Review Reports - D1-Like and D2-Like Dopamine Receptors in the Rat Prefrontal Cortex: Impacts of Genetic Generalized Epilepsies and Social Behavioral Deficits

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript adds valuable information of the expression pattern of D1/D2-like receptors in the cortex in the context of epilepsy. The manuscript is well formulated. However, I have some minor points that the authors should address to add a more comprehensive understanding.

1. Is it possible to add analysis of D1/D2 signal in sublayers in the insula and ACC?

2. Can the authors comment on the implications of the lack of correlation between D1DR and D2DR densities?

3. Add in the discussing dopaminergic innervation in epileptic models or patients? is there a difference?, how does that relate ti the differences in receptor expression?

Minor points:

1. Figure 6,7,8 please add color bar for comparison.

2. Figure 7, is it relevant to include the Wistar rats? or would be enough to include only WAG/Rij and WAG/Rij-AGS?, would the results change?

Text:

1. Line 40, Add reference for DA as neuromodulator

2. Line 55, Add reference

3. Figure 8 caption, the authors refer to lateral prefrontal cortex, instead of medial?

4. Line 459, interoception, instead of interception.

Comments on the Quality of English Language

Sufficient

Author Response

The authors are grateful for the Reviewer’s comments, extensive and interesting. We do hope, that the question raised were answered and that the manuscript has been improved.

“The manuscript adds valuable information of the expression pattern of D1/D2-like receptors in the cortex in the context of epilepsy. The manuscript is well formulated. However, I have some minor points that the authors should address to add a more comprehensive understanding.”

-1. Is it possible to add analysis of D1/D2 signal in sublayers in the insula and ACC?

- yes, such a comparison would be a good additional study. Especially for ACC, where the lamination is rather distinct; the sublayers of insular cortex are not clearly separated one from another, forming rather a mild gradiental pattern. We’ll think of analyzing separately “upper layers” and ‘deep layers” in those subgerions. Here, it would run us out of the few weeks, given for the revision.

-2. Can the authors comment on the implications of the lack of correlation between D1DR and D2DR densities?

Previously, we saw strong correlations between local D1DR, D2DR as measured in the striatal territories, where dopamine plays a major role in regional neurochemistry. Here, in prefrontal regions, the correlations are much weaker, probably to a functional mosaic pattern in the cortices, and /or supplementary role of dopamine here.

-we have added the following paragraph in Discussion:

“Surprisingly, the lateral and medial prefrontal regions were poorly inter-correlated (Table 2A,B). Previously, we saw significant inter-correlations of D1DR and D2Dr receptor densities within the territories of the dorsal and ventral striatum of normal Wistar rats, as well as in WAG/Rij-nAGS rats [Tsyba et al, 2023}. The same effect was expected for the prefrontal subregions, if one considers a common source of DA-ergic innervations for prefrontal regions, namely the ventral tegmental area {REF?, REF?}. A possible explanation for this lack of correlation, would be an additive role of DA-ergic neuromodulation within the prefrontal cortex, in its turn influenced by …, and far more complex functional fragmentation of prefrontal regions. Within the striatal complex, DA plays a dominating role {REF?}, so the functional coupling of the subregions are less masked by other neuromodulators/neuromediators.”

-3. Add in the discussing dopaminergic innervation in epileptic models or patients? is there a difference?, how does that relate to the differences in receptor expression?We have added the corresponded paragraph. Actually, the question is rarely addressed, since the majority of studies are related to the regions, directly affected by seizures. Indirect hypothesis, as old ones as “epileptic personalities” from XIX century, let one suggest that there should be also remote, epilepsy-related, and long-term aminergic effects in seizure-free limbic structures as well. There are several papers in animals, which we have cited in the manuscript, as the following:

“Usually, research concerning DR density and epilepsy focuses mainly on brain areas directly involved in seizure generation and attributes densities’ changes to comorbidities [Rocha et al, 2012; Alcantara-Gonzalez et al, 2013].”
“In general, our results are in line with known DR distribution in the prefrontal cortex. The lower DR density in the prefrontal cortex than in basal ganglia can explain a lower responsiveness to DA agonist and antagonist substances in that brain region [Fuster, 2015 – chapter 3].”
“The reduced dopaminergic activity was associated with the tendency to develop aggression in patients with epilepsy treated with levetiracetam [Brodie et al, 2016; Elnazer and Agrawal, 2016]. Therefore, it would be interesting to study the aggressive behavior in epileptic rats with different antiepileptic therapy.
Cognitive impairment, the most common side effect of anticonvulsants, especially with poly-therapy, was associated with the lower D2DR activity in the prefrontal cortex in paediatric patients [Zhu et al, 2017]. Also, low D2DR function in the prefrontal cortex is shown to be related to social anxiety or social phobia whereas abnormally high D2DR function – with the symptoms of attention deficit hyperactivity disorder [Kodama&Watanabe, 2017].”

- Minor points:

- 1. Figure 6,7,8 please add color bar for comparison.

-done

-2. Figure 7, is it relevant to include the Wistar rats? or would be enough to include only WAG/Rij and WAG/Rij-AGS?, would the results change?

Wistar is the mother strain for KM rats, so it is at least reasonable to compare KM and Wistar rats; however, the epilepticity is another factor of KM-Wistar difference. WAG/Rij strain is also a Wistar-derived one, the rats are epileptic, but do not display social deficits. Concerning to Your question: the white bars stand for the pooled group of “socially normotypic” rats. The Supplementary Tables S1 and S2 were added, to give the precise regional values for each rat group, at each level. As one can see, there are lateral regions, where the “autistic” groups strikingly differs from all the three others (f.e., in D1DR values of the dysgranular cortex and pirirform cortex; D2DR in the dysgranular and dorsal agranular cortices, see Tables S1B,S2B). Unfortunatelly, the number of animals used does not let us estimating the AGS*AUT interaction; but this would be an interesting question.

- Text:

- 1. Line 40, Add reference for DA as neuromodulator

done

Line 55, Add reference

done

- 3. Figure 8 caption, the authors refer to lateral prefrontal cortex, instead of medial?

-corrected

- 4. Line 459, interoception, instead of interception.

corrected

Reviewer 2 Report

Comments and Suggestions for Authors

Comments are listed here for the author’s consideration to further improve the quality and overall impact of the manuscript.

1. English edition is required, as there are too many errors in the manuscript.

2. Authors must include references immediately after finishing a sentence, throughout their full manuscript. The lack of references throughout the whole manuscript is concerning.

3. Authors use too many abbreviations in the manuscript. Try to use the minimum necessary.

4. It is recommended to include the following references:

Alcantara-Gonzalez D, Floran B, Escartin E, Rocha L. Changes on D2-like receptor induced Gi protein activation and hippocampal dopamine release in kindled rats. Prog Neuropsychopharmacol Biol Psychiatry. 2013 Jan 10;40:246-51. doi: 10.1016/j.pnpbp.2012.10.007.

Rocha L, Alonso-Vanegas M, Villeda-Hernández J, Mújica M, Cisneros-Franco JM, ET AL. Dopamine abnormalities in the neocortex of patients with temporal lobe epilepsy. Neurobiol Dis. 2012 Jan;45(1):499-507. doi: 10.1016/j.nbd.2011.09.006

5. Section 1 (Introduction) should be shortened. The introduction is long and unconcise.

6. The dopaminergic system include “D1-like” D1 and D5 receptor subtypes and “D2-like”: D2Short (S), D2Long (L), D3 and D4 receptor subtypes. Authors mention through the manuscript the levels of D1-like receptors and D2-like receptors, Does this mean that they determined any dopamine receptor subtype indistinctly by autoradiography? Clarify it in the methods section.

