Effect of Voluntary Wheel-Running Exercise on the Endocrine and Inflammatory Response to Social Stress: Conditioned Rewarding Effects of Cocaine

Round 1

Reviewer 1 Report

The present study by Ferrer-Perez investigates the modulatory role of physical activity (voluntary wheel running [VWR]) in the endocrine, inflammatory and reinforcing cocaine (conditioned place preference [CPP]) effects produced by intermittent episodic social defeat (SD) stress in mice. The study also explores the reinforcing effects of VWR by using the CPP paradigm. The results show that VWR counteracts the effects of SD on corticosterone increases depending on housing conditions. In contrast, VRW does not reduce the effects of SD increasing IL-6 and BDNF in the striatum and hippocampus of the mice. In fact, confirming previous studies, VWR itself increases the levels of these molecular markers. The results also show that SD increases CPP induced by a low dose of cocaine and that this effect is not significantly changed by VWR. Interestingly, VWR (intermittent) seems to promote the extinction of drug-associated memories after SD, but to worsen it in non-stressed animals. Finally, the study also shows that VWR and cocaine seem to have comparable reinforcing effects by using the CPP paradigm. These results are significant and relevant to better understand how physical activity and social stress interact. The methods and analytical tools utilized are sound. There are some methodological aspects that should be clarified in order to enhance the impact of the study.

1/ Stress protocol. The authors state that the modulatory effects of physical activity on stress are controversial and that this controversy could be due to differences in stress intensity and protocol. In the current study, they used repeated intermittent (episodic) social defeat which can have opposite effects compared to chronic (continuous) social defeat in the context of cocaine sensitization and seeking (Miczek et al., J Neurosci, 2011). Given the expertise of the authors and the literature cited, it will help the Introduction/discussion to be more specific and clarify as far as possible whether this controversy regarding the counteractive effects of exercise (CPP, inflammation, etc) could be related to using episodic or chronic social defeat.

-  Also, in Studies 1 and 2, why there are 3 weeks between the last SD episode and CPP?

 2/ VWR protocol. The authors emphasized the importance of optimizing the designs of physical interventions to provide the most beneficial regime of exercise. In this context, it is critical to clarify as far as possible the VWR method utilized in the study:

- Is there any measure of the time that animals are running or the running distance? Could it be individual differences depending on this?

- The authors should state how long animals are housed in the described conditions. It looks that animals are housed in the corresponding conditions 5 days before the beginning of the SD protocol. Are these housing conditions maintained until the end of the experiment? In this case, animals did more exercise before the CPP experiments compared to the evaluation of biological samples? Figure 2 is particularly confusing at this respect.

- It is not clear the rational of using 1 vs 4 wheels housing since the 1-wheel environment could add more stress (i.e., competition) to the protocol.

 3/ Aims. The aims as described at the end of the Introduction section and at the beginning of the Discussion section do not match. It would help the reader to align the aims in both sections

Author Response

1/ Stress protocol. The authors state that the modulatory effects of physical activity on stress are controversial and that this controversy could be due to differences in stress intensity and protocol. In the current study, they used repeated intermittent (episodic) social defeat which can have opposite effects compared to chronic (continuous) social defeat in the context of cocaine sensitization and seeking (Miczek et al., J Neurosci, 2011). Given the expertise of the authors and the literature cited, it will help the Introduction/discussion to be more specific and clarify as far as possible whether this controversy regarding the counteractive effects of exercise (CPP, inflammation, etc) could be related to using episodic or chronic social defeat.

After reading reviewer's recommendation, we have emphasized in the introduction that our focus is on intermittent episodic experiences of social defeat. In addition, we have briefly pointed out the different consequences on the response to cocaine of chronic versus intermittent stress experiences (lines 79-82 and 86-93).

-  Also, in Studies 1 and 2, why there are 3 weeks between the last SD episode and CPP?

The protocol followed in studies 1 and 2 aimed to evaluate the long-lasting behavioral consequences of intermittent Social Defeat Stress over cocaine response, rather than acute effects. Therefore, there is a delay of three weeks between the last social defeat experience and cocaine exposure. In this regard, previously published studies considered a delay of two weeks enough to evaluate long-term behavioral consequences of social defeat experiences (Okamura et al., 2022).

Okamura, H., Yasugaki, S., Suzuki-Abe, H., Arai, Y., Sakurai, K., Yanagisawa, M., ... & Hayashi, Y. (2022). Long-term effects of repeated social defeat stress on brain activity during social interaction in BALB/c mice. Eneuro, 9(3). 10.1523/ENEURO.0068-22.2022

 2/ VWR protocol. The authors emphasized the importance of optimizing the designs of physical interventions to provide the most beneficial regime of exercise. In this context, it is critical to clarify as far as possible the VWR method utilized in the study:

- Is there any measure of the time that animals are running or the running distance? Could it be individual differences depending on this?

