Review Reports

Round 1

Reviewer 1 Report

1. The rats were 6 weeks old at the beginning of experiments. Why the authors chose adolescent rats to perform their research? Stress at adolescence could produce somatic, HPA axis, and neuropeptide changes. Could developmental factor play an important role in the final results?    
2. In the Introduction, the authors claimed that the studies are limited due to the scare of animal model of social stress. Crowding stress model is effective to introduce social stressors, which could mimic the pathogenesis of stress-related disorders in human. However, what is the behavioral results of crowding stress? Could crowding stress induce any depressive-like symptoms in rats? 

 

Author Response

Response to Reviewer 1

Because Reviewer no.1 pointed out the that English style/grammar of the manuscript needs corrections, we would like to explain that the last version of the manuscript was edited for English language, grammar, punctuation, spelling, and overall style by our proofreader specializing in scientific English before its submission to Biomolecules. Confirmation of the English correction we attach to letter we send to Editorial Office.

1. “The rats were 6 weeks old at the beginning of experiments. Why the authors chose adolescent rats to perform their research? Stress at adolescence could produce somatic, HPA axis, and neuropeptide changes. Could developmental factor play an important role in the final results?”

There were two reasons we used middle/late adolescent rats. The first reason was based on data showing that social stressors during adolescence increase the risk for stress-related mental disorders in adulthood, and most experimental procedures in animals aiming to understand neural mechanism underlying social stress exposure are applied in adolescence (e.g., Burke AR, Impact of adolescent social experiences on behavior and neural circuits implicated in mental illnesses. Neurosci Biobehav Rev 2017 May;76(Pt B):280-300.). Second, we wanted to relate obtained electrophysiological results to our previous data showing that experimentally induced cortisolemia enhanced basal excitatory transmission in the frontal cortex (M1 part), and antidepressant treatment normalized it, which was performed using the same strain and age of rats (see Bobula et al., Imipramine counteracts corticosterone-induced enhancement of glutamatergic transmission and impairment of long-term potentiation in the rat frontal cortex, Pharmacol Rep, 63 (2011) 1404-1412).
Answering the question about the importance of the developmental factor to our final results, we are aware that adolescence is a dynamic developmental period for neural pathways implicated in stress response (e.g., Jankord R et al., Stress vulnerability during adolescent development in rats. Endocrinology 2011 Feb;152(2):629-38.). Therefore, studying the effects of crowding stress applied in adulthood or early adolescence on glutamate synapse functioning and signal transduction in the frontal cortex could have produced different results than we present in the manuscript (obtained in rats in the developmental stage of middle/late adolescence). From this point of view, the present study results can be considered an important background for future research aimed at understanding the influence of developmental periods on glutamate signaling alterations in the frontal cortex induced by crowding stress. On the other hand, we are not convinced that an animal's developmental state would significantly alter stress effects on glutamate synapse functioning and signal transduction in the frontal cortex. We think so because of several reports describing similar to our results obtained in animal models with stress applied in various developmental stages (e.g. Toth E et al. Age-dependent effects of chronic stress on brain plasticity and depressive behavior. J Neurochem. 2008 Oct;107(2):522-32; Yuen EY et al. Repeated stress causes cognitive impairment by suppressing glutamate receptor expression and function in the prefrontal cortex, Neuron, 73 (2012) 962-977; Chocyk A et al. Early-life stress affects the structural and functional plasticity of the medial prefrontal cortex in adolescent rats, Eur J Neurosci, 38 (2013) 2089-2107; Sowa E et al. Prenatal stress enhances excitatory synaptic transmission and impairs long-term potentiation in the frontal cortex of adult offspring rats, PLoS One, 10 (2015) e0119407). Based on the above analysis, we did not discuss the influence of the development factor on the manuscript results, considering it as potentially confusing for readers.

2. “In the Introduction, the authors claimed that the studies are limited due to the scare of animal model of social stress. Crowding stress model is effective to introduce social stressors, which could mimic the pathogenesis of stress-related disorders in human. However, what is the behavioral results of crowding stress? Could crowding stress induce any depressive-like symptoms in rats?”

Reviewer no.1 is concerned about the lack of behavioral results in the manuscript and the lack of information on whether crowding stress induces any depressive-like symptoms in rats. We did not perform any behavioral test on rats utilized to obtain biochemical and electrophysiological data presented in the manuscript, and therefore we cannot provide additional information about behavioral effects of applied stress procedure.

