In vivo antidepressant efficacy of 3-substituted thietane-1, 1-dioxide derivative - a preliminary study for novel anti-depression therapy in neurological disorders.

BACKGROUND
Psychosocial stress-induced depressive behavior is linked to etiology of several neurological diseases viz., PTSD, and neurodegenerative disease like Alzheimer's disease (AD). The repeated bouts of social stress defeat can be induced using Resident-Intruder-Paradigm (RIP) and chronic mild social stress (CMSS) animal models to assess the stress-induced depressive behavioral patterns.


OBJECTIVES
The aim of this study to examine the anti-depressive efficacy of 3-methoxythietane-1,1-dioxide (N-14) in RIP models of behavioral alterations.


METHODS
In this study, we have used Sprague-Dawley rats in Resident-Intruder-Paradigm (RIP), where intruders interacted with residents Day 0 to Day +5 for 10 minutes to invoke CMSS in intruders and became defeated/submissive rats due to the depressive-like behavioral alterations in social activity, explorations, grooming, defense, aggressive behavior, and social interaction, freeze, and rearing etc., with residents. Group I is control intact animals, group II received N-14 alone; group III received CMSS, and group IV received cotreatment of N14 with CMSS. N-14 (2 mg/kg) was administered intraperitoneally from Day 0 to Day +5 to intact animals and intruder animals under conditions of CMSS.


RESULTS
Several behavioral tests viz., forced swim test, open field test, and elevated-plus maze test were used to examine the above behavioral dynamic parameters. The dynamic interaction between Residents and Intruders during the study showed substantial alterations in exploratory activity, aggressiveness, and defensive behavior, body weight, and thymus mass in stressed animals. N-14 cotreatment has mitigated sociability, exploratory activity, and aggressiveness and increased social adaptability and defensive behavior. Extensive rise in active forms of defense and submission latency indicate that N-14 has induced antidepressant activity with a psycho-sedative component of action.


CONCLUSION
Serendipitously, we observed the ameliorative capability of N-14 cotreatment to mitigate depressive-behavioral symptoms in intruders.

their usage is constrained by the additional comorbid cognitive decline, and nausea, vomiting, diarrhea, anorexia, weight loss, dizziness, constipation, and dry mouth, anxiety, and insomnia etc., Therefore, the selection of a particular antidepressant must encompass the adverse effects to treat comorbid symptoms such as behavioral alterations in depression [5,23]. However, therapeutic effectiveness of antidepressants varies by individual and may have serious behavioral abnormalities and other sideeffects. Therefore, there is an urgent need for the development of new and more e cacious antidepressants [5]. Unfortunately, a large proportion of promising molecules in preclinical studies have been shown to be ineffective in clinical trials stage. The actual consequences for clinical failure of several antidepressant molecules are associated to the lack of assessment of existing models of depressive-like states [5,26,27].
In addition, we have examined the safety pro le and activity of new promising lead compounds from the 3-substituted thiethan-1,1-dioxides class (Ex., 3-methoxyethane-1,1-dioxide, laboratory code N- 14). First time, we have examined the e cacy of heterocyclic compound N-14 and there is no current literature available. N-14 showed antidepressant effects and low toxicity in the in vivo model [28,29]. Alternatively, the models based on "social stress & resident-intruder paradigm" are predictive and can be validated. These model methods are e ciently bene cial to invoke behavioral and homeostatic behaviors in animal models in a semi-natural environment. In addition, these methods signi cantly bene cial for examining the e cacy of antidepressants to reverse abnormal behaviors associated with depression [27]. We used modi ed RIP that enable the assessment of aggressive and defensive behaviors [30]. The present research is a 'preliminary study' to examine the psychopharmacology of "3-substituted thietane-1, 1dioxide derivative (N-14)" in vivo models of invoking depression through social stress; this could be a 'prospective novel animal model' to determine the effects of stress on adolescent neurobehavioral phenomenon consequently bene cial to develop novel nontoxic therapeutic intervention against comorbid depression using in vivo models of neurodegenerative diseases including AD.

