.

Please provide the effect size where available.

Response: We provided the effect size in the revised manuscript.

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

This study presents compelling evidence for the potential sleep-enhancing effects of GABALAGEN (GBL), a product rich in γ-aminobutyric acid (GABA) derived from the fermentation of collagen by specific Lactobacillus strains. Through receptor binding assays and EEG analysis in a pentobarbital-induced sleep model in rats, GBL demonstrated significant affinity for the 5-HT2C receptor and increased serum 5-HT levels, particularly at higher doses. These findings suggest that GBL's sedative effects may be linked to the activation of the serotonergic system, making it a promising candidate as a novel sleep-inducing compound in natural products.

Major concerns:

Why did the authors choose to further evaluate the effects of GBL on sleep regulation using a pentobarbital-induced sleep model, given that pentobarbital alone already induces sleep? What is the underlying rationale for this choice? What considerations led to this decision instead of simply administering GBL to normal rats?

Minor concerns：

1. The abbreviation "GBL" is introduced in the abstract without clarification that it stands for GABALAGEN.

2. In Figure 2, it is unclear what the groups labeled "Nor" and "Con" represent, as this is not explained in the text or figure legend.

3. It is recommended that the section headings and figure titles in the Results section be written to directly reflect the specific conclusions drawn from the data presented.

4. The abbreviation "DZP" is not clearly defined.

Author Response

Dear Editor;

We hope the manuscript is now in a form suitable for publication in CIMB.

Major concerns:

Response: The rationale for selecting the pentobarbital-induced sleep model to evaluate the sleep-regulating effects of GBL, despite pentobarbital itself being a sleep-inducing agent, lies in the model's ability to provide a standardized and quantifiable assessment of sleep enhancement effects. The pentobarbital-induced sleep model is commonly used to evaluate the sedative and sleep-inducing properties of compounds by measuring specific parameters wake, REM, and NREM times are measured through EEG, which is widely used to evaluate the sedative and sleep-inducing properties of compounds. This model helps to minimize the natural variability in sleep patterns that might occur when GBL is administered alone in normal rats, allowing for a more precise evaluation of whether GBL genuinely improves or alters sleep patterns. Thus, using this model allows us to clearly identify GBL’s contribution to sleep enhancement and facilitates comparisons with other known sedative agents. This approach enables a clearer and more reliable understanding of GBL's pharmacological profile in sleep regulation.

Minor concerns：

The abbreviation "GBL" is introduced in the abstract without clarification that it stands for GABALAGEN.

Response: We have revised the abstract to clarify that the abbreviation "GBL" stands for GABALAGEN, as suggested.

In Figure 2, it is unclear what the groups labeled "Nor" and "Con" represent, as this is not explained in the text or figure legend.

Response: Thank you for pointing out the lack of clarity regarding the group labels in Figure 2. We have revised the figure legend to clearly define the groups as follows:

Nor: Normal group, untreated and naïve. Con: Control group, injected with pentobarbital and vehicle without any additional treatment.

It is recommended that the section headings and figure titles in the Results section be written to directly reflect the specific conclusions drawn from the data presented.

Response: We have revised the section headings and figure titles to more accurately reflect the specific conclusions drawn from the data presented. The heading "2.1. Binding affinity GBL to 5-HT_2C receptor" has been revised to "2.1. GBL shows high binding affinity to 5-HT_2C receptor, supporting its sedative potential." Figure titles have been updated from general descriptions to specific outcomes, such as changing "Effect of GBL on EEG sleep architecture and profile" to "GBL enhances NREM sleep and reduces Wake Time in pentobarbital-Induced rat model."

The abbreviation "DZP" is not clearly defined.

Response: We have added the abstract to include the following sentence as “Diazepam (DZP) was used as a positive control to compare the efficacy of GBL."

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I found this manuscript very confusing and apparently lacking in certain information.

Page 1: Abstract states “GABALAGEN(GBL), a low-molecular-weight collagen infused with GABA”. Is GABA bound covalently to the collagen? What else is produced in the two fermentation processes? Has the product been assayed for 5HT?

Page 2: What does statement really mean? “GABALAGEN (GBL) is a type of collagen with a low molecular weight that contains GABA, which has the same structure as collagen molecules.” How does the product have the3 same stricture as collagen?

