Effects of Head-Only Exposure to 900 MHz GSM Electromagnetic Fields in Rats: Changes in Neuronal Activity as Revealed by c-Fos Imaging without Concomitant Cognitive Impairments

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study is well designed…but some few comments need clarification:

1) Why the Effect of subchronic GSM 900 MHz electromagnetic field exposure on working memory performance was investigated over 10 days while on the reference memory performance was over 14 days

2) Why the authors decided to use only one behaviour test for each test for memory performance (working and reference)

3) The authors need to elaborate/discuss on the mechanism(s) involved in the significant increases in c-Fos expression as a result of the changes in neuronal activation due to exposure to 900MHz GSM EMF

4) The authors need to include a sketch for the apparatus/devise used for the Electromagnetic field exposure system. It can be added to section 2.1. in the methods

5) Figure 3 and Figure 7: the magnification is not mentioned and the scale bar is only represented on 1 sub-figure for each (3B & 7B)…Also, it is recommended to include a high power magnification image that may be situated inset of each sub-figure

6) The paper needs serious updating. The latest reference used is 2011. No references were cited from 2012 to 2024..!!

Author Response

 

We thank Reviewers 1 and 2 for their constructive comments and suggestions which have helped us improving the clarity of our manuscript.

 

Reply to Reviewer 1 :

 

The study is well designed…but some few comments need clarification:

1) Why the Effect of subchronic GSM 900 MHz electromagnetic field exposure on working memory performance was investigated over 10 days while on the reference memory performance was over 14 days.

The different duration of training between the working and reference memory paradigms is due to the differential level of task difficulty of the two paradigms. In the working memory test, 10 days were required for the rats to reach asymptotic performance whereas in the reference memory test, rats needed 14 days to reach a comparable asymptotic level of performance. This is now mentioned in the text of the manuscript on pages 4-5, lines 191-195.

 

2) Why the authors decided to use only one behaviour test for each test for memory performance (working and reference)

During exposure, the rats were restrained in rockets made of transparent Plexiglas designed to fit rats of a maximal weight of 250 g. At the end of the 14 days of exposure (for the reference memory task), some rats were already at the maximal body size that can fit in the rockets which prevented us from expanding behavioural testing any longer. Also, because our primary objective was to pinpoint the GSM-induced changes in activity of brain circuits using c-Fos imaging, we were constrained to select an experimental design in which independent groups of naive rats were submitted to only one single behavioural event instead of a battery of successive cognitive tasks which, otherwise, would have compromised the specificity of our c-Fos imaging approach. This is now mentioned on page 5, lines 195-197.

 

3) The authors need to elaborate/discuss on the mechanism(s) involved in the significant increases in c-Fos expression as a result of the changes in neuronal activation due to exposure to 900MHz GSM EMF.

We agree and thank the reviewer for this relevant comment. Several factors can indeed contribute to increases in c-Fos expression following exposure to 900 MHz GSM EMF. We now allude to some potential mechanisms in a dedicated paragraph on page 17, lines 591 to 612. We also provide corresponding references, including recent ones throughout the manuscript, as judiciously recommended by Reviewer 1 (see point 6 below).

 

4) The authors need to include a sketch for the apparatus/devise used for the Electromagnetic field exposure system. It can be added to section 2.1. in the methods.

We acknowledge this pertinent suggestion. We now include a sketch depicting the apparatus used for the EMF exposure (page 3, figure 1).

 

5) Figure 3 and Figure 7: the magnification is not mentioned and the scale bar is only represented on 1 sub-figure for each (3B & 7B)…Also, it is recommended to include a high power magnification image that may be situated inset of each sub-figure.

We now provide the magnification of the images of figure 3 and 7 (now figures 4 and 8) in the form of scale bars listed in the figure legends. A large view of each brain region of interest was chosen to ensure visualization of a sufficiently high number of Fos-positive nuclei to illustrate between-group differences in c-Fos density without compromising the resolution of individual Fos-positive nuclei which remain clearly visible.

Our immunohistochemical procedure used specific primary and secondary antibodies directed against c-Fos as we previously described (Lesburguères et al., 2011, Science, 331, 924-928) and is based on visible single-staining which does not justify higher magnification as for instance double-staining using immunofluorescence. We therefore feel that higher power magnification images would not provide additional useful information.