Experimental design
Animals: All animal procedures were carried out in accordance with the International Recommendations of the European convention for the protection of vertebrate animals used for experimental and other scienti c purposes [31]. These procedures were approved by the Ethical Committee for animal care of the Bashkir State Medical University, Ufa, Russian Federation. The experimental subjects were white male Sprague Dawley rats [32]. They were kept in standard vivarium conditions with a 12/12 h light/dark cycle (lights off at 20:00) and provided water and food ad libitum.
Before experiment, all male rats were categorized into "Residents" and "Intruders" [32]. Resident male rats weighing 250-350 g were housed in large individual cages (90*60*50 cm) for more than 2 weeks to facilitate the development of territoriality. Each male was provided a sterilized companion female one week before the test to enhance territoriality and prevent effects of social isolation. Two days before the test, Residents interacted with small non-experimental male rats for 10 minutes twice a day to develop dominance behavior.
The idea of the experiment is to develop depressive-like symptoms in Intruders due to repeated bouts of social defeat incurred by chronic mild social stress (CMSS) during Resident-Intruder interactions and N-14 cotreatment used to reverse these symptoms.
Drug administration LD-50 of N-14 was determined as per Ivanova, O.A., Nikitina, I.L et al (2011) [28]. N-14 was suspended in Tween 80 and dissolved in 0.9% NaCl solution and prepared 2 mg/kg concentration. Rats were treated daily with N-14 or saline intraperitoneally in a volume of 2 ml/kg on days from 0 to +5.

Procedures
Chronic bouts of social defeat were induced in intruders from chronic mild stress (CMSS) when interacting with residents in the cage [32,33]. Intruders interacted with Residents for 10 minutes daily for 6 days (i.e., from Days 0 through +5). Interactions occurred during the dark phase. Interactions between residents and intruders were recorded using a Panasonic V760 camera.
The behavior of the Intruders was analyzed by computing the duration of the patterns and types of behaviors using BrainTest 2.0 program [34]. Trained observers collected data on social behaviors as follows:  three liters of water and maintained them at room temperature for 15 minutes. Subsequently, the procedure was repeated again next day for 5 minutes. Here, immobility is referred as the time spent motionless on the water surface without any submerged limb movement in the water was recorded for every rat using Zeiss (ZSD-808) Stopwatches. Higher immobility was considered as the depressive-behavior, which can be ameliorated by the administration of anti-depressants [37]; Mean ± SEM immobile time during Day 2 (in 5 min) was noted for all the intruders.
Open Field (OF) Open eld activity of intruders experiencing CMSS was recorded using Opto-Varimex-3 Activity Meter (Columbus Instruments, Columbus, OH, USA) coupled with a standard, open, Plexiglas arena. The animal movement was recorded using infrared sensors by placing them 3 cm above the oor as described by Wei S et. al., (2014) [33]. Each rat was placed in the centers of apparatus prior to the testing consequently allowed it to explore for at least 3 minutes. At this time, the open-eld activity was recorded and the other exploratory dynamics viz., rearing etc., were observed with care and quanti ed these parameters [33].

Elevated Plus Maze (EPM) test
EMP is composed of plus-shaped platform situated above 80 cm from the oor. It consists of two open arms (50×10 cm; 100 lux) and two closed arms (50×10×40 cm; 20 lux). Intruder rats were placed across the center square towards the closed arm. The following parameters were recorded for 5 minutes EPM test using Plus-maze version 2.0, [38], Ernst Fricke software; For instance, the parameters are, Additionally, the weight gain of and the amount of food consumed by Intruders (comparing Days -1 to +5) were measured and coded in terms of 100 g of animal weight. At the end of the experiment, the animals were euthanized ethically. To assess the anti-depressant effect of N-14 against stress-induced behavioral alterations, we have analyzed several behavioral patterns like social inactivity, exploration, grooming, defensive behavior, and aggressive behavior. In addition, we have examined the masses of the liver, spleen, thymus, and adrenal glands were measured.

Grooming
Grooming pattern was signi cantly modulated in Groups III and IV. This effect was evident on all experimental days in Group III where maximum values were observed on Days + 1 and + 4, p = 0.08 and p = 0.075 respectively. This total duration of pattern was declined signi cantly with N-14 cotreatment in Group IV on Days + 1, +4, and + 5 when compared to Group III (p < 0.05 on Day + 3).