As stated by the reviewer, an important variable that may explain individual differences in the effect of VWR is exercise intensity. As the reviewer suggested, differences in physical activity could have been assessed though a monitoring system that allowed to obtain running data such as distance. Unfortunately, the traditional vertical running wheels that we used inside the home cages lack of any monitory system. This is a limitation of our study, thanks to the reviewer's comment, we have included a sentence in the discussion highlighting this problem (Lines 651-653). However, through observation we were able to confirm that, after a period of habituation to low profile and vertical running wheels, all animals displayed intense running activity and even competed for the new resource.

- The authors should state how long animals are housed in the described conditions. It looks that animals are housed in the corresponding conditions 5 days before the beginning of the SD protocol. Are these housing conditions maintained until the end of the experiment? In this case, animals did more exercise before the CPP experiments compared to the evaluation of biological samples? Figure 2 is particularly confusing at this respect.

We appreciate the reviewer's comment and agree with the fact that the methods writing requires more clarity for a full understanding of the physical activity intervention. To increase the replicability of our study, we have introduced several changes in section 2.3. In addition, Figure 1 and Figure 2 have been modified.

Regarding the questions raised, as reviewer stated, part of the animal’s groups of study 1 were housed with one (1x4) or four (4x4) running wheels from their arrival to our laboratory and until the end of the study. The two remaining groups were housed under standard condition until the end of the study (SED and INT), with the difference between them that animals within the Intermittent exercise group (INT) had three sessions per week (Mondays, Wednesdays and Fridays) of 1 hour of individual access to a cage with a voluntary running wheel. As reviewer spotted, animals within 1x4 and 4x4 groups have 5 days of running wheels access prior to the social defeat protocol, while animals within the INT group had only one session of 1-hour training. Regarding the final number of days of exercise, animals in 1x4 and 4x4 groups had 49 days of continuous access to running wheels before the extinction of conditioned place preference, while animals within the INT condition had 19 sessions of 1 hour of running.

On the other hand, all animals in study 2 were housed throughout the study under standard condition. They underwent a repeated social defeat protocol and a conditioned place preference protocol induced by 10 mg/kg cocaine. It was only after the evaluation of the establishment of conditioned place preference that half of the animals began the intermittent exercise program with three sessions of running per week. Animals within the EXP-INT.post condition had 40 sessions of VWR and animals in RSD-INT.post group had 32 sessions until the CPP extinction and the obtention of biological samples.

- It is not clear the rational of using 1 vs 4 wheels housing since the 1-wheel environment could add more stress (i.e., competition) to the protocol.

As reviewer pointed out, in the manuscript it has been discussed the fact that having only one activity wheel per cage can trigger a competition for the resource that can induce stress. When we made the experimental design, we decided to have a group of mice with continuous access to VWR, which means introduce the running wheels inside the cage. As the access to wheels have been classically used as a way to generate environmental enrichment (e.g. see review Ratuski and Weary, 2022), we designed a group with only 1 wheel per cage and another with 4 wheels per cage, as mice were housed 4 per cage. As previous studies have shown, limited environmental enrichment resources can promote aggressive competition (Howerton et al., 2008; Nevison et al., 1999). In this regard, as soon as the study was initiated, we realized by observation that mice competed for the running wheel in the 1x4 experimental group. We decided to include the results of the 1x4 condition as they are significant an could be useful for other researchers when designing their own studies.

Howerton, C. L., Garner, J. P., & Mench, J. A. (2008). Effects of a running wheel-igloo enrichment on aggression, hierarchy linearity, and stereotypy in group-housed male CD-1 (ICR) mice. Applied Animal Behaviour Science, 115(1-2), 90-103.

Nevison, C. M., Hurst, J. L., & Barnard, C. J. (1999). Strain-specific effects of cage enrichment in male laboratory mice (Mus musculus). Animal Welfare, 8(4), 361-379

Ratuski AS, Weary DM. Environmental Enrichment for Rats and Mice Housed in Laboratories: A Metareview. Animals (Basel). 2022 Feb 9;12(4):414. doi: 10.3390/ani12040414. PMID: 35203123; PMCID: PMC8868396.

 3/ Aims. The aims as described at the end of the Introduction section and at the beginning of the Discussion section do not match. It would help the reader to align the aims in both sections

As reviewer recommended, aims of the introduction section and discussion have been matched for a better understanding of the manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

CPP studies have pointed (this should be painted) a similar picture, socially defeated animals are more sen-                          Line 72

Author Response

Following reviewer recommendation, the sentence has been corrected (line 73).

Reviewer 3 Report

The authors have studied the correlation between physical exercise and its impact on mice’s response to stress and cocaine. The experimental dataset with different classes of mice was well designed. The ANOVA statistics were used to analyze the results across various mice groups. The manuscript is well written with an appropriate discussion of the results. We have the following comments-

1. Line 141-143: On what basis the authors distinguished these animal groups is not clear.

2.     Although other gene markers for signal transduction (AK1, HPCAL1, APP, etc.), central dogma (DEAF1, RPS10, RPS9, EYA1, etc.), synaptic function (CLTB, CART, PDYN, etc.), ion channels, and immune responses that lead to cocaine seeking are also present, only BDNF expression was specifically examined by the authors. Is there a particular justification for choosing BDNF.