6) The paper needs serious updating. The latest reference used is 2011. No references were cited from 2012 to 2024..!!

We sincerely apologize for the lack of recent references. We have now incorporated 16 recent studies (listed below) in the discussion to provide an updated view of this field of research.

 

1. Sienkiewicz, Z. & van Rongen, E. Int J Environ Res Public Health 16, doi:10.3390/ijerph16091607 (2019).
2. McNamee, J. P. et al. Int J Radiat Biol 92, 338-350, doi:10.3109/09553002.2016.1159353 (2016).
3. Occelli et al. Neuroscience. 2018 Aug 10;385:11-24. doi: 10.1016/j.neuroscience.2018.06.002.
4. Lameth, J. et al. Neurotox Res 38, 105-123, doi:10.1007/s12640-020-00191-3 (2020).
5. Yang, H. et al. Bioelectromagnetics 43, 106-118, doi:10.1002/bem.22388 (2022).
6. Bektas, H. et al. J Chem Neuroanat 126, 102168, doi:10.1016/j.jchemneu.2022.102168 (2022).
7. Dasgupta, S. et al. Environ Sci Technol Lett 9, 327-332, doi:10.1021/acs.estlett.2c00037 (2022).
8. Wang, Y. et al. Environ Pollut 283, 117087, doi:10.1016/j.envpol.2021.117087 (2021).
9. Souffi S, et al. Sci Rep. 2022 Mar 8;12(1):4063. doi: 10.1038/s41598-022-07923-9.
10. Lara Aparicio, S.Yet al. NeuroSci 2022, 3, 687–702. https://doi.org/10.3390/neurosci3040050.
11. Lagroye I., et al. Radioprotection, 43 5 (2008) 066. DOI: https://doi.org/10.1051/radiopro:2008741.
12. El Khoueiry C. et al. J Neurophysiol. 2018 Dec 1;120(6):2719-2729. doi: 10.1152/jn.00589.2017.
13. Canovi A. et al. Front Public Health. 2023 Aug 7;11:1231360. doi: 10.3389/fpubh.2023.1231360.
14. Bertagna F. et al. (2021) Ann N Y Acad Sci.;1499(1):82-103. doi: 10.1111/nyas.14597.
15. Narayanan SN, et al. (2019) Environ Sci Pollut Res Int. 2019 Oct;26(30):30693-30710.

 

 

Reply to Reviewer 2:

 

The article evaluating the effects of EMF (cell phone simulation) on neuronal activity/stimulation in rats via cFos expression and behavioral experiments is well written and the conclusion is supported by the data provided. In the methodology section, it has been mentioned to refer previously published articles by the authors for the set-up. It will be useful if a simple image to show the setup is included in the current article. Previous articles show the images but including a summary of experiments and the setup in a diagram or picture form will be useful to understand. The change in cFos levels has been evaluated by immunocytochemistry, inclusion of the protein expression using Wester Blots will be supportive to the data.

 

1. It will be useful if a simple image to show the setup is included in the current article. Previous articles show the images but including a summary of experiments and the setup in a diagram or picture form will be useful to understand.

We acknowledge this pertinent suggestion. We now include a diagram depicting the apparatus used for the EMF exposure (page 3, figure 1).

 

2. Only male rats were used in this study, why female rats were not included in this study.

Our study was restricted to male rats due to the impossibility of housing separately male and female rats during GSM 900 MHz exposure and behavioral testing. Also, all of our behavioural tasks were validated using male rats and we have no experience with females. Whether female rats could be more sensitive to GSM 900 MHz exposure would deserve further investigation, although data from animal and human studies have not pinpointed an apparent gender effect. This limitation of our study is now acknowledged in the discussion of the manuscript, on page 19, lines 713-717.

 

 

3. There are various limitations, and some have been mentioned in the conclusion section. Investigating the effect on EMF on neuronal activity, various channels, and the suitability of using cFos in systemic blood as a biomarker of transcriptional activity will be of translational aspects and this has not been discussed.