Page 3: Stated “The study demonstrated that GBL exhibits robust binding activity to the 5-HT2C receptor, characterized by its efficacy as a 5-HT2C receptor agonist.” How is it shown to be an agonist? Were any 5-HT receptors tested or only 5-HT2C?

Page 6: Stated “In the screening test for GBL, it exhibited the highest binding affinity to the 5-HT2C receptor” Highest compared to what else?

Page 8: Stated “the GABA receptor was sourced from rat brain tissue.” How was the GASA receptor used? Can’t find any other mention of it in the manuscript other that on Page 9 “Tryptamine and muscinol were used as negative controls. GBL was used to evaluate the sedative effect of the GABA receptor and 5-HT2c receptor binding assays.”

Page 8 and 9: “muscinol” should be “muscimol”.

Does GABA itself influence 5-TH2C receptors? This would be an important control.

What about 5-HT3 receptors? These are ionotropic receptors that have significant sequence homology with ionotropic GABA(A) receptors.

Author Response

Dear Editor;

Attached please find a revised copy of our manuscript cimb-3059418 entitled “GABALAGEN Facilitates Pentobarbital-Induced Sleep by Modulating the Serotonergic System in Rats” which we have modified in response to the critiques of your referees. All changes in this revision are highlighted (yellow text).

We hope the manuscript is now in a form suitable for publication in CIMB.

Reviewer 1

I found this manuscript very confusing and apparently lacking in certain information.

Response: We have clarified defined GABALAGEN as follows: 'GABALAGEN is the product of fermented collagen by Lactobacillus brevis BJ20 (L. brevis BJ20) and Lactobacillus plantarum BJ21 (L. plantarum BJ21), enriched with GABA and characterized by low molecular weight. In order to achieve a low-molecular-weight collagen (275 dalton), fish collagen is first hydrolyzed at 55℃±2℃ for 12h with prozyme then go through two consecutive fermentations via Lactobacillus brevis BJ20 (accession No. KCTC 11377BP) and Lactobacillus Plantarum BJ21 (accession No. KCTC 18911P). GABA in GABALAGEN is by product of fermentation with Lactobacillus brevis and plantarum.

Regarding covalent biding of GABA to the collagen, GABA is not bound covalently to collagen. GABA binds to specific receptors on their surfaces. It is not typically associated with collagen, known as a structural protein found in connective tissues such as skin and bones. These proteins provide strength and structure to tissues rather than participating in neurotransmission like GABA. Therefore, there is no known biological mechanism for GABA to be covalently bound to collagen so far.

In addition, other products have been produced and found through the two fermentation processes except GABALAGEN containing high-concentration of GABA with low-molecular-weight collagen peptide and postbiotics.

Response: We have defined GABALAGEN for clarity as follows 'GABALAGEN is the product of fermented collagen enriched with GABA and characterized by low molecular weight. GABALAGEN is a form of fermented collagen which has a similar structure and properties to chemically treated collagen.

Response: In 5-HT2 receptor binding assay, GBL binds very highly and competitively to 5-HT binding sites on receptors. We do not directly test for agonist or antagonist activity against 5-HT2 serotonin receptors in the present study. However, we assumed that GBL may be 5-HT2c receptor antagonist since it was very highly competitive with tryptamine, a basic neurotransmitter systems which confirming its selectivity for serotonin receptors. Therefore it may be acting 5-HT2c antagonistic effects on behavior. Therefore we assumed its efficacy as a 5-HT2C receptor agonistic effects on behaviors. Previous studies have shown that antagonists of the 5-HT2C receptor increase NREM sleep [36], indicating effectiveness in promoting sleep. Consistent with this, our results demonstrate that GBL enhances NREM sleep, thus improving sleep quality.

As the reviewer pointed out, we need to conduct functional assays to test whether it is agonist or antagonist activity in order to comprehensively understand GBL's pharmacological profile using a variety of 5-HT2c agonist and antagonists in the further study. These points were discussed in the revised one.

We did not investigate GBL on other 5-HT receptor systems in this study.

Page 6: Stated “In the screening test for GBL, it exhibited the highest binding affinity to the 5-HT2C receptor” Highest compared to what else?