3.     IL-6 is already linked to neurological conditions like schizophrenia, multiple sclerosis, anxiety, and stress. Is there a relationship between the aforementioned ailments and social distress, and may exercise help with these conditions as well?

4.     Men and women typically respond to stress in psychologically and biologically distinct ways. Although references 16, 80, and 100 are research conducted on female rats or mice only, but the authors used only male animals for their experiments. How do the authors interpret this prejudice towards females when it comes to corticosterone?

5. Line 152: Please add more explanation on PND 56 to make it clear to readers.
6. Line 218-219: What is the basis of these criteria "33%" and "67%"
7. Line 271: "Determination of corticosterone, IL-6, and BDNF levels....", Needs sentence reformulation. A small subheading can be used for this paragraph.
8. Line 346-347: "Intermittent ex-346 exercise (INT)"... INT should be expanded when it was used first time in the text. Authors should check the same with other abbreviations e.g. PND used in the manuscript.
9. Line 31 -33: In this regard, …… needs to be paraphrased. 
10. Line 36 – 37: Requires more clarification and rephrase the sentence to say “Physical activity has been shown to be valuable in assisting with cessation of tobacco and drug use” 
11. Line 45 – 47: All three references exclusively support the notion for cocaine, and none of them specifically address meth and heroin. Perhaps drop the last two drugs? 
12. Line 58: Explain the word “drug-related learning”. 
13. Line 118: The exact use of 50 OF1 is not very clear here, make sure to elaborate on this. 
14. Line 126: Directive 2010/63/EU, consider adding a reference 
15. Line 168 - 170: “free drug extinction” needs clarification and how many of these sessions? 
16. Line 178 – 183:  

·        Related to the notion of cocaine in the second instance((3mg/kg-cocaine, n=15), it is misleading the reader to assume cocaine was administered within both compartments. Instead, I suggest moving the number to the very beginning – One group ((3mg/kg-cocaine, n = 15). 

·        The inactive wheel needs to be elaborated on somewhere above. It does make sense, however, might confuse the reader. 

17.  Line 204 – 210: The reference style is inconsistent. Please place a numerical reference instead. Check this throughout the manuscript. 

18.  Line 438 – 440: Have you mentioned any results from Kaplan-Meier analysis to support the statement? It will be interesting to see the hazard ratio or any other parameter measurement in this context. 

19.  Line 499 - 500: “Previous hypothesis” - Needs reference. 

20.  Line 512 – 514: “levels were higher in those animals that compete for the use of the exercise wheel” -. How did mice compete for the wheel in single wheel cages, in the presence of the mesh? 

21.  Line 521- 524: “last agonistic encounter” – do you mean violent? 

22.  Line 311: Make sure all p-s are italicized, and a space is maintained following each p in “p-value”. 

23.  It might be much more helpful to refer to a supplement table for statistics values, as per APA style of presenting data: 6-20 statistics => try a table, more than 20 statistics => opt for a graph (https://www.scribbr.com/apa-style/results-section/). 

Comments for author File: Comments.pdf

Author Response

1. Line 141-143: On what basis the authors distinguished these animal groups is not clear.

In the first study, we evaluated four different experimental groups with differential access to activity wheels. One group consisted of a total of 24 animals housed in groups of four with one activity wheel inside their cage (named in the manuscript as 1x4). Another group of animals (n = 20) were housed with four activity wheels per cage (maned as 4x4). The remaining animals, 48 were housed under standard condition (with no activity wheel inside their cages). Half of these animals housed under standard condition (n = 24) were included in a program of intermittent exercise with individual access to running wheels three times per week (named Intermittent Exercise, INT). The other 24 animals housed under standard condition did not increase their physical activity and had no access to running wheels, therefore were considered as sedentary and named as SED.

We are grateful that reviewer spotted that the writing of the 2.3 section should be improved to increase the replicability of the study. As a consequence, we have partially rewritten the section to improve the clarity of the text Lines 145-205.

2. Although other gene markers for signal transduction (AK1, HPCAL1, APP, etc.), central dogma (DEAF1, RPS10, RPS9, EYA1, etc.), synaptic function (CLTB, CART, PDYN, etc.), ion channels, and immune responses that lead to cocaine seeking are also present, only BDNF expression was specifically examined by the authors. Is there a particular justification for choosing BDNF.