Although overcrowding is considered an important social stressor for induction of stress-related behaviors (see, e.g., Deslauriers J. et al., Current Status of Animal Models of Posttraumatic Stress Disorder: Behavioral and Biological Phenotypes, and Future Challenges in Improving Translation. Biol Psychiatry. 2018 May 15;83(10):895-907) literature data (as we mentioned in the introduction) showing behavioral and molecular effects of crowding stress exposure are scarce. Among few data showing neurobiological effects of overcrowding, only two papers (to the best of our knowledge) demonstrated behavioral effects of overcrowding applied to mice (Lin E-J D et al. Social overcrowding as a chronic stress model that increases adiposity in mice. Psychoneuroendocrinology. 2015 Jan;51:318-30; Reiss D et al. Effects of social crowding on emotionality and expression of hippocampal nociceptin/orphanin FQ system transcripts in mice. Behav Brain Res. 2007; 184:167–173). In both cases, repeated overcrowding exposure evoked anxiety behavior, and this effect depended on mice strain. We did not find behavioral data showing crowding stress effects in rats. Therefore, we discussed our results to literature data obtained in different social stress models.

Even though we did not present behavioral data in the manuscript, we monitored rats' body weight, measured plasma level of ACTH, CORT, IL-3, performed adrenal morphometry and checked the weight of the thymus and spleen. Overall, we evidenced profound physiological changes evoked by stress and the presence of stress reaction after prolonged exposure to overcrowding. In our opinion, these results allow us to discuss electrophysiological and biochemical data as crowding stress-related changes. Because repeated stress exposure in animal models is considered a model of stress pathology, we concluded that we obtained molecular data related to this pathomechanism. Behavioral data, which we plan to perform in the future, would improve our understanding of whether molecular data are specific to, e.g., depressive-like behavior. At this point, we can only indicate that our electrophysiological results highly suggest that crowding stress evoked changes are implicated in the pathology of depression because similar data obtained in adolescent/young adult animals exposed to stress prenatally or early after birth was accompanied by depressive-like behavior (Sowa J et al. Prenatal stress enhances excitatory synaptic transmission and impairs long-term potentiation in the frontal cortex of adult offspring rats, PLoS One, 10 (2015) e0119407; Chocyk A. et al. Early-life stress affects the structural and functional plasticity of the medial prefrontal cortex in adolescent rats, Eur J Neurosci, 38 (2013) 2089-2107). Moreover, the same effect evoked by chronic corticosterone treatment was reversed by antidepressant treatment (Bobula et al., Imipramine counteracts corticosterone-induced enhancement of glutamatergic transmission and impairment of long-term potentiation in the rat frontal cortex, Pharmacol Rep, 63 (2011) 1404-1412).

Reviewer 2 Report

The manuscript describes interesting molecular changes during physiological exposure of animals to crowding stress. In view of molecular context, it is strongly recommended to underline throughout the text, but especially in the title and abstract, that the animal crowding is meant, as crowding is also employed for protein crowding whose changes may also cause cellular stress. Therefore, better use "animal crowding model" etc.

Please, explain why “After the stress procedure all rats were transferred into separate cages for one hour”. Wouldn’t such an experimental design address the relaxation from crowding rather than the crowding effect per ce?

Why 10 mM glucose and 95% oxygen are used upon the brain slices perfusion? Please, discuss in relation to physiological concentrations in the blood. Were not conditions of hyperoxia and high glucose contributing to the observed changes? Indeed, the latter could manifest the ability of the brain to adapt to the former. Hence, the difference between the physiological and experimental O2 & glucose must be admitted.

The sentence covering the four lines, 380-384, must be edited and divided. Shortening the Discussion will allow to focus reader’s attention on significance of the major findings of this work.

Because in the figures and abstract you show a biphasic response of many parameters at the synapses, the conclusion “changes in synaptic activity evoked by CS are not blunted alongside prolonging exposure to homotypic stress, “ must be similar to that in the abstract, i.e. more specific, than mentioning  just “synaptic activity”.

Author Response

Response to Reviewer 2:

1. “The manuscript describes interesting molecular changes during physiological exposure of animals to crowding stress. In view of molecular context, it is strongly recommended to underline throughout the text, but especially in the title and abstract, that the animal crowding is meant, as crowding is also employed for protein crowding whose changes may also cause cellular stress. Therefore, better use "animal crowding model" etc.”