7. Indicate: [3H]SCH 23390, what type of receptor ligand is this? D1 or D5 receptor subtype? or both? [3H]spiperone, what type of receptor ligand is this? D2, D3 or D4 receptor subtype? or all of them? Indicate it in the methods.

8. Line 213-217. The putative effects of AGS were checked by comparing the pooled group of KM and WAG/Rij-AGS rats with the pooled group of AGS-unsusceptible rats (i.e., WS and WAG/Rij).

The effects of AbS’ proneness were estimated by comparing the pooled groups of WAG/Rij and WAG/Rij-AGS rats with the pooled groups of WS and KM rats.

Here, there is a problem with the pooled groups.

For one side, AGS-unsusceptible rats (i.e., WS and WAG/Rij): although both groups did not present audiogenic seizures, one group are control animals and other are animals with absence seizures. Absence seizures produce changes in the levels of dopamine receptors. So, it is not possible to pooled together in one group.

The same occurs with the animals from pooled groups of WS and KM, it is not possible to pooled together in one group. Audiogenic convulsions produce changes in the levels of dopamine receptors.

So, all these comparisons are not reliable. Authors should correct this part of the results.

9. Indicate values of receptor binding (pmol/g) for each group and brain area evaluated with p values.

10. Figure 3 and 4. Axes of the figure are not clearly seen. Correct them. The graphs should be improved in their image resolution. Indicate also the color scale of the image processed with thermal imager. Indicate in the figure caption that abbreviations of the evaluated areas are indicated in table 1.

11. Figure 6 and 8. Indicate color scale of both cuts and include examples of 3 groups with epilepsy for both receptors.

12. Improve tables 2A and 2B.

Comments on the Quality of English Language

English edition is required, as there are too many errors in the manuscript.

Author Response

The authors are grateful for the detailed questions, raised by the Reviewer. We do hope, that we’ve improved the readability and lucidity of the manuscript, following Your points.

English edition is required, as there are too many errors in the manuscript.

- we asked a native speaker to read the manuscript and correct errors

Authors must include references immediately after finishing a sentence, throughout their full manuscript. The lack of references throughout the whole manuscript is concerning.

references were added

Authors use too many abbreviations in the manuscript. Try to use the minimum necessary.

we tried to reduce abbreviations, although the most of them are necessary

It is recommended to include the following references:

thanks a lot, the references were cited in the manuscript.

Section 1 (Introduction) should be shortened. The introduction is long and unconcise.

-we have shortened and clarified the Introduction, and hope it increased the readability of the paper.

The dopaminergic system include “D1-like” D1 and D5 receptor subtypes and “D2-like”: D2Short (S), D2Long (L), D3 and D4 receptor subtypes. Authors mention through the manuscript the levels of D1-like receptors and D2-like receptors, Does this mean that they determined any dopamine receptor subtype indistinctly by autoradiography? Clarify it in the methods section.

- we indeed analyzed only the integral signals for the D1-like and D2-like dopamine receptor families. Although we couldn’t point the precise DA receptor subtypes to be changed in each particular pathology, the resulted general landscape of DA receptors alterations warrants the next studies, to specify the receptor subtypes, and also to look at the source nuclei, supplying the cortical regions with dopamine.

the requested information has been added to the method and discussion.

Indicate: [3H]SCH 23390, what type of receptor ligand is this? D1 or D5 receptor subtype? or both? [3H]spiperone, what type of receptor ligand is this? D2, D3 or D4 receptor subtype? or all of them? Indicate it in the methods.

- the ligands used bind to the whole families of D1-like (D1 and D5 subtypes) and D2-like (D2,D3,D4 subtypes) receptors, thus providing an integral picture. In a future, it will be possible to dissect the effects observed, by local applications of specific ligands. The requested information has been added to the method section.

Line 213-217. The putative effects of AGS were checked by comparing the pooled group of KM and WAG/Rij-AGS rats with the pooled group of AGS-unsusceptible rats (i.e., WS and WAG/Rij).