Response: In a pilot study, we tested biding affinity of GBL on the GABA_A receptor and 5-HT_2C receptor binding assays. GBL exhibits higher binding activity to 5-HT_2C receptors than to GABA_A receptors, and we only included results of 5-HT_2C receptors, and deleted data of GABA_A receptor, and accordingly errors were corrected in the revised manuscript.

Response: We modified the 5-HT2c receptor binding assay in material and methods section as “The 5-HT2c receptor (serotonin 5HT2c membrane preparation in HEK293 cells) was procured from PerkinElmer (Waltham, MA, USA). The Protein Chip was obtained from Proteogen, and Cy5-labeled tryptamine were acquired from Peptron. The stock buffer for the 5-HT2c receptor comprised 50 mM Tris-HCL (pH 7.4), 0.5 mM EDTA, 10 mM MgCl2, and 10% sucrose. The 5-HT2c receptor-binding assay buffer comprised 50 mM Tris-HCl, 10 mM MgCl, 1 mM EDTA, and 0.1% bovine serum albumin (BSA) at pH 7.4. The 5-HT2c receptor (50 µg/mL) was immobilized on the Protein Chip, serving as a substrate to capture protein, for 16 h at 4 °C. After double washing in 0.05% phosphate-buffered saline containing 0.2% Triton X-100 (PBST) for 10 min and drying with Nitrogen gas (N2), the Protein Chip underwent a 1-hour blocking step at room temperature using 3% BSA. Following three washes with PBST and drying, Cy5-labeled tryptamine (500 µM, with 30% glycerol in PBS as a buffer) and GBL (using 30% glycerol in PBS as a buffer) were applied to the Protein Chip and incubated for 1 h at 37 °C. Subsequently, the Protein Chip was rinsed with PBST and DW and dried under a stream of N2 gas. GBL, dissolved in ethanol and diluted to the desired concentration using PBS, covered a con-centration range from 1,000 µM to 15.625 µg/mL. Tryptamine was used as negative controls. GBL was used to evaluate the sedative effect of the 5-HT2c receptor binding assays.”

Page 8 and 9: “muscinol” should be “muscimol”.

Response: We corrected typos in current revised manuscript.

Does GABA itself influence 5-TH2C receptors? This would be an important control.

What about 5-HT3 receptors? These are ionotropic receptors that have significant sequence homology with ionotropic GABA(A) receptors.

Response: As far as we have known, there is no direct interaction or influence of GABA on 5-HT2C receptors. They operate independently, each responding to their respective neurotransmitter. However, it should be noted that neurotransmitter systems in the brain can interact indirectly through complex neural circuits, but these interactions involve broader networks rather than direct receptor-to-receptor interactions between GABA and 5-HT2c receptors. For example, there is evidence that GABA can influence serotonin receptors, including 5-HT receptors such as 5-HT1A and 5-HT2A [37]. Therefore it is possible that GABAlagen, which increase in GABA activated serotonin systems through 5-HT2c receptors and these connections can modulate the activity of serotonergic neurons and thereby influence the release of serotonin.

In addition, GABA may can indirectly affect the activity of 5-HT2C receptors. It is known that increased GABAergic inhibition via GABAA receptors can enhance the activity of 5-HT2C receptors in the hypothalamus, influencing functions such as sleep regulation, appetite suppression or temperature regulation. Therefore, the interaction between the GABAergic system and serotonin receptors like 5-HT2C plays a crucial role in sleep control. These issues are discussed in the revised manuscript.

Regarding study on 5-HT3 receptors, it is known that 5-HT2C receptors have a greater impact on sleep rather than 5-HT3C receptors, therefore we did not conduct experimental studies specifically targeting 5-HT3C receptors.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Major Concerns:

1) Introduction lacks supporting references. Please add appropriate citations to the review of the literature.

2) As mentioned in the introduction, "However, research on the impact of GBL on sleep is limited despite various research," please cite any existing work on the impact of GBL on sleep.

3) The title suggests a "Pentobarbital-Induced Sleep" model; however, there is no mention of such a group in the study design.

4) The methods section needs to be rewritten to clearly delineate the different groups in the study design. For example:

Normal: What does "normal" mean? Did this group receive any drug candidate?