Social Exploration
N-14 cotreatment also reduced Social Exploration pattern on all days of the experiment when compared to Group III (p < 0.05 on Days 0 and + 3).

Latency of First Interaction
Latency of First Interaction was increased in Groups III and IV when compared to Day 0 (p < 0.05 on Day + 1, Group III); N-14 cotreatment enhanced the pattern value in Group IV on Days + 1 (161%), + 4 (340%) and + 5 (225%) when compared to Group III.

Defense
Defensive behavior was higher in the group of stressed animals cotreated with N-14 (p < 0.05, only on Day + 3) than in Group III due to the Freeze pattern. Freeze was higher on all experimental days (p < 0.05, only on Day + 3) compared to the stressed animals of Group III (the proportion was 87-94%, Figure- In case of Group III stressed animals, there was a tendency to decrease in defensive behavior on all days; which might be due to reduction in the Submission Posture on Days + 1, +2, + 3, +5 (statistically insigni cant) and Freeze on Day + 4.
During the experiment, passive forms of defense viz., Freeze and Submissive Posture prevailed in the behavior structure of animals of both groups. This was caused by individual behavioral alterations of the Intruders rather than speci c response to CMSS [40]. The largest proportion of passive defense in animals treated with N-14 was Freeze (87-94%, Figure-

Latency of Submission
The antidepressant activity of N-14 was con rmed by an increase in the Latency of Submission when compared to Group III (1.5-10 times on Days + 2 to + 5), and in the dynamics relative to Day 0 (1.4-9 times on Days + 1, +3, + 4, +5, p < 0.05), with the behavioral pattern reaching its maximum on Day + 5 (Me = 488.3 s).
The results of Resident-Intruder interaction are shown in Table-1. Additionally, we calculated the proportions of each pattern in the structure of Intruders behavior on Days 0 to + 5. They are given in Fig. 2 2) The asterisk (*) indicates statistical signi cance (p<0.05) for the Mann-Whitney test relative to Group III.
3) The double asterisk (**) indicates statistical signi cance (p<0.05) for the Wilcoxon test relative to Day 0. There was a slight increase in the Open Arms time in Group III by 47% compared to the intact control Group I. There was reduction in Open and Closed Arms entries by 33% and 25%, respectively; concomitantly, reduction in Closed Arms Returns (by 33%) and Head Dipping over the sides of the maze (by 150%) was observed when compared to the control Group I. There parameters were signi cantly altered by the cotreatment of N-14 to CMSS animals. N-14 cotreatment invoked reduction in the Open Arms time in the Group IV-stressed animals, near normal to Group I intact animals. The number of Closed Arms Returns and Closed Arms entries signi cantly increased with N-14 when compared to Group I Intact (by 167% and 25%) and Group III Stressed (by 100% and 67%). In intact animals, N-14 induced signi cant reduction in the center time and closed arms entries with simultaneous increase in the open arms time, open arms entries, and closed arms returns.
Effect of N-14 cotreatment on Weight gain and food consumption CMSS signi cantly induced weight gain of the Intruders (Δ 0−+6 = 28.5 g, p = 0.009) but did not affect the amount of food consumed by Intruders. Moreover, N-14 did not affect the weight of intact animals or their food consumption; these parameters were reduced by 29 g (p = 0.004) and 4.5 g respectively in the Group IV stressed animals. Group IV individual food consumption was lower than the Group III signi cantly on Days + 4 and + 5.

Effect of N-14 cotreatment on the mass of internal organs viz., thymus, and liver
Interaction between Residents and Intruders caused profound increase in the mass of thymus (135%) in Group III stressed animals. A similar trend was observed in the group of animals treated with N-14 (Group II). The administration of N-14 to animals that underwent CMSS (Group IV) induced profound decline in the mass of thymus by 55% compared to Group III, (p = 0.065); similarly, the liver mass was also signi cantly decreased by 13% in Group IV (p = 0.026) compared to Group III.