The effects of AbS’ proneness were estimated by comparing the pooled groups of WAG/Rij and WAG/Rij-AGS rats with the pooled groups of WS and KM rats.

Here, there is a problem with the pooled groups.

The same occurs with the animals from pooled groups of WS and KM, it is not possible to pooled together in one group. Audiogenic convulsions produce changes in the levels of dopamine receptors.

So, all these comparisons are not reliable. Authors should correct this part of the results.

In a way, we can directly address the question of AGS and AbS effects, the study was designed just for this purpose (and general effects of epilepsies, as well as of the “autistic” phenotype, were not foreseen). For this purpose, the regional effect interactions (AGS*AbS) were assessed by ANOVA’s, and post-hoc tests were done if we saw a significant AGS*AbS interaction (or a tendency to it). For each particular region, the pooling was done, if the groups to be pooled didn’t differ significantly in their regional values of D1DR or D2DR receptor bindings. So, the question of between-group difference within the groups to be pooled was directly concerned and checked. As one can see from Figures 3 and 4 (as well as from the added Supplementary Tables S1 and S2), there is a number of regions were the pooling was quite natural. The approach has been used in a number of our published papers (Midzyanovskaya et al, 2016; 2021, 2022, 2023). We believe that such a comparative analysis help us to reduce false-positive results (when between-strain differences obtained do not actually refer to the pathology studied), and increase translatability, since in a clinic we do not have inbreed or homogeneous cohorts.

We have extended the description of statistical method used, to clarify the question.

Indicate values of receptor binding (pmol/g) for each group and brain area evaluated with p values.

this was done by adding Supplementary Tables S1A-F, S2A-F, with local values for each region measured, with p-values and the percentages of changes for the main effects.

Figure 3 and 4. Axes of the figure are not clearly seen. Correct them. The graphs should be improved in their image resolution. Indicate also the color scale of the image processed with thermal imager. Indicate in the figure caption that abbreviations of the evaluated areas are indicated in table 1.

the suggestions were followed.

Figure 6 and 8. Indicate color scale of both cuts and include examples of 3 groups with epilepsy for both receptors.

-done

Improve tables 2A and 2B.

-The caption was done to be more detailed.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript presents a thorough investigation into the role of the prefrontal cortical dopaminergic system in the psychopathology of epilepsies and their associated comorbid conditions, notably autism spectrum disorder (ASD). By utilizing autoradiography, the authors meticulously studied the D1-like (D1DR) and D2-like (D2DR) receptor binding densities in different strains of Wistar rats, effectively portraying the localized changes in the dopaminergic system. These shifts, observed in both the medial and lateral prefrontal regions, potentially signal localized dopaminergic dysfunctions associated with generalized epilepsies and/or ASD.

Below are my comments that I urge the authors to address to improve the quality of the manuscript.

1. In the legend for Figure 1, please clarify that the color scheme mentioned applies to subsequent figures and is not relevant to Figure 1 itself.

2. The results section (section 3) is well organized under the different subsections. However, there could be more detailed descriptions while comparing the different strains of Wiser rats. For example, instead of just saying “AGS-related decrease”, specify the magnitude or percentage of the decrease where possible. This will reduce ambiguity and allow a grasp of the true extent of the observed changes.

3. In continuation to point 2, along with a more detailed description of the comparisons, providing an explanation as to how these findings compare to expected outcomes/previous studies will be helpful.

4. There is a lot of data in figures 3 and 4. However, only some of the subplots are discussed in the result section. Can the authors include an explanation for the other plots in the result section as well?

5. Representation of data in figures 3 and 4 can be improved to make it more reader friendly for the audience. Some of the suggestions include –

i. All text, including axis labels and legends is large enough to be easily read when printed or viewed on a screen.

ii. Include a label for the y-axis (even if the units of the measurement are arbitrary)

iii. Consider using annotations or arrows to point out significant findings or trends within the graphs since there is a lot of information in this figure.

iv. Consider moving some of the plots which are not very significant to the results/discussion session to make the necessary plots more reader friendly.

v. Explain the abbreviations used in the figure in the figure legent (consider providing a succinct summary of the abbreviations used so readers don’t have to jump around)

vi. Remove any elements that don’t add value to the figure.