The reviewer raised the interesting possibility that EMF exposure could trigger transcriptional activity in multiple cell types, with for some of them, could be examined in in blood cells. Recent advances have provided evidence that c-Fos expression is not limited to neurons and can be expressed in other cell types, such as glial cells. We now mention this possibility in the discussion (page 17, line 592-613). However, we have not performed double-staining in our study and cannot determine whether c-Fos is differentially expressed as a function of the cell phenotype, including endothelial cells. Further investigation is needed to unravel in which cell types Fos expression occurs following EMF exposure. A dedicated transcriptomic analysis using single-cell RNA sequencing from blood cells would be needed and is beyond the scope of the objectives of the present manuscript. This is now acknowledged on page 19 lines 706-713.

 

 

4. EMF also affects the immune response of the brain and the immune response in rats is different than of humans, please discuss how the findings may be translated.

We agree with the Reviewer that EMF exposure can also affect the immune response of the brain and that this immune response can differ between rats and humans. This possibility is now discussed on page 17, lines 585-589.

 

5. Further, increase in cFos may be due to neuronal activation or due to damage, inclusion of hematoxylin and eosin images will support that the change in cFos is due to neuronal activity and not due to damage.

We cannot exclude that c-Fos increases might also result from cellular damage as a contributing factor. We, however, think that such a possibility is unlikely. In previous studies (Watilliaux et al 2011; Lameth et al 2017, Occelli et al 2018, Souffi et al 2022) performed both in developing and adult rats, we did not observe tissue damage and no microglial or astroglial activation after a 2 h period of exposure to GSM in healthy animals [29, 66, 71, 72]. We have included a sentence to mention this lack of tissue damage and glial reaction in the discussion on page 17, lines 623-626. Note also that in experiment 1, it is not the highest SARs which led to increases in c-Fos activation, further making unlikely that tissue damage acts as a main contributing factor to the observed changes in c-Fos expression.

 

6. There may be a multitude of effects on various systems, this article investigated only cFos, which organ system the authors wanted to focus is not clear.

The reviewer is correct in the sense that we focused here on c-Fos expression but we agree that many other effects can also be detected, specifically at the level of gene expression. We now cite 5 recent studies (references 62-66), which have reported changes in gene expression after EMF exposures (page 17, lines 583-584).

 

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The article evaluating the effects of EMF (cell phone simulation) on neuronal activity/stimulation in rats via cFos expression and behavioral experiments is well written and the conclusion is supported by the data provided. In the methodology section, it has been mentioned to refer previously published articles by the authors for the set-up. It will be useful if a simple image to show the setup is included in the current article. Previous articles show the images but including a summary of experiments and the setup in a diagram or picture form will be useful to understand. The change in cFos levels has been evaluated by immunocytochemistry, inclusion of the protein expression using Wester Blots will be supportive to the data. 

Only male rats were used in this study, why female rats were not included in this study.

There are various limitations, and some have been mentioned in the conclusion section. Investigating the effect on EMF on neuronal activity, various channels, and the suitability of using cFos in systemic blood as a biomarker of transcriptional activity will be of translational aspects and this has not been discussed. 

EMF also affects the immune response of the brain and the immune response in rats is different than of humans, please discuss how the findings may be translated.

Further, increase in cFos may be due to neuronal activation or due to damage, inclusion of hematoxylin and eosin images will support that the change in cFos is due to neuronal activity and not due to damage.

There may be a multitude of effects on various systems, this article investigated only cFos, which organ system the authors wanted to focus is not clear.

Author Response

 

We thank Reviewers 1 and 2 for their constructive comments and suggestions which have helped us improving the clarity of our manuscript.

 

Reply to Reviewer 1 :

 

The study is well designed…but some few comments need clarification:

1) Why the Effect of subchronic GSM 900 MHz electromagnetic field exposure on working memory performance was investigated over 10 days while on the reference memory performance was over 14 days.

The different duration of training between the working and reference memory paradigms is due to the differential level of task difficulty of the two paradigms. In the working memory test, 10 days were required for the rats to reach asymptotic performance whereas in the reference memory test, rats needed 14 days to reach a comparable asymptotic level of performance. This is now mentioned in the text of the manuscript on pages 4-5, lines 191-195.