Discussion
Currently, there are no signi cant molecular therapies against the depression induced in several neurological diseases, neurodegenerative diseases like AD, and trauma conditions. Short-term "modernlife stress", "chronic isolation stress", "chronic mild/variable stress", "chronic mild social stress" are reported to be increase the pathophysiology of AD and comorbid depressive symptoms with cognitive impairment [41][42][43][44][45][46]. The present preliminary study examined the anti-depressive e cacy of 3-substituted thietane-1,1-dioxide using RIP-stress induced models. It was concluded that the adolescent Sprgue-Dawley male rats exposed to chronic mild stress exhibit a depressive phenotype eventually exempli ed by the hippocampal fear-conditioning, and synaptic plasticity [32,47,48]. In order to examine whether the RIP-induced behavioral alterations are accompanied by depression during mild social stress, we have used Sprague-Dawley rat models of daily exposure to mild social stress from day +0 to day +5 [33]. Sheng Wei et al (2014) delineated that the RIP induced aggressive behavior in the intruders [33]. Reverse-Resident-Intruder-Paradigm (rRIP) is reported to be an e cient model to examine the effects of stress on adolescent neurobehavioral phenomenon [32]. The major ndings of our study is that the CMSS has induced 'depressive-like behavior' in male adult intruder rats, which was relieved by the cotreatment of N-14 in the group of animals which underwent chronic mild stress. There was an initial dynamic interaction between residents and intruders during the experiment, which was exempli ed by the increase in the exploratory activity through nonsocial exploration. The social defeat model from chronic social stress summarized the association between behavioral and physiological effects relevant to depression in many neuropsychiatric and neurodegenerative diseases. Fiona Hollis et al (2014) have reviewed the suitability of the social defeat to the understanding of psycho-neurobiology of depression and the potential avenues to develop novel therapies against behavioral alterations during depression in neurodegenerative diseases [49]. Normally, the intruders in the RIP exhibit defensive and aggressive behavior in response to offensive attacks by residents [50]. Hence, this paradigm is signi cant model to ascertain the behavioral aspects like defense in relation to social stress using intruder as the major experimental animal. We have not introduced social stress in the same room where the non-stressed controls were housed because control animals may experience major stress when they witnessing social stress [51,52]. However, the intruder may face dire consequences during defense against residents and experience severe depression, anorexia, loss of body weight due to social defeat from chronic social stress [30]. Our study vividly reported a signi cant decrease in body weight and increase in the weight of thymus of intruders due to the recurrent depressive symptoms due to social defeat from residents. N-14 cotreatment did not affect either weight or amount of food consumption in the intruders but mitigated the mass of thymus.
The forced-swimming test is predominantly suitable to determine the anti-depressant activity of novel lead molecules [53][54][55][56][57]. The reduced immobility time in this test was indicated for anti-depressive e cacy that countered the behavioral expression of depressive-symptoms in rats [58]. Previous reports from Koolhaas J et al (1990), Bielajew C et al (2003) delineated the behavioral alterations from social defeat from chronic mild social stress [58,59]. In our study, CMSS mitigated the social interaction of intruders both in the dynamics compared to Day 0 and in comparison with Group III. This data indicate a decrease in aggressiveness of animals due to the development of an Avoiding Con ict strategy and increasing social adaptability. In Group IV, N-14 cotreatment signi cantly mitigated the level of aggressiveness than in stressed animals of Group III alone. N-14 cotreatment induced the extensive social interaction of Group IV animals throughout the experiment compared with Group III stressed animals, although there was a decrease in this type of behavior in Group III on Days +3 & +5 whereas on Days +4 and +5 in group IV.
In the forced swimming test, CMSS invoked reduction in the DIM (duration of immobilization) FST. N-14 did not affect DIM in FST of stressed animals signi cantly, which may be due to test de ciencies: it has been shown that interventions affecting the locomotor activity of animals led to a change in DIM and, accordingly, to false positive or negative ndings [60]. Therefore, in order to evaluate the antidepressant effect of the compounds with a sedative component, it is advisable to use a depression index i.e. the ratio of the number of short [less than 6 s] immobilization periods to the number of active swimming periods [29].
Social Inactivity was decreased in group IV rats due to extensive decline in the exploratory activity N-14 cotreatment. This nding may be due to the psycho-sedative effect of N-14. The Non-Social Exploration pattern also re ects the level of exploratory activity, which was changed unidirectional with Social Inactivity. N-14 signi cantly alleviated its total duration than stressed animals. This kind of behavioral patterns with N-14 cotreatment vividly delineated its ability to mitigate the development of behavioral despair similar to other anti-depressants [56,61,62]. In our study, the cotreatment of N14 with animals underwent social stress have resulted in the higher motivation and impaired behavioral despair.