Control: Was any vehicle given to this group?

DZP: What is the rationale for including this group?

5) Given that rats are nocturnal, why were sleep recordings conducted after 8 PM (during the 12-hour dark cycle)?

6) Drugs were administered for 5 consecutive days. Whether a single dosage insufficient to mitigate the effect?

7) The immunohistochemistry methodology is poorly described. The authors should clearly explain how the analysis was conducted. For example, how many brain sections from the rostral to caudal extent were used for counting? What software were used and how the data was normalized. The authors need to show low magnification images with brain landmarks.

8) It is highly unlikely that there is a reduction in orexin cell number, but orexin intensity may vary, which is visible in the figure. Please quantify orexin intensity in the LHA to conclude the results.

9) Given that GABALAGEN shows strong binding affinity to 5-HT, the authors could also show 5-HT neurons in the brain to indicate cell expression patterns to confirm there finding

10) Please discuss the likely mechanism of action for the differential effects of low and high doses of GL_L and GL_H, especially regarding the decrease in orexin levels in the LHA.

Comments on the Quality of English Language

Editing errors are present.

Author Response

1) Introduction lacks supporting references. Please add appropriate citations to the review of the literature.

Response: We added the references in the introduction.

Riemann, D.; Espie, C. A.; Altena, E.; Arnardottir, E. S.; Baglioni, C.; Bassetti, C. L. A.; Bastien, C.; Berzina, N.; Bjorvatn, B.; Dikeos, D.; Dolenc Groselj, L.; Ellis, J. G.; Garcia-Borreguero, D.; Geoffroy, P. A.; Gjerstad, M.; Goncalves, M.; Hertenstein, E.; Hoedlmoser, K.; Hion, T.; Holzinger, B.; Janku, K.; Jansson-Frojmark, M.; Jarnefelt, H.; Jernelov, S.; Jennum, P. J.; Khachatryan, S.; Krone, L.; Kyle, S. D.; Lancee, J.; Leger, D.; Lupusor, A.; Marques, D. R.; Nissen, C.; Palagini, L.; Paunio, T.; Perogamvros, L.; Pevernagie, D.; Schabus, M.; Shochat, T.; Szentkiralyi, A.; Van Someren, E.; van Straten, A.; Wichniak, A.; Verbraecken, J.; Spiegelhalder, K., The European Insomnia Guideline: An update on the diagnosis and treatment of insomnia 2023. J Sleep Res 2023, 32, (6), e14035.3. Bollu, P. C.; Kaur, H., Sleep Medicine: Insomnia and Sleep. Mo Med 2019, 116, (1), 68-75.
Bendaoud, I.; Etindele Sosso, F. A., Socioeconomic Position and Excessive Daytime Sleepiness: A Systematic Review of Social Epidemiological Studies. Clocks Sleep 2022, 4, (2), 240-259.
Jiang, Y.; Jiang, T.; Xu, L. T.; Ding, L., Relationship of depression and sleep quality, diseases and general characteristics. World J Psychiatry 2022, 12, (5), 722-738.
Do, D., Trends in the use of medications with insomnia side effects and the implications for insomnia among US adults. J Sleep Res 2020, 29, (4), e13075.
Joung, H. Y.; Ye, M.; Lee, M.; Hong, Y.; Kim, M.; Kim, K. S.; Shim, I., Sedative-Hypnotic Activity of the Water Extracts of Coptidis Rhizoma in Rodents. Clocks Sleep 2022, 4, (1), 145-159.
Cho, S. M.; Shimizu, M.; Lee, C. J.; Han, D. S.; Jung, C. K.; Jo, J. H.; Kim, Y. M., Hypnotic effects and binding studies for GABA and 5-HT receptors of traditional medicinal plants used in Asia for insomnia. Journal of Ethnopharmacology 2010, 132, (1), 225-232.
Huang, J. H.; Xu, F.; Yang, L. P.; Tuolihong, L.; Wang, X. Y.; Du, Z. B.; Zhang, Y. Q.; Yin, X. L.; Li, Y. J.; Lu, K. R.; Wang, W. S., Involvement of the GABAergic system in PTSD and its therapeutic significance. Frontiers in Molecular Neuroscience 2023, 16.
Boonstra, E.; de Kleijn, R.; Colzato, L. S.; Alkemade, A.; Forstmann, B. U.; Nieuwenhuis, S., Neurotransmitters as food supplements: the effects of GABA on brain and behavior. Front Psychol 2015, 6, 1520.
Johnston-Cox, H. A.; Yang, D.; Ravid, K., Physiological implications of adenosine receptor-mediated platelet aggregation. J Cell Physiol 2011, 226, (1), 46-51.