 

2) Why the authors decided to use only one behaviour test for each test for memory performance (working and reference)

During exposure, the rats were restrained in rockets made of transparent Plexiglas designed to fit rats of a maximal weight of 250 g. At the end of the 14 days of exposure (for the reference memory task), some rats were already at the maximal body size that can fit in the rockets which prevented us from expanding behavioural testing any longer. Also, because our primary objective was to pinpoint the GSM-induced changes in activity of brain circuits using c-Fos imaging, we were constrained to select an experimental design in which independent groups of naive rats were submitted to only one single behavioural event instead of a battery of successive cognitive tasks which, otherwise, would have compromised the specificity of our c-Fos imaging approach. This is now mentioned on page 5, lines 195-197.

 

3) The authors need to elaborate/discuss on the mechanism(s) involved in the significant increases in c-Fos expression as a result of the changes in neuronal activation due to exposure to 900MHz GSM EMF.

We agree and thank the reviewer for this relevant comment. Several factors can indeed contribute to increases in c-Fos expression following exposure to 900 MHz GSM EMF. We now allude to some potential mechanisms in a dedicated paragraph on page 17, lines 591 to 612. We also provide corresponding references, including recent ones throughout the manuscript, as judiciously recommended by Reviewer 1 (see point 6 below).

 

4) The authors need to include a sketch for the apparatus/devise used for the Electromagnetic field exposure system. It can be added to section 2.1. in the methods.

We acknowledge this pertinent suggestion. We now include a sketch depicting the apparatus used for the EMF exposure (page 3, figure 1).

 

5) Figure 3 and Figure 7: the magnification is not mentioned and the scale bar is only represented on 1 sub-figure for each (3B & 7B)…Also, it is recommended to include a high power magnification image that may be situated inset of each sub-figure.

We now provide the magnification of the images of figure 3 and 7 (now figures 4 and 8) in the form of scale bars listed in the figure legends. A large view of each brain region of interest was chosen to ensure visualization of a sufficiently high number of Fos-positive nuclei to illustrate between-group differences in c-Fos density without compromising the resolution of individual Fos-positive nuclei which remain clearly visible.

Our immunohistochemical procedure used specific primary and secondary antibodies directed against c-Fos as we previously described (Lesburguères et al., 2011, Science, 331, 924-928) and is based on visible single-staining which does not justify higher magnification as for instance double-staining using immunofluorescence. We therefore feel that higher power magnification images would not provide additional useful information.

6) The paper needs serious updating. The latest reference used is 2011. No references were cited from 2012 to 2024..!!

We sincerely apologize for the lack of recent references. We have now incorporated 16 recent studies (listed below) in the discussion to provide an updated view of this field of research.

 

1. Sienkiewicz, Z. & van Rongen, E. Int J Environ Res Public Health 16, doi:10.3390/ijerph16091607 (2019).
2. McNamee, J. P. et al. Int J Radiat Biol 92, 338-350, doi:10.3109/09553002.2016.1159353 (2016).
3. Occelli et al. Neuroscience. 2018 Aug 10;385:11-24. doi: 10.1016/j.neuroscience.2018.06.002.
4. Lameth, J. et al. Neurotox Res 38, 105-123, doi:10.1007/s12640-020-00191-3 (2020).
5. Yang, H. et al. Bioelectromagnetics 43, 106-118, doi:10.1002/bem.22388 (2022).
6. Bektas, H. et al. J Chem Neuroanat 126, 102168, doi:10.1016/j.jchemneu.2022.102168 (2022).
7. Dasgupta, S. et al. Environ Sci Technol Lett 9, 327-332, doi:10.1021/acs.estlett.2c00037 (2022).
8. Wang, Y. et al. Environ Pollut 283, 117087, doi:10.1016/j.envpol.2021.117087 (2021).
9. Souffi S, et al. Sci Rep. 2022 Mar 8;12(1):4063. doi: 10.1038/s41598-022-07923-9.
10. Lara Aparicio, S.Yet al. NeuroSci 2022, 3, 687–702. https://doi.org/10.3390/neurosci3040050.
11. Lagroye I., et al. Radioprotection, 43 5 (2008) 066. DOI: https://doi.org/10.1051/radiopro:2008741.
12. El Khoueiry C. et al. J Neurophysiol. 2018 Dec 1;120(6):2719-2729. doi: 10.1152/jn.00589.2017.
13. Canovi A. et al. Front Public Health. 2023 Aug 7;11:1231360. doi: 10.3389/fpubh.2023.1231360.
14. Bertagna F. et al. (2021) Ann N Y Acad Sci.;1499(1):82-103. doi: 10.1111/nyas.14597.
15. Narayanan SN, et al. (2019) Environ Sci Pollut Res Int. 2019 Oct;26(30):30693-30710.