6. Figure 3 and 4 – provide color map for I, II and III

7. Figure 6 and 8 – provide color maps

8. For data on Table 2A and B, what is a strong correlation? How is this defined? What is the basis for it?

9. Line 57 – development of both – “of” is missing in this sentence.

By implementing these suggestions, the text can offer readers a more in-depth understanding of the data and its implications, making the research more accessible and impactful.

Comments on the Quality of English Language

The English language quality is generally good. The content is comprehensible, and the scientific details are conveyed effectively.

Author Response

The authors are deeply grateful for the positive remarks and detailed comments of the Reviewer. We tried to follow closely the suggestions.

“The manuscript presents a thorough investigation into the role of the prefrontal cortical dopaminergic system in the psychopathology of epilepsies and their associated comorbid conditions, notably autism spectrum disorder (ASD). By utilizing autoradiography, the authors meticulously studied the D1-like (D1DR) and D2-like (D2DR) receptor binding densities in different strains of Wistar rats, effectively portraying the localized changes in the dopaminergic system. These shifts, observed in both the medial and lateral prefrontal regions, potentially signal localized dopaminergic dysfunctions associated with generalized epilepsies and/or ASD.

Below are my comments that I urge the authors to address to improve the quality of the manuscript. “

-1. In the legend for Figure 1, please clarify that the color scheme mentioned applies to subsequent figures and is not relevant to Figure 1 itself.

- done

The results section (section 3) is well organized under the different subsections. However, there could be more detailed descriptions while comparing the different strains of Wiser rats. For example, instead of just saying “AGS-related decrease”, specify the magnitude or percentage of the decrease where possible. This will reduce ambiguity and allow a grasp of the true extent of the observed changes.

- We have added the whole dataset to the Supplementary Tables (S1A-F,S2A-F), with the main effects highlighted in red, with corresponded p-values and the magnitudes of changes.

In continuation to point 2, along with a more detailed description of the comparisons, providing an explanation as to how these findings compare to expected outcomes/previous studies will be helpful.

-the discussion was completed, major changes highlighted in grey.

“As a modulator, DA cannot be ascribed any particular prefrontal function but it plays a critical role in three main areas: movement, cognition (especially memory), and affect. It was postulated that DA facilitates the working memory through D1DR with the inverted-U mode of action: both excessive and insufficient D1DR activation can harm prefrontal cortical functions [Fuster, 2015 – chapter 3].

In general, our results are in line with known DR distribution in the prefrontal cortex. The lower DR density in the prefrontal cortex than in basal ganglia can explain a lower responsiveness to DA agonist and antagonist substances in that brain region [Fuster, 2015 – chapter 3].

Surprisingly, D1DR and D2DR were poorly inter-correlated in normal and epileptic rats (Table 2A,B). Previously, we saw significant inter-correlations of D1DR and D2DR receptor densities within the territories of the dorsal and ventral striatum of normal Wistar rats, as well as in WAG/Rij-nAGS rats [Tsyba et al, 2023]. The effect was broken in AGS-susceptible rats (KM and WAG/Rij-AGS), which was suggestive for additional regulation of DAergic neurotransmission, related to AGS experience [Tsyba et al, 2023]. A similar effect was expected for the prefrontal subregions, if one considers a common source of DA-ergic innervations for the both prefrontal regions, namely the ventral tegmental area [Islam et al, 2021]. Instead, here, the local D1DR and D2DR binding densities represented rather independent datasets, which suggested that levels of regionally available DA would not be the major factor to set the levels of D1DR and D2DR binding. Indeed, a functional significance of other, non-DAergic systems for the regional expression of prefrontal DA receptors is widely discussed in the literature. An intriguing exception is the D2DR binding density in the granular insular cortex of epileptic (but not control) rats, which was positively correlated with the majority of other prefrontal regions (bold numbers in Table 2B). DA neurotransmission in the granular insular cortex is known to reduce various types of pain [rev. in Labrakakis, 2023]. So, the increased coupling between the granular insular cortex and other prefrontal regions might hint that a processing of pain-related stimulus in epileptic brain would affect also other prefrontal functions. Further studies will help to clarify this question. “