2) As mentioned in the introduction, "However, research on the impact of GBL on sleep is limited despite various research," please cite any existing work on the impact of GBL on sleep.

Response: Research on GBL has been conducted minimally. So, we changed the sentence in the introduction as “Although, there are no current studies investigating the effect of GBL on sleep, its high GABA and glycine content could lead to a positive impact on sleep efficacy for those with sleep complaints.”

3) The title suggests a "Pentobarbital-Induced Sleep" model; however, there is no mention of such a group in the study design.

Response: We added the study design in the material and method section as “Rats were randomly assigned to five groups: untreated, naïve (Nor, n = 8); pentobarbital injected with vehicle (CON, n = 8); pentobarbital injected along with 100 mg/kg GBL (GBL_L, n = 6); pentobarbital injected along with 250 mg/kg GBL (GBL_H, n = 6) and pentobarbital injected along with 10 mg/kg DZP (DZP, n = 7) in this study.

4) The methods section needs to be rewritten to clearly delineate the different groups in the study design. For example:

Normal: What does "normal" mean? Did this group receive any drug candidate?

Control: Was any vehicle given to this group?

DZP: What is the rationale for including this group?

Response: We stated clearly grouping names in the study design follows as; “Rats were randomly assigned to five groups: untreated, naïve (Nor, n = 8); pentobarbital injected with vehicle (CON, n = 8); pentobarbital injected along with 100 mg/kg GBL (GBL_L, n = 6); pentobarbital injected along with 250 mg/kg GBL (GBL_H, n = 6) and pentobarbital injected along with 10 mg/kg DZP (DZP, n = 7) in this revised one. "Normal" refers to the typical or standard condition. We administered saline to both the 'Normal' and 'Control' groups.

The reason for using DZP as a positive control in sleep experiments is that it is widely recognized as a stable and well-established sleep inducer. DZP is known for its high stability and effectiveness in inducing sleep, making it a suitable standard for comparison with other experimental conditions. We added rationale for using DZP in the material and method section as “Diazepam is widely recognized as a sedative drug and was used as a positive control in this experiment.”

5) Given that rats are nocturnal, why were sleep recordings conducted after 8 PM (during the 12-hour dark cycle)?

Response: Despite being nocturnal animals, rats primarily engage in activities during the night and rest during the day. Therefore, to accurately observe and analyze the natural sleep patterns and activity cycles of rats, EEG recordings were conducted during the night. This approach ensures that the data collected reflect the physiological rhythms of the rats, thereby enhancing the reliability and validity of the research findings.

6) Drugs were administered for 5 consecutive days. Whether a single dosage insufficient to mitigate the effect?

Response: The drugs were administered for 5 consecutive days rather than a single dosage for several reasons. A single dosage may not maintain the desired therapeutic effect due to rapid metabolism or excretion of the drug. Treatment with a single dosage is not enough to produce sleeping promoting effect in this pentobarbital-induced model, and five repeatedly subchronic injections of the drug were chosen in the present study. Administering the drug over 5 consecutive days ensures a sustained blood concentration, enhancing the overall efficacy. If the drug's efficacy is dose-dependent, a single dosage may not deliver an adequate amount. Consecutive dosages ensure a stable and sufficient supply of the drug to achieve the desired effect. Administering a high dose in a single instance can increase the risk of side effects. Dividing the total dosage into 5 administrations can minimize adverse effects while maintaining therapeutic efficacy.