In the present manuscript we evaluate the animal response to cocaine administration through a conditioned place preference (CPP) paradigm. This paradigm allows to evaluate the potential of different doses of a drug to establish sustained associative learning process between the interoceptive response to the drug and the environmental cues. The potential of cocaine to establish drug-associated memories (conditioned rewarding properties of cocaine) is dependent on associative learning. In this regard, changes in cognitive performance and memory consolidation could modulate the development of CPP. In this vein, physical activity has been reported to exert pro-cognitive effects and modulate learning that has been linked to changes on BDNF expression. Therefore, we decided that a great target to evaluate if physical activity exerted modulatory effects over CPP establishment was changes in BDNF expression in structures that are key for associative learning such as the hippocampus or the striatum.

Baj, G., D'alessandro, V., Musazzi, L., Mallei, A., Sartori, C. R., Sciancalepore, M., ... & Tongiorgi, E. (2012). Physical exercise and antidepressants enhance BDNF targeting in hippocampal CA3 dendrites: further evidence of a spatial code for BDNF splice variants. Neuropsychopharmacology, 37(7), 1600.

Baruch, D. E., Swain, R. A., & Helmstetter, F. J. (2004). Effects of exercise on Pavlovian fear conditioning. Behavioral neuroscience, 118(5), 1123.

Erickson, K. I., Voss, M. W., Prakash, R. S., Basak, C., Szabo, A., Chaddock, L., ... & Wojcicki, T. R. (2011). Exercise training increases size of hippocampus and improves memory. Proceedings of the National Academy of Sciences, 108(7), 3017-3022.

Vaynman, S., Ying, Z., & Gomez‐Pinilla, F. (2004). Hippocampal BDNF mediates the efficacy of exercise on synaptic plasticity and cognition. European Journal of Neuroscience, 20(10), 2580-2590.

3. IL-6 is already linked to neurological conditions like schizophrenia, multiple sclerosis, anxiety, and stress. Is there a relationship between the aforementioned ailments and social distress, and may exercise help with these conditions as well?

As the reviewer stated, over the past few decades, there has been growing evidence for a link between immune system deregulation and a number of mental illnesses (Chistyakov et al., 2018). For instance, patients diagnosed with major depression have increased levels of circulating pro-inflammatory cytokines such as IL-1ß, IL-6, Interleukin-8 (IL-8), Interleukin-12 (IL- 12), interferon-y and TNF-α (Hodes et al., 2014). Besides mood disorders, it is widely accepted that alterations in inflammatory parameters are linked to the vulnerability to different mental illnesses such as autism, schizophrenia and even substance use disorder, which is the focus of interest in our research group (Fiedorowicz et al., 2015; Ménard et al., 2017). In this regard, it has been found that social stress experiences induce alterations of the immune system, including elevations of IL-6 levels (Ménard et al., 2017), therefore it is likely that these negative experiences worsen prior inflammatory dysregulations. We have previously found using animal models that social stress experiences increased plasmatic and central IL-6 levels of socially defeated mice (Ferrer-Pérez et al., 2018). We have also found that the administration of an anti-inflammatory drug reverted the impact of social stress over drug response, but did not protect against the anxiogenic effects of social stress.

We have a solid trajectory studying social stress consequences over several parameters such as drug response, cognitive performance and anxiety- and depression-like behavior, among others. Therefore, is very difficult to us to give an answer scientifically based on our research to the reviewer question on whether physical exercise may be useful in other conditions such as schizophrenia or multiple sclerosis. As we discussed in the manuscript, physical exercise exerts an anti-inflammatory post-effect that has been proven to be effective to regulate inflammatory patterns in states with elevated chronic inflammation such as in depression. However, as we have replicated in our study, the volume of exercise and the intensity determines whether physical exercise exerts an anti-inflammatory or an inflammatory effect (Balducci, et al., 2010). For instance, we cited a study where it was found that, while moderate exercise reduced inflammatory markers and improved mood, high intensity exercise promoted higher levels of perceived stress and inflammation (Paolucci, et al., 2018). Therefore, our conclusion is that physical exercise may be beneficial for these conditions, but the intensity of exercise has to be taken into consideration in order to obtain a possible anti-inflammatory effect.

Balducci, S., Zanuso, S., Nicolucci, A., Fernando, F., Cavallo, S., Cardelli, P., ... & Fallucca, F. (2010). Anti-inflammatory effect of exercise training in subjects with type 2 diabetes and the metabolic syndrome is dependent on exercise modalities and independent of weight loss. Nutrition, Metabolism and Cardiovascular Diseases, 20(8), 608-617.

Chistyakov, D. V., Astakhova, A. A., & Sergeeva, M. G. (2018). Resolution of inflammation and mood disorders. Experimental and molecular pathology, 105(2), 190-201

Ferrer-Pérez, C., Martinez, T. E., Montagud-Romero, S., Ballestín, R., Reguilón, M. D., Miñarro, J., & Rodríguez-Arias, M. (2018). Indomethacin blocks the increased conditioned rewarding effects of coc

Fiedorowicz, J. G., Prossin, A. R., Johnson, C. P., Christensen, G. E., Magnotta, V. A., & Wemmie, J. A. (2015). Peripheral inflammation during abnormal mood states in bipolar I disorder. Journal of affective disorders, 187, 172-178.