Thank Reviewer no.2 for the comment about possible confusion in understanding the meaning of “crowding stress.” To avoid confusion with the understanding of crowding stress, we added the “Psychosocial” word at the beginning of the title. Further, in order to improve understanding, in the abstract (row 11) and the introduction (row 36), where we introduced the abbreviation CS for the first time, we added the word “psychosocial”. Additionally, a list of abbreviations was added after the Conclusion section, where we explained CS abbreviation – an animal model of psychosocial crowding stress.

2. “Please, explain why “After the stress procedure all rats were transferred into separate cages for one hour”. Wouldn’t such an experimental design address the relaxation from crowding rather than the crowding effect per ce?”

The reason of rats separation for one hour after crowding stress procedure was to calm down basal hypothalamic-pituitary-adrenocortical activity. In our previous, control experiment we measured the concentration of corticosterone 15, 30, 60 and 120 min after i.c.v. saline injection. Although the i.c.v. saline injection significantly raised the serum corticosterone concentration at 15-30 min, it declined to a basically resting level by 60 min (Bugajski J, Gadek A. Central Hi- and H2-Histaminergic Stimulation of Pituitary-Adrenocortical Response under Stress in Rats(1983) Neuroendocrinology 36:424-430;  Bugajski J, Gadek A. The Effect of Adrenergic and Cholinergic Antagonists on Central Histaminergic Stimulation of Pituitary-Adrenocortical Response under Stress in Rats. Neuroendocrinology (1984) 38: 447-452). Therefore in our study, to study long term effects of crowding stress procedure, after stress application rats were transferred into separate cages for one hour and left undisturbed. After this time animals were rapidly decapitated to obtain presented in manuscript results.

3. “Why 10 mM glucose and 95% oxygen are used upon the brain slices perfusion? Please, discuss in relation to physiological concentrations in the blood. Were not conditions of hyperoxia and high glucose contributing to the observed changes? Indeed, the latter could manifest the ability of the brain to adapt to the former. Hence, the difference between the physiological and experimental O2 & glucose must be admitted.”

Reviewer no.2 is concerned that 10 mM glucose and 95% oxygen were used for the incubation of brain slices and that these factors may produce conditions of hyperoxia and high glucose in the tissue.

10 mM is indeed higher than the physiological concentration of glucose in the cerebrospinal fluid which is estimated to lie between 2.5 and 4.4 mM. However, it should be noted that 10 mM glucose is routinely used the artificial cerebrospinal fluid (ACSF) in many laboratories worldwide in experiments involving extracellular electrophysiological recording from brain slices. The use of 10 mM glucose in the ACSF is based on long-term experience that includes experiments which revealed abnormal electrophysiological activity at lower glucose concentration in the ACSF (e.g. An et al., Effects of glucose and glutamine concentration in the formulation of the artificial cerebrospinal fluid (ACSF). Brain Res. 2008 Jul 7;1218:77-86.). It has been suggested that in the conditions of slice incubation a diffusion of glucose from the ACSF into the several hundred micrometer thick slice is limited as blood vessels in the tissue are non-functional. Therefore, a higher concentration of glucose in the ACSF is necessary to achieve a close to normal concentration of glucose in the tissue during an in vitro brain slice experiment.

The use of a mixture of 95% O2/5% CO2 (carbogen gas) to bubble and saturate the ACSF is also a standard in experiments involving extracellular electrophysiological recording from in vitro brain slices. Again, this apparently higher oxygen supply in the ACSF is necessary to achieve a level of the gas within the slice that enables a close to normal neuronal activity patterns (see e.g. Hajos et al., Maintaining network activity in submerged hippocampal slices: importance of oxygen supply. Eur J Neurosci. 2009 Jan; 29(2): 319–327.)

4. “The sentence covering the four lines, 380-384, must be edited and divided. Shortening the Discussion will allow to focus reader’s attention on significance of the major findings of this work.”

According to Reviewer no.2 suggestion we shortened and corrected lines 380-384. After correction this fragment sounds as follows:

However, in our study CS effects seem to be specific to the membrane AMPA and NMDA receptors containing GluA1 and GluN2B subunits because CS similarly affected the phosphorylation level of these subunits, which takes place only at the plasma membrane [46].

5. “Because in the figures and abstract you show a biphasic response of many parameters at the synapses, the conclusion “changes in synaptic activity evoked by CS are not blunted alongside prolonging exposure to homotypic stress, “ must be similar to that in the abstract, i.e. more specific, than mentioning  just “synaptic activity”.”