Response: We described the immunohistochemistry methodology in detail in the material and method section as follows. “After transcranial perfusion, the brains were dissected, then post-fixed in 4% formaldehyde overnight, and placed in 30% sucrose solution for 24 h at 4 °C. Samples were cut into 30 μm thickness and the sections were kept at −20 °C. The brain sections were washed in PBS three times for 10 min and then quenched for 10 min at RT 3% H2O2 in PBS. Samples were rinsed in PBS three times for 10 min and blocked for 1 h at RT 0.2% triton X-100 (Sigma) and 1.5% bovine serum albumin (BSA) (Sigma, MA, USA) in PBS. Sections were rinsed in PBS containing 0.5% BSA three times for 10 min. Primary rabbit polyclonal antibodies against orexin (Abcam, Cambridge, MA, USA) were diluted to 1:800. The sections underwent a 12-hour incubation at 4 °C with constant agitation. After rinsing with PBST, a 2-hour incubation at room temperature was per-formed using a biotinylated goat anti-rabbit antibody (Vector Laboratories, Inc., Burlin-game, CA, USA) diluted to 1:200 in PBST with 2% v/v normal goat serum. Subsequently, the sections were exposed to an avidin-biotin-peroxidase complex reagent (Vector Laboratories) for 2 h at room temperature. After further rinsing with PBST, the tissues were developed using a DAB substrate kit (Vector Laboratories). The final steps included mounting the sections on slides, air-drying, and covering them for microscopic observation. The images were captured using a DP2-BSW imaging system (Olympus, CA, USA). Cells testing positive for Orexin were counted on a grid that was placed on LH. The number of cells was counted at a magnification of 100×, using a rectangular microscopic grid measuring 200×200 ㎛².”

Response: We changed the figure 4 and evaluated orexin intensity in the LHA.

9) Given that GABALAGEN shows strong binding affinity to 5-HT, the authors could also show 5-HT neurons in the brain to indicate cell expression patterns to confirm there finding

Response: We tested GBL treatment on 5-HT-positive neurons using immunohistochemistry and provided their corresponding results.

10) Please discuss the likely mechanism of action for the differential effects of low and high doses of GL_L and GL_H, especially regarding the decrease in orexin levels in the LHA.

Response: We added the sentence in the discussion section as “GBL_H may decrease Orexin in the LH in a dose-dependent manner, leading to a reduction in arousal and potentially enhancing the quality of sleep.”

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

“GABALAGEN Facilitates Pentobarbital-Induced Sleep by Modulating the Serotonergic System in Rats”（ cimb-3059418）

This manuscript aimed to elucidate the effects of GBL through receptor binding assays and validate its efficacy in improving sleep by conducting experiments on animal sleep models. The results revealed that GBL displayed binding affinity to the 5-HT2C receptor. Administration of a low dose of GBL (GBL_L; 100 mg/kg) increased non-rapid eye movement sleep time and decreased wake time based on EEG data in pentobarbital-induced rats. Administration of a high dose of GBL (GBL_H; 250 mg/kg) increased NREM sleep time. Additionally, GBL groups significantly increased concentration of 5-HT level in the serum. GBL_H decreased orexin expression in the lateral hypothalamus. All these results suggest that GBL holds promise as a novel compound for inducing sleep in natural products. Overall, this topic is very interesting and the findings hold great practical implications. The usage of multiple methods further strengthens the quality of the current manuscript. However, some concerns appeared after reading the whole manuscript.

1. How did you determine the sample size? Did you calculate the sample size needed before formal study? The current sample size seems too little to get reliable results.

2. Some important articles need to be reviewed and discussed, such as,

Oishi, Y., Saito, Y. C., & Sakurai, T. (2023). GABAergic modulation of sleep-wake states. Pharmacology & Therapeutics, 108505.

Chowdhury, S., Matsubara, T., Miyazaki, T., Ono, D., Fukatsu, N., Abe, M., ... & Yamanaka, A. (2019). GABA neurons in the ventral tegmental area regulate non-rapid eye movement sleep in mice. Elife, 8, e44928.