Hodes, G. E., Pfau, M. L., Leboeuf, M., Golden, S. A., Christoffel, D. J., Bregman, D., ... & Russo, S. J. (2014). Individual differences in the peripheral immune system promote resilience versus susceptibility to social stress. Proceedings of the National Academy of Sciences, 111(45), 16136-16141.aine induced by repeated social defeat. PloS one, 13(12), e0209291.

Ménard, C., Pfau, M. L., Hodes, G. E., Kana, V., Wang, V. X., Bouchard, S., ... & Janssen, W. G. (2017b). Social stress induces neurovascular pathology promoting depression. Nature neuroscience, 20(12), 1752.

Paolucci, E. M., Loukov, D., Bowdish, D. M., & Heisz, J. J. (2018). Exercise reduces depression and inflammation but intensity matters. Biological psychology, 133, 79-84.

4. Men and women typically respond to stress in psychologically and biologically distinct ways. Although references 16, 80, and 100 are research conducted on female rats or mice only, but the authors used only male animals for their experiments. How do the authors interpret this prejudice towards females when it comes to corticosterone?

The social defeat (SD) protocol has been developed for male, as it is based in the motivation to obtain the dominance of a territory. Therefore, in general is no suitable for female rodents. However, a number of laboratories, including ours, have developed different procedures to induce social stress in females trying to mimic a social defeat experience. In our model, called vicarious social defeat (VSD), female mice are exposed to social defeat episodes of male mice, therefore female indirectly experience the defeat as they are exposed to non-physical sensory stimulus (visual, olfactory, and chemosensory). For that, aggressive male mice residents are housed in cages containing clear perforated Plexiglass separators, which divide the cage into two separate compartments. For each social defeat session (25-min/day), intruder male mice were placed into the same compartment as the resident, while VSD female mice were placed in the neighboring compartment allowing only a vicarious experience (i.e., visual, olfactory, auditory) of the aggressive encounter.

Females are exposed to four episodes of social defeat vicariously, and following each session, the female mouse stays housed for 24h with the aggressive male resident (separated from the resident with a perforated Plexiglas wall in between of both areas). Females are physically protected from the male encounter but not from visual, olfactory and auditory threats, which are part of the vicarious episode. After 24h, the female is taken back to her home cage until the following encounter.

Our results (actually under review) confirmed that vicariously stressed female experience a significant increase in corticosterone after the SD episodes, and similarly to males show an increased in the conditioned rewarding effects of cocaine and also an increased in ethanol intake. Unfortunately, we have not tested the protective effects of VWR yet in females, as the development and validation of the female model is still in progress. Our recent results using the VSD model in females makes us believe that a similar decrease of corticosterone observed in males would be observed in females.

5. Line 152: Please add more explanation on PND 56 to make it clear to readers.

At the request by the reviewer, explanation of acronyms has been included in the text.

6. Line 218-219: What is the basis of these criteria "33%" and "67%"

There are two main protocols of CPP: biased, and unbiased. In a biased protocol, the drug is always associated with the same compartment (generally that which was not preferred in a pre-conditioning test), while the other compartment is paired with administration of the vehicle. There can be confusion in the interpretation of CPP expression/ reinstatement when using the biased method. For example, anxiolytic or anxiogenic drugs can alter the time spent in a white or black chamber independently of conditioned drug reward.

In an unbiased protocol, there is no clear preference for any compartment before conditioning, and drug and vehicle are administered in randomly assigned compartments. It is necessary that an ANOVA of the time spent during the pre-C test in the compartment associate with saline and in the compartment associate with cocaine (or other drug) did not revealed statistical differences between them. Therefore, we know that values higher or lower than 66 o 33% will produce differences. Besides, this is a measure to avoid that animals with extreme preferences could be incorporate in the study and induce false results. We believe that our protocol is extremely cautious to evaluate only the rewarding effect of the drug.

7. Line 271: "Determination of corticosterone, IL-6, and BDNF levels....", Needs sentence reformulation. A small subheading can be used for this paragraph.

We have reformulated the sentence as reviewer suggested. However, no new subheading was added as the journal Style Guide indicates that “Any headings used in a fourth level may simply appear as a paragraph with no indentation.”

8. Line 346-347: "Intermittent ex-346 exercise (INT)"... INT should be expanded when it was used first time in the text. Authors should check the same with other abbreviations e.g. PND used in the manuscript.

At the request of the reviewer, explanation of acronyms has been included in the text.

9. Line 31 -33: In this regard, …… needs to be paraphrased. 

Following reviewer recommendation, the sentence has been rephrased

10. Line 36 – 37: Requires more clarification and rephrase the sentence to say “Physical activity has been shown to be valuable in assisting with cessation of tobacco and drug use” 

The sentence has been rephrased as “During drug of abuse cessation, physical activity has been useful helping in tobacco treatment by decreasing craving and withdrawal symptoms, therefore reducing the relapse rate [4,5]” Lines 36-38.