There is a lot of data in figures 3 and 4. However, only some of the subplots are discussed in the result section. Can the authors include an explanation for the other plots in the result section as well?

- we have included (and highlighted in grey) several paragraphes discussing regional effects; however the whole pattern seen as a core effect and same tendencies in the neighboring regions, is also informative. We think, that such a pattern suggest some insufficiency of DA supply from the fibers, running from VTA to prefrontal cortex. The corresponded paragraph has been added.

Representation of data in figures 3 and 4 can be improved to make it more reader friendly for the audience. Some of the suggestions include –
All text, including axis labels and legends is large enough to be easily read when printed or viewed on a screen.
Include a label for the y-axis (even if the units of the measurement are arbitrary)

iii. Consider using annotations or arrows to point out significant findings or trends within the graphs since there is a lot of information in this figure.

Consider moving some of the plots which are not very significant to the results/discussion session to make the necessary plots more reader friendly.
Explain the abbreviations used in the figure in the figure legend (consider providing a succinct summary of the abbreviations used so readers don’t have to jump around)
Remove any elements that don’t add value to the figure.

Thanks a lot for the suggestions; we tried to follow at least some of them. The areas of interest have been highlighted by blue shadings to easy the flow of attention; letters were enlarged; units were added, colour bars supplied; abbreviations listed in the enlarged legends. The Supplementary Tables S1-S2 also may help the reader to see and compare the regional values, and catch the main effects.

Figure 3 and 4 – provide color map for I, II and III

- done

Figure 6 and 8 – provide color maps

done

For data on Table 2A and B, what is a strong correlation? How is this defined? What is the basis for it?

significant correlations were determined automatically by the software used (STATISTICA), according to the Spearman method of rank order correlations. The explanation was included in the legend of Table 2

Line 57 – development of both – “of” is missing in this sentence.

-done

By implementing these suggestions, the text can offer readers a more in-depth understanding of the data and its implications, making the research more accessible and impactful.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Upon review of the revisions and your responses to the comments, the Reviewer is pleased to report that the manuscript has significantly improved. The thoroughness of your revisions and the clarity of the authors' responses to the earlier comments are commendable. I especially appreciate the additional discussion points you have incorporated, which enrich the manuscript and strengthen its contribution to the field.

I therefore endorse it for publication. The improvements made have clearly elevated the quality of your work, making it a valuable addition to the scientific literature.

Author Response

- Upon review of the revisions and your responses to the comments, the Reviewer is pleased to report that the manuscript has significantly improved. The thoroughness of your revisions and the clarity of the authors' responses to the earlier comments are commendable. I especially appreciate the additional discussion points you have incorporated, which enrich the manuscript and strengthen its contribution to the field.

I therefore endorse it for publication. The improvements made have clearly elevated the quality of your work, making it a valuable addition to the scientific literature.

- The authors are grateful for the suggestions to improve the manuscript, erased earlier, as well as for the current positive comments.

Reviewer 2 Report

Comments and Suggestions for Authors

I have few comments for the author’s consideration to further improve the quality and overall impact of the manuscript.

1. Title: “D1-like and D2-like dopamine receptors of rat prefrontal cortex: impacts of genetic generalized epilepsies and social behavioral deficits”, It is necessary to change it through " D1-like and D2-like dopamine receptors of rat prefrontal cortex: evaluation in three animal models of genetic epilepsies”.