3. In the discussion part, the most-related empirical study (reference [8]) needs to be discussed with current findings.

4. A limitation part needs to be added at the end of discussion part.

5. Some facts about insomnia need to be updated.

Riemann, D., Espie, C. A., Altena, E., Arnardottir, E. S., Baglioni, C., Bassetti, C. L. A., Bastien, C., Berzina, N., Bjorvatn, B., Dikeos, D., Dolenc Groselj, L., Ellis, J. G., Garcia-Borreguero, D., Geoffroy, P.A., Gjerstad, M., Gonçalves, M., Hertenstein, E., Hoedlmoser, K., Hion, T., … Spiegelhalder, K. (2023). The European Insomnia Guideline: An update on the diagnosis and treatment of insomnia 2023. Journal of Sleep Research, 32(6), e14035. https://doi.org/10.1111/jsr.14035

Comments on the Quality of English Language

Minor editing of English language required

Author Response

Reviewer 3

How did you determine the sample size? Did you calculate the sample size needed before formal study? The current sample size seems too little to get reliable results.

Response: We understand that determining the sample size for an EEG study is very important to ensure reliable and statistically valid results. We have considered determining an appropriate sample size based on our pilot study, which informed our estimates of variability and effect size for the current study. Basically sample size is calculated, on the basis of our research hypothesis and the nature of data, and statistical test using ANOVA and Tukey's post-hoc test, in which very significant results were obtained with sample size.

Some important articles need to be reviewed and discussed, such as,

Response: We added the contents of articles in the discussion section as “Several studies have examined the role of GABA in sleep regulation, focusing on various brain regions [29]. The functions of the GABAergic system in these areas have high-lighted their significant roles in the regulation of the sleep/wake cycle. While VLPO neurons may predominantly promote wakefulness, specific subsets of GABAergic neurons induce sleep [30]. Recent studies suggest that selective excitation of the POAGABA → LH pathway increases wakefulness [31]. Previous research has also implicated VTAGABA neuron terminals in the LH in regulating GABA's role in sleep [29]. Recent advances in understanding the functions of the GABAergic system in brain regions have further clarified their significant roles in sleep/wake regulation. These findings can provide a crucial foundation for understanding the complex neurobiological mechanisms through which GBL affect sleep impairment and sleep disorder.”

Oishi, Y., Saito, Y. C., & Sakurai, T. (2023). GABAergic modulation of sleep-wake states. Pharmacology & Therapeutics, 108505.

In the discussion part, the most-related empirical study (reference [8]) needs to be discussed with current findings.

Response: We added the contents of article in the discussion section as “GABA-rich foods have recently been recognized as effective and safe bioactive sub-stances. High doses of GABA-containing black tea have been shown to reduce anxiety. In a previous study, a fermented rice germ extract containing GABA normalized caffeine-induced sleep disorders. Moreover, in another study, the combination of GABA and L-theanine not only reduced sleep latency but also prolonged sleep duration in a pentobarbital-induced sleep model. Also, in the caffeine-induced arousal model, combined GABA and l-theanine is an attractive NREM sleep-promoting regimen as it increases delta wave oscillations. Therefore, it has been suggested that GABA-enriched foods may have beneficial effects on sleep behaviors.”

A limitation part needs to be added at the end of discussion part.

Response: We added a limitation part in revised manuscript as “This study had some limitations. First, the number of subjects included was too small. However, we believe that the quality of the EEG signal is good enough since we recorded EEG through electrode implantation, and it was not contaminated by motion artifacts. Second, the specific mechanisms underlying GBL's effects on wake/REM/NREM sleep were not explored, necessitating further research. Third, the long-term effects and safety of GBL when used alone have not been thoroughly evaluated. Fourth, the study was conducted on animal models, and therefore, the results may not be directly applicable to humans. To address these limitations, future research should evaluate various dosages and long-term effects, and conduct clinical trials to investigate the effects of GBL on human subjects.”

Some facts about insomnia need to be updated.

Response: We modified about insomnia in the introduction as “In Europe, up to 10% of the adult population experiences sleep disorders [1]. Since insomnia becomes more common as people age, this issue becomes particularly concerning in countries like China with growing older populations [2]. Insomnia is a sleep disorder characterized by difficulty falling asleep or staying asleep [3].”

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I have made comments on the authors reply that they provided with the revised manuscript - see file with comments highlighted in yellow.

Comments for author File: Comments.docx

Reviewer 2 Report

Comments and Suggestions for Authors

All comments are addressed.

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GABALAGEN Facilitates Pentobarbital-Induced Sleep by Modulating the Serotonergic System in Rats

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