11. Line 45 – 47: All three references exclusively support the notion for cocaine, and none of them specifically address meth and heroin. Perhaps drop the last two drugs? 

Missing references regarding heroine and methamphetamine have been included (line 48).

12. Line 58: Explain the word “drug-related learning”.

The word was used to describe associative learning process between the interoceptive response to the drug and the environmental cues. The writing has been changed to “literature points that it may be ineffective or even enhance the formation of contextual drug-associated memories [25].”

13. Line 118: The exact use of 50 OF1 is not very clear here, make sure to elaborate on this. 

The statement has been changed as follows: “In addition to the experimental animals, 50 OF1 adult male mice were single-housed in order to enhance their territorial aggressiveness and were used as aggressors for the social defeat encounters.” (line 124-127).

14. Line 126: Directive 2010/63/EU, consider adding a reference

As reviewer suggested, the directive has been included in the reference section.

15. Line 168 - 170: “free drug extinction” needs clarification and how many of these sessions? 

The complete explanation of the conditioned place preference protocol followed, including extinction sessions, is included in the 2.4.3 subsection.

Specifically, the experimental groups that develop conditioned place preference for drug-paired compartment, undergo two extinction session per week (Tuesdays and Thursdays). These extinction sessions consist of placing the mice in the conditioned place preference apparatus for 15 minutes without the administration of cocaine or saline and analyze the time spent in each compartment. This procedure is repeated until the time spent in the drug-paired compartment by each group is similar (statistically equal) to the one spent prior to drug-conditioning during the Pre-C test.

16. Line 178 – 183:  Related to the notion of cocaine in the second instance((3mg/kg-cocaine, n=15), it is misleading the reader to assume cocaine was administered within both compartments. Instead, I suggest moving the number to the very beginning – One group ((3mg/kg-cocaine, n = 15). The inactive wheel needs to be elaborated on somewhere above. It does make sense, however, might confuse the reader. 

Following reviewer suggestion, the paragraph has been rewritten as follows: “In the third Study (depicted in Figure 3), the rewarding potential of VWR to establish a CPP and to compete against cocaine reinforcement was analyzed. One group of animals (VWR group, n = 21) underwent a CPP protocol in which an active running wheel was paired to one compartment and the opposite compartment was paired with a blocked wheel (inactive) that did not allow running. A second group of animals (3mg/kg-cocaine, n = 15) underwent a traditional CPP protocol with the pairing of a dose of 3 mg/kg cocaine (pictured in orange) with one compartment and an injection of saline (pictured in blue) in the other (3mg/kg-cocaine, n = 15). Finally, in the third group (3mg/kg-cocaine-vs-VWR, n = 17), an active running wheel and a saline injection was paired with one compartment while a dose of 3 mg/kg cocaine and an inactive wheel was paired to the other compartment.”.

17. Line 204 – 210: The reference style is inconsistent. Please place a numerical reference instead. Check this throughout the manuscript. 

Citation has been corrected.

18. Line 438 – 440: Have you mentioned any results from Kaplan-Meier analysis to support the statement? It will be interesting to see the hazard ratio or any other parameter measurement in this context. 

Kaplan-Meier results are included in the body of the 3.5 section. It can be read that: “All groups were subjected to extinction sessions, and the Kaplan–Meier analysis revealed that within animals housed under standard activity condition more time was required to achieve extinction in those with previous social defeat experiences (RSD-SED.post) than in non-stressed animals (EXP-SED.post) (χ2 = 4.035 p = 0.045). Kaplan–Meier analysis also revealed that the increase of physical activity through a regiment of intermittent exercise (INT.post) modulate the extinction of drug-associated memories. Non-stressed animals in the condition of intermittent exercise (EXP-INT.post) required more extinction sessions than sedentary counterparts (EXP-SED.post) (χ2 = 4.373 p = 0.037). However, extinction was achieved earlier in physically-active socially defeated animals (RSD-INT.post) when compared to their sedentary counterparts (RSD-SED.post) (χ2 = 4.307 p = 0.038).

Upon the reviewer request, we have performed a Cox regression and obtained the hazard ratio for cocaine extinction in experiment 2. The HR is different depending on the stress experience of the animals. The results have been added to 3.5 section as follows:

For non-stressed mice, a negative hazard ratio of 0.42 (95% CI 0.16-1.08) was found, as VWR decreased in a 68% the probability that mice extinguish drug-induced place preference. On the other hand, in socially defeated mice, VWR exhibited an opposite effect with a positive hazard ratio of 2.25 (95% CI 0.53-9.046) favoring the probability of extinction in a 125%.

19. Line 499 - 500: “Previous hypothesis” - Needs reference. 

The sentence “Conversely to our previous hypothesis” was referred to the fact that when we designed the study, we expected VWR to exert a protective effect on the establishment of CPP. However, as the reviewer pointed, the writing can be confusing as the reader may understand that we were referring to a previously stated hypothesis, therefore we have rewritten the sentence. (line 534).