Initial activity of the rats located in any new environment (for ex. an open eld) can be considered as an indicator of its emotion and motivation state [63][64][65]. Inescapable open led often trigger stress and reward behavior of novelty consequently induces impaired locomotor and exploratory activity in new environment [66,67]. However, the reduced exploration of a novel environment may be related to higher anxiety levels in stressed animals. We have not examined whether the observed changes in locomotor activity were due to altered anxiety level [33]. Social Inactivity and Non-Social Exploration are indicators of the social passivity of animals along with the Grooming pattern; The grooming pattern was also decreased under the cotreatment of N-14 in stressed animals. N-14 signi cantly reduced the sociability of animals, which was evident by its activity at the patterns of social exploration and latency of rst interaction. This may indicate an increase in the sociability of animals although there was substantial rise of latency of rst interaction.
A report by Sheng Wei et al (2017) described the ability of residents to defend intruders due to their emotional aggressiveness; the administration of uoxetine to the intruder groups mitigated the aggressive behavior and resident-intruder stress [68]. It was well established that the RIP confer aggressive behavior in male rats; which can be considered as the model of depression to evaluate the aggressive behavior upon anti-depressant therapy [33,69]. In our study, N-14 signi cantly increased Defense behaviors throughout the experiment in the both dynamics when compared to Group III. N14 cotreatment enhanced adaptive survival strategy which was evident from the Passive forms of Defense behavior prevailed in Intruders' behavior, where the predominant pattern observed was Freeze behavior (87-94%). In addition, N14 invoked reduction in Move Away and Defensive Upright Posture compared to the Stress Group, which is correlated to the decline in aggressiveness by N-14. There was an increase in the active forms of Defense behaviors: Flight & Latency of Submission when compared to Group III. This is apparently a signi cant evidence for the antidepressant activity of the compound N-14 with psychosedative properties. However, the dynamics (Days 0 to +5) showed a tendency for the Defense behaviors to increase: Day +1 (48.6%) and Day +5 (120.7%). This is due to the reduction in the duration of Freeze on all days of the experiment and Submissive Posture on Days 0, +2, +3 compared to Group III. Previously it was reported hat uoxetine has reduced the defensive behavior of animals eventually inhibiting 'passive form' and stimulating 'active form' of defensive behavior [68,70].
Lipid extract Channa Striatus have proven ameliorative effect against depression induced from Chronic Unpredictable Mild Stress Model in rats [71]. Similarly, another report depicted the e cacy of curcumin to alleviate the depression-induced memory defects by modulating oxidative stress & cholinergic activity using chronic unpredictable mild stress-induced depression models [72]. In our study, both N-14 stimulate active forms of defensive behavior exhibiting antidepressant properties. N-14 cotreatment reduced the aggressiveness of animals. N-14 also enhanced defensive behavior due to the passive form and simultaneously reduced the sociability and exploratory activity of animals, which is the opposite effect to uoxetine [https://cyberleninka.ru/article/n/izuchenie-antidepressivnoy-aktivnosti-i-pro lya-bezopasnostinovyh-proizvodnyh-tietan-1-1-dioksida].
In the OF test, it was shown that N-14 cotreatment signi cantly reduced Exploratory Activity and patterns Moving and Sitting in animals subjected to CMSS compared to Group III. The obtained results have con rmed the reduction in Social Inactivity and Non-Social Exploration, which was evident from the decrease in exploratory and motor activity of animals treated with N-14 as revealed from the analysis of the RIP. In groups exposed to CMSS (Groups III and IV), the level of Exploratory Activity was higher than in the group of intact animals (Group I). The level of Emotional Anxiety was slightly lower. N14 cotreatment invoked sedative properties evident from the decrease in behavioral patterns of Exploratory Activity, Movement on the Spot, and Moving compared to the stressed rats. Therefore, our results are laying pavement for the future studies to address the molecular and neurobiological signaling underlying the anti-depressive behavioral patterns with N14 in rat models and in vivo AD models.
A report by Sergio D. Iñiguez et al (2014) delineated that the social defeat stress in adolescent male c57BL/6 mice confer depression like phenotype [73]. The administration of SSR149415 (a rst selective and orally active non-peptide antagonist of vasopressin V1b receptors) mitigated the physical state of the coat of socially stressed animals during RIP and induced anti-depressant effect [74]. In addition, the ndings concluded that SSR149415 administration normalized grooming during CMSS [74]. In our study, EPM tests described an extensive rise in the Open Arms time, and reduction in the number of Open and Closed Arms entries, as well as an increase in the number of Closed Arms Returns and Head Dippings over the sides of the maze in the Stress group was observed compared to controls (Group I). These are may be due to manifestation of Risk Assessment behavior rather than an indicator of anxiety in animals [36]. N-14 cotreatment signi cantly invoked decline in the Open Arms Time near normal to intact control animals; however, the number of Closed Arms Returns and Closed Arms entries substantially increased with N-14 in Group IV compared to both intact and stressed groups. This may indicate a normalization of the behavioral structure of animals subjected to CMSS. Thus, our reports for the antidepressant activity of N14 are in line with above studies.
The reduction in the thymus mass, body weight was evident in rodent models during CMSS-induced depression behavior [75,76]. CMSS fostered reduction in the weight gain of the Intruders. N-14 cotreatment signi cantly reduced the weight of Group IV animals and their feed consumption. Interaction between Residents and Intruders led to the increase in mass of thymus of stressed animals (Group III). N-