 

 

Reply to Reviewer 2:

 

The article evaluating the effects of EMF (cell phone simulation) on neuronal activity/stimulation in rats via cFos expression and behavioral experiments is well written and the conclusion is supported by the data provided. In the methodology section, it has been mentioned to refer previously published articles by the authors for the set-up. It will be useful if a simple image to show the setup is included in the current article. Previous articles show the images but including a summary of experiments and the setup in a diagram or picture form will be useful to understand. The change in cFos levels has been evaluated by immunocytochemistry, inclusion of the protein expression using Wester Blots will be supportive to the data.

 

1. It will be useful if a simple image to show the setup is included in the current article. Previous articles show the images but including a summary of experiments and the setup in a diagram or picture form will be useful to understand.

We acknowledge this pertinent suggestion. We now include a diagram depicting the apparatus used for the EMF exposure (page 3, figure 1).

 

2. Only male rats were used in this study, why female rats were not included in this study.

Our study was restricted to male rats due to the impossibility of housing separately male and female rats during GSM 900 MHz exposure and behavioral testing. Also, all of our behavioural tasks were validated using male rats and we have no experience with females. Whether female rats could be more sensitive to GSM 900 MHz exposure would deserve further investigation, although data from animal and human studies have not pinpointed an apparent gender effect. This limitation of our study is now acknowledged in the discussion of the manuscript, on page 19, lines 713-717.

 

 

3. There are various limitations, and some have been mentioned in the conclusion section. Investigating the effect on EMF on neuronal activity, various channels, and the suitability of using cFos in systemic blood as a biomarker of transcriptional activity will be of translational aspects and this has not been discussed.

The reviewer raised the interesting possibility that EMF exposure could trigger transcriptional activity in multiple cell types, with for some of them, could be examined in in blood cells. Recent advances have provided evidence that c-Fos expression is not limited to neurons and can be expressed in other cell types, such as glial cells. We now mention this possibility in the discussion (page 17, line 592-613). However, we have not performed double-staining in our study and cannot determine whether c-Fos is differentially expressed as a function of the cell phenotype, including endothelial cells. Further investigation is needed to unravel in which cell types Fos expression occurs following EMF exposure. A dedicated transcriptomic analysis using single-cell RNA sequencing from blood cells would be needed and is beyond the scope of the objectives of the present manuscript. This is now acknowledged on page 19 lines 706-713.

 

 

4. EMF also affects the immune response of the brain and the immune response in rats is different than of humans, please discuss how the findings may be translated.

We agree with the Reviewer that EMF exposure can also affect the immune response of the brain and that this immune response can differ between rats and humans. This possibility is now discussed on page 17, lines 585-589.

 

5. Further, increase in cFos may be due to neuronal activation or due to damage, inclusion of hematoxylin and eosin images will support that the change in cFos is due to neuronal activity and not due to damage.

We cannot exclude that c-Fos increases might also result from cellular damage as a contributing factor. We, however, think that such a possibility is unlikely. In previous studies (Watilliaux et al 2011; Lameth et al 2017, Occelli et al 2018, Souffi et al 2022) performed both in developing and adult rats, we did not observe tissue damage and no microglial or astroglial activation after a 2 h period of exposure to GSM in healthy animals [29, 66, 71, 72]. We have included a sentence to mention this lack of tissue damage and glial reaction in the discussion on page 17, lines 623-626. Note also that in experiment 1, it is not the highest SARs which led to increases in c-Fos activation, further making unlikely that tissue damage acts as a main contributing factor to the observed changes in c-Fos expression.

 

6. There may be a multitude of effects on various systems, this article investigated only cFos, which organ system the authors wanted to focus is not clear.

The reviewer is correct in the sense that we focused here on c-Fos expression but we agree that many other effects can also be detected, specifically at the level of gene expression. We now cite 5 recent studies (references 62-66), which have reported changes in gene expression after EMF exposures (page 17, lines 583-584).

 

 

 

Author Response File: Author Response.pdf