20. Line 512 – 514: “levels were higher in those animals that compete for the use of the exercise wheel” -. How did mice compete for the wheel in single wheel cages, in the presence of the mesh? 

In 1x4 and 4x4 groups, animals housing setups include one or four vertical activity wheels placed inside their cages. In these housing conditions mice have continuous access to running wheels. Additionally, this housing includes another standard housing items that allow to fulfill the basic comfort for mice and to fulfill legal requirements of animal welfare such as two different nesting materials and wooden chew sticks.

The wire mesh is only present during social defeat episodes. In these episodes, experimental animals are taken out of their cages (where the running wheels are located) and placed inside the homecage of an aggressive resident male mouse, that has the wire mesh. First, experimental animal is protected by the wire mesh from direct attack for the aggressive mice, but after 10 minutes the wire mesh is removed allowing direct contact of the two mice. Therefore, the wire mesh is only present during social defeat episodes that take place in a different cage (the resident aggressive mouse).

The sentence pointed out by the reviewer makes reference to the fact that in the 1x4 condition, the four mice housed together in their home cage only have one wheel to run. This induces a competition among them to access the wheel and increase the level of stress and consequently the corticosterone levels.

21. Line 521- 524: “last agonistic encounter” – do you mean violent? 

An agonistic encounter in the conditions of our experimental procedure is not violent, as indeed ritualized and lack of intense physical damage. In fact, we have not observed wounds in the experimental animals (those intruders that lost the encounter) that required of veterinary treatment.

The term agonistic encounter or agonistic interaction has been traditionally used to refer to social defeat episodes. This expression has been widely used in the scientific literature, for instance:

Golden, S. A., Covington, H. E., Berton, O., & Russo, S. J. (2011). A standardized protocol for repeated social defeat stress in mice. Nature protocols, 6(8), 1183-1191.

McCann, K. E., & Huhman, K. L. (2012). The effect of escapable versus inescapable social defeat on conditioned defeat and social recognition in Syrian hamsters. Physiology & behavior, 105(2), 493-497.

Murra, D., Hilde, K. L., Fitzpatrick, A., Maras, P. M., Watson, S. J., & Akil, H. (2022). Characterizing the behavioral and neuroendocrine features of susceptibility and resilience to social stress. Neurobiology of stress, 17, 100437.

22. Line 311: Make sure all p-s are italicized, and a space is maintained following each p in “p-value”. 

All p-s have been italicized and spaces between p values have been checked.

23. It might be much more helpful to refer to a supplement table for statistics values, as per APA style of presenting data: 6-20 statistics => try a table, more than 20 statistics => opt for a graph (https://www.scribbr.com/apa-style/results-section/). 

We completely agree with the reviewer that a table with the statistical values is a great resource to present the results of a paper. However, the present research has a very complex experimental design with three different studies and multiple experimental groups. Therefore, we have decided to present the results in a narrative manner with the aim of increasing the understanding of the manuscript.

Author Response File: Author Response.pdf

Reviewer 4 Report

The manuscript entitled, “Effect of voluntary wheel running exercise on the endocrine and inflammatory response to social stress: conditioned rewarding effects of cocaine”, assesses the impact of neuroinflammatory and neuroendocrine changes, promoted by physical exercise and social stress, on mouse sensitivity to cocaine. The manuscript is well written, the figures are well presented, the results are well described, and the data analysis was executed using proper statistical approaches. The reviewers comments are as follows:

1.     Both sexes were included in the experiments. It would be interesting to investigate differences in cocaine sensitivity and social stress response between male and female.  

2.     The authors must discuss potential underlying mechanisms for results showing a decrease in IL-6 and BDNF levels in the hippocampus in the “1h intermittent exercise (INT)” group. 

3.     The authors address the neuroendocrine response by measuring corticosterone levels. I recommend supplementing these results with studies measuring circulating adrenocorticotropic hormone (ACTH). Because it is produced by the pituitary gland and it stimulates corticosterone release from the adrenal cortex, ACTH is often studied with corticosterone to assess neuroendocrine response to stress. 

Author Response

.     Both sexes were included in the experiments. It would be interesting to investigate differences in cocaine sensitivity and social stress response between male and female.  

We did not include both sexes in the study. Only male mice were used. The social defeat protocol used in the present manuscript has been developed and validated only for male rodents. It is based on the instinctive territorial aggression that it is motivated by the obtention of the dominance of a territory or resource. This territorial aggression is a sexually dimorphic behavior that does not manifest in female rodents.

However, a number of laboratories, including ours, have developed different procedures to induce social stress in females that is related to social defeat experiences. In our model, called vicarious social defeat (VSD), female mice are exposed to social defeat episodes of male mice, therefore female indirectly experience the defeat as they are exposed to non-physical sensory stimulus (visual, olfactory, and chemosensory). For that, aggressive male mice residents are housed in cages containing clear perforated Plexiglass separators, which divide the cage into two separate compartments. For each social defeat session (25-min/day), intruder male mice were placed into the same compartment as the resident, while VSD female mice were placed in the neighboring compartment allowing only a vicarious experience (i.e., visual, olfactory, auditory) of the aggressive encounter.