Conclusions
The intake of current anti-depressants has been associated with severe adverse effects in depression patients and AD patients' comorbid with depression. The current study concluded that the dynamic interaction between Residents and Intruders over the course of the experiment showed substantial "increase in exploratory activity (non-social exploration)", "decrease in aggressiveness and defensive behavior", as well as a "decrease in body weight gain" and an "increase in thymus mass" in stressed animals. N-14 cotreatment did not affect the weight gain of the intruders, food consumption. N-14 has proven e cacy to reduce thymus mass in rats subjected to CMSS. N-14 cotreatment has "reduced sociability, exploratory activity and aggressiveness" and "increased social adaptability and defensive behavior of animals" due to the passive forms of defense with concomitant "decrease in exploratory and motor activity. Increased active forms of defense and submission latency indicate that N-14 has antidepressant e cacy with a psycho-sedative component of action; this psychopharmacology study can be further extrapolated for in vivo & clinical studies to delineate the molecular signaling for N-14 e cacy.

Limitations
Our study is a preliminary study, previously, the current paradigm has proved as a well established challenging models to study the effects of stress-induced depression despair for the models of social defeat, which characteristically rely on male aggression not readily displayed by female subjects [77][78][79]. Reports by Holly et al. 2012; Jacobson-Pick et al. 2013 declared that the social defeat from CMSS models elicit aggressive behavioral displays by resident females; this is making observers very di cult to record the symptoms because the social defeat in female rats appears to invoke several symptoms but only after a delay [49,79]. Another signi cant limitation of this study is that we have not determined the in uence of anxiety level on the results. Instead we have signi cantly focused on the predominant differences in the exploratory behaviors between the groups. Our choice of paradigm more broadly focused on to detect the behavioral alterations invoked from the novel anti-depressant molecule as a preliminary step for further future understating of the depression in AD-induced in vivo models, and clinical situation that mimics AD-like pathology and behavioral sign .

Future Studies
We will extend our future studies in the forthcoming research project for the understating of the AD- The experimental subjects were white outbred rats, males and females. They were kept in standard vivarium conditions with a 12/12 h light/dark cycle (lights off at 20:00). They had free access to water and food. Cervical dislocation was used to euthanize the animals.

Consent for publication
Not applicable.

Competing interests
The authors declare no competing nancial and non-nancial interests.