2. Section 1 (Introduction) should be shortened. In the first version, Introduction was from line 30-120. Right now, Introduction is from line 30 to 111. It was not shortened.

3. For non-specific binding why authors used 10⁻⁵M ketanserin (high-affinity non-selective antagonist of 5-HT2 receptors)? Authors should mention the reason in the methodology section and not just at the end of the discussion.

4. Section 2.5: “The brain structures were identified according to [29,34]. Recognizing the difficulty of transitioning from the old nomenclature to the new one, we decided to use both versions in the text, figures and tables.” Authors should use only one nomenclature, because it is confusing for readers, especially those who are not familiar with the topic. I recommend to use the new nomenclature.

5. Table 1. Indicate which is the old and the new nomenclature.

6. Avoid the following abbreviations throughout the manuscript: Abs=Absence seizures; ASD=autism spectrum disorder; AGS=audiogenic seizures.

Comments on the Quality of English Language

Minor editing of English language required.

Author Response

The authors are deeply thankful for the attentive reading of our manuscript, and also for the suggestions how to improve the paper. Below, there are the point-to-point reply to the questions raised.

-I have few comments for the author’s consideration to further improve the quality and overall impact of the manuscript.

Title: “D1-like and D2-like dopamine receptors of rat prefrontal cortex: impacts of genetic generalized epilepsies and social behavioral deficits”, It is necessary to change it through " D1-like and D2-like dopamine receptors of rat prefrontal cortex: evaluation in three animal models of genetic epilepsies”.

- The authors are thankful for the suggestion, and we’ll use the proposed title for one of the next papers, derived from the same dataset. For this particular manuscript, we prefer to keep the present title. We do think that the result on the changes in prefrontal D1-like and D2-like DA receptors, which corresponds to the social behavioral deficits typical for KM rats, deserves its mention in the title.

-2. Section 1 (Introduction) should be shortened. In the first version, Introduction was from line 30-120. Right now, Introduction is from line 30 to 111. It was not shortened.

- The Introduction has been shortened from 1211 words (the 1^st version) to 744 words(as in the present version).

-3. For non-specific binding why authors used 10⁻⁵M ketanserin (high-affinity non-selective antagonist of 5-HT2 receptors)? Authors should mention the reason in the methodology section and not just at the end of the discussion.

- Ketanserin was used to block the potential binding of [3H]spiperone to the D2 –like receptor, according to { Janowsky et al, Extrastriatal Dopamine D2 Receptors: Distribution, Pharmacological Characterization and Region-Specific Regulation by Clozapine. J. Pharmacol. Exp. Ther. 1992, 261, 1282–1290; Szűcs, et al. Characterization of dopamine D2 receptor binding, expression and signaling in different brain regions of control and schizophrenia-model Wisket rats. 2020, Brain Research, 1748, 147074; Tyler, et al High intensity interval training exercise increases dopamine D2 levels and modulates brain dopamine signaling. 2023, Frontiers in Public Health, 11}. A short mention was added the Method Section.

-4. Section 2.5: “The brain structures were identified according to [29,34]. Recognizing the difficulty of transitioning from the old nomenclature to the new one, we decided to use both versions in the text, figures and tables.” Authors should use only one nomenclature, because it is confusing for readers, especially those who are not familiar with the topic. I recommend to use the new nomenclature.

- We have replaced the mentions of old nomenclature for the new one, throughout the manuscript. The old nomenclature has been left in the tables, for quick references – a lot of basic researchers are used to the old nomenclature, and a huge scientific literature has been written in these terms.

-5. Table 1. Indicate which is the old and the new nomenclature.

- These mentions have been added.

-6. Avoid the following abbreviations throughout the manuscript: Abs=Absence seizures; ASD=autism spectrum disorder; AGS=audiogenic seizures.

- The abbreviations have been replaced throughout the manuscript.

Comments on the Quality of English Language

Minor editing of English language required.
The additional English revision has been done.