In accordance with the Reviewer no.2 recommendation, we have rewritten not precise fragment of the summary. After correction, the sentence reads as follows:

We have shown for the first time that evoked by CS, the enhancement of basal excitatory synaptic transmission and the attenuation of long-term synaptic plasticity are not blunted alongside prolonging exposure to homotypic stress, suggesting that the procedure of psychosocial stress does not enable the habituation of animals to stress conditions.

Reviewer 3 Report

This is an interesting approach in revealing the brain mechanisms of social stress. My comments are related to the formulations of the manuscript.

1. Introduction: sentences in rows 29-34 are unnecessary. The first sentence should be: Data showing effects of social stress... You can also delete the sentences related RS (rows 67-70), because RS will not be mentioned in the rest of the manuscript.
2. Materials: O2, CO2 etc.. subscript must be used! Row 133: (i.e., 21) what is this? The names of the manufacturers (rows 170-176) are not correct and not precise.
3. Results: the Figure displaying body weights is not necessary. Mentioning them in the text is enough. Fig. 4: the symbols of the graphs are not explained (which is black and what is white). Tables 1-3 are not necessary, because the text and Fig. 4 gives the information.
4. Discussion: the Discussion must follow the order of the Results. The discussion of the Western results must be the last - after discussing the electrophysiological results.
5. A list of abbreviations could help the understanding of the text. 

Author Response

Response to Reviewer 3:

1. “Introduction: sentences in rows 29-34 are unnecessary. The first sentence should be: Data showing effects of social stress... You can also delete the sentences related RS (rows 67-70), because RS will not be mentioned in the rest of the manuscript.”

Thank you for indicating information not related directly with presented results. We removed unnecessary sentences according to Reviewer no.3 suggestion.

2. “Materials: O2, CO2 etc.. subscript must be used! Row 133: (i.e., 21) what is this? The names of the manufacturers (rows 170-176) are not correct and not precise.”

Thank you for pointing out not precise methodological description and mistakes in Materials.

Corrected text in the bracket in row 133 is:

“(total volume of 21 sections as every 21st section was stained)”

Corrected text in rows 170-176 is:

“Antibody binding was detected using an enhanced chemiluminescence kit (ECL Plus 32106, Pierce^TM, USA). Equal loading proteins were further confirmed by probing with anti-calnexin antiserum (1:5000, ADI-SPA-865-F, Enzo Life Sciences, USA) or anti-β-actin antiserum (1:5000, A5441, MilliporeSigma, USA). The following antibodies were used in the experiment: p(Y1472) GluN2B (1:1000, M2442, MilliporeSigma, USA), GluN2B (1:1000, 610416, BD Biosciences, USA), mGluR5/1a (1:2000, 2032-mGluR5/1a, PhosphoSolutions, USA), GluA1 (1:2000, ab31232, abcam, Great Britain), VGLUT1 (1:1000, MAB5502, MilliporeSigma, USA) or VGLUT2 (1:1000, D7D2H, Cell Signaling Technology, USA). All western blot analyses were performed minimum twice to confirm the results. Chemiluminescence of specific signals was visualized with the Multi-Application Gel Imaging System, Immunoreactive bands were quantified by an image analyzer (MultiGauge V3.0, Fujifilm, Japan).”

3. “Results: the Figure displaying body weights is not necessary. Mentioning them in the text is enough. Fig. 4: the symbols of the graphs are not explained (which is black and what is white). Tables 1-3 are not necessary, because the text and Fig. 4 gives the information.”

Since body weight results we described in the text and present in Figure 1 have been the typical reaction of the organism to stress, we removed Figure 1. from the manuscript according to Reviewer no.3 suggestion. Similarly, because field potential data are described in Figure 4 and the result section, we removed indicated by Reviewer no.3 Tables 1-3 from the manuscript. After removing Figure 1 from the manuscript, Figures 2-6 were reordered throughout the manuscript.

Symbol description in Figure 4 has been explained. Moreover, we improved the appearance and description of Figures 4-6 (axis names, figure legends, type of symbols for groups has been standardized).

4. “Discussion: the Discussion must follow the order of the Results. The discussion of the Western results must be the last - after discussing the electrophysiological results.”

According to Reviewer no.3 recommendation, we moved two paragraphs with discussion of electrophysiological results (rows 406-447) before discussing the western blot data (rows 359-405) to perform Results discussion in order of their presentation in Figures.

5. “A list of abbreviations could help the understanding of the text.”

A list of abbreviations has been added to the manuscript after Conclusion section.

Round 2

Reviewer 1 Report

The authors fully addressed my concerns.