Females are exposed to four episodes of social defeat vicariously, and following each session, the female mouse stays housed for 24h with the aggressive male resident (separated from the resident with a perforated Plexiglas wall in between of both areas). Females are physically protected from the male encounter but not from visual, olfactory and auditory threats, which are part of the vicarious episode. After 24h, the female is taken back to her home cage until the following encounter. The female control group undergoes the same protocol, but without the presence of a male RSD encounter in the cage and without the presence of the resident during the 24 hours of housing.

Our results (actually under review in another journal) confirmed that vicariously stressed female experience a significant increase in corticosterone after the SD episodes, and similarly to males show an increased in the conditioned rewarding effects of cocaine and also an increased in ethanol intake. Unfortunately, we have not tested the protective effects of VWR yet in females. As the development and validation of the female model is still in progress, but we agree with the reviewer that I would be interesting to carry out this research in the future.

2. The authors must discuss potential underlying mechanisms for results showing a decrease in IL-6 and BDNF levels in the hippocampus in the “1h intermittent exercise (INT)” group. 

In our opinion, there is a misunderstanding or mistake in the reviewer’s comment, as the results of our study showed increases but not decreases in BDNF levels in the hippocampus

With respect to the IL-6 levels in the hippocampus, the results of our study showed that the increase of physical activity in the three different modalities analyzed (1x4, 4x4 and INT) promoted an elevation of hippocampal levels of the pro-inflammatory cytokine IL-6 (similar results were found in the striatum). Regarding the possible underlying mechanisms of this increase of IL-6, studies show that during physical effort, there is a peak of release of IL-6 by muscles that rapidly decrease after exercise. This increase is followed by a transient anti-inflammatory state caused by the induction of anti-inflammatory cytokines such as IL-10 and by the inhibition of the synthesis of other pro-inflammatory cytokines. However, it seems that the volume of exercise and the intensity determines whether it exerts an anti-inflammatory or an inflammatory effect. Increases in intensity and volume are translated into increases in IL-6 plasmatic concentrations, which can modify the net result of this response, turning an initial anti-inflammatory effect into an inflammatory effect. In our case, we observed an intense activity of wheel running behavior in our mice. Therefore, it is likely that the enhancement of central and peripheral IL-6 levels registered in animals with access to voluntary running wheels is caused by a pattern of excessive exercise. Therefore, our results did no showed a decrease in hippocampal activity in the condition of INT access to VWR, quite the opposite, an increased.

With respect BDNF, our results also showed an increased in hippocampal BDNF levels, which is congruent with vast previous literature that describes this increase after wheel running (e.g. Baj et al., 2012). However, the exact molecular mechanisms that undelay the increase of BDNF after physical activity are not unraveled (review in Walsh and Tschakovsky, 2018). Which is known, is that the enhancement of BDNF gene and protein expression leads to the activation of signaling pathways that enhance learning processes and memory formation as we found in our study. Although in the INT condition, the observed increased in the hippocampal BDNF levels is lower than that presented in the 4x4 condition, there is a significant increase in those mice exposed to intermittent VWR with respect to those in the sedentary condition.

Baj, G., D'alessandro, V., Musazzi, L., Mallei, A., Sartori, C. R., Sciancalepore, M., ... & Tongiorgi, E. (2012). Physical exercise and antidepressants enhance BDNF targeting in hippocampal CA3 dendrites: further evidence of a spatial code for BDNF splice variants. Neuropsychopharmacology, 37(7), 1600.

Walsh, J. J., & Tschakovsky, M. E. (2018). Exercise and circulating BDNF: mechanisms of release and implications for the design of exercise interventions. Applied Physiology, Nutrition, and Metabolism, 43(11), 1095-1104.

3. The authors address the neuroendocrine response by measuring corticosterone levels. I recommend supplementing these results with studies measuring circulating adrenocorticotropic hormone (ACTH). Because it is produced by the pituitary gland and it stimulates corticosterone release from the adrenal cortex, ACTH is often studied with corticosterone to assess neuroendocrine response to stress. 

We completely agree with the reviewer that a complementary evaluation of the adrenocorticotropic hormone would have been allowed a more complete characterization of the neuroendocrine response to social defeat stress. However, is it impossible for us to carry out this analysis as we have already used all the available biologicals samples to corticosterone analysis. Blood obtention for the hormonal analysis of social defeat stress was carried out using the tail-nick procedure, which allows to obtain only a small amount of blood (50 μl) than then is processed for plasma extraction.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Thanks to authors for addressing the comments and clarifying the doubts. 

Reviewer 4 Report

I agree with the changes made by the authors.