Influence of social isolation and aggressive behavior in the appearance of Depression-like in Experimental model

The study of animal behavior in the laboratory environment aims to promote welfare and minimize the discomfort of animals during scientifi c tests. This knowledge is important to understand social interactions and develop methods of environmental enrichment, thus increasing the quality of life of animals housed in the laboratory. The goal this study was evaluated the individual and social behavior and infl uence in the anxiety or depression-like condition in mouse during aggressive behavior and compared with social isolation in the animal facilities. Observing their daily routine, the presence of aggressive behavior patterns of individual mice, mainly male adult mouse. We used two ethological methods: i) Openfi eld and ii) Elevated plus-maze test in the three categories of behavior observed, dominant, subordinate and isolated mice. Our results showed that in groups where there was the presence of aggressive behavior patterns (in both ethological tests) motor and exploratory activity in dominant and subordinate categories was inferior when compared with to harmonic interaction group. Furthermore, subordinate and isolated mice showed a signifi cant decrease in activity, compatible with depressive behavior described in the literature for experimental models. Concluded, from these results we were able to observe that social disturbance and aggressive behavior during social and individual interaction promoted discomfort and stress in mice lab and depression-like state. Research Article Infl uence of social isolation and aggressive behavior in the appearance of Depression-like in Experimental model Gabriel Melo de Oliveira* and Cleiton Felizardo Brito Gabriel Melo de Oliveira* and Cleiton Felizardo Brito Received: 11 July, 2019 Accepted: 04 August, 2019 Published: 06 August, 2019 *Corresponding author: Gabriel Melo de Oliveira, Laboratório, Biologia Celular, Instituto Osvaldo Cruz, FIOCRUZ, AV, Brasil 4365, Manguinhos, Rio de Janeiro, RJ. CEP: 21045-900, Brazil, Tel: +55-21-25621366; E-mail:


Introduction
Actually, there is intense discussion related to the laboratory animal welfare in biomedical testing. Ethical parameters in the use of laboratory animals are, mainly, based in the 3Rs principles [1]. Briefl y, these principles suggest: i) substitution of animals by alternative methods; ii) reduction in the number of animals used and iii) refi nement of housing and manipulation techniques. In non-human primate model various studies have addressed the question of promoting an environment for individuals to develop their exploratory activity and increase the quality of social interaction [2]. Accordingly, studies recommend trying to fi nd better techniques for the maintenance of laboratory animals to promote the welfare of individuals and minimize animal discomfort [2,3]. For rodents there are also several studies related to environmental enrichment [4,5]. However, indexed literature analysis described (Pub Med -August 2019) a relatively small amount of research into the individual and social behavior of mice. About 49,763 studies have evaluated the issue of welfare in non-human primates, whereas only 316 corresponding works related on mice [6].
Hence, the perceived necessity for the study of mice behavior in the laboratory in order to promote knowledge that could be used in the development of new equipment, procedures and techniques, improving the welfare of laboratory mice. Furthermore, we observe that this knowledge in the animal behavior is also important to improve the empathy of the manipulator in relation to the animal, increasing the quality in the procedures performed and minimizing the occurrence of biological accidents [6].
Territorialim, sexual selection and social interaction was intrinsic characteristic of the mouse lab and dominant behavior can be observed in both male and female mice. In the adult male mice, the aggressive behavior, there is high prevalence of behavior between adult individuals. The appearance of these fi ghts is easily observable during routine cleaning and handling of these animals [7]. The aggression between male mice may have several causes: genetic [8], hormonal [9,10] or neurochemical [11,12] among others. It´s suggested that this aggression could be related to the struggle of individuals for social domination within the structural hierarchy [13].
However, some studies suggest that the presence of aggression occurs because of the failure of the harmonic hierarchy [14]. The presence of dominant and subordinate (attacked) individuals was observed in several male groups. We can identify mice that promote aggression and others who suffer attacks [15].
It is likely that mice suffering attacks are those that are in an inferior position in social dominance [16]. The social isolation of the aggressor animal is the main attitude in the management of aggressive animals [16].
The aim this study is to investigated the infl uence of the aggressive behavior in emotional state of the mice lab during housing in the animal facilities. We use motor and exploratory activity and pattern of aggressive behavior (PAB) in the all groups (and categories), and social isolation animal. Using ethological methods, normally employed for the evaluation of animal models of depression and anxiety [16], we measured the level of motor and exploratory activity in dominant, subordinate and isolated mice [17]. Then our proposal was to compare among the categories studied the presence of the state of anxiety and depression, mainly in the management of social isolation and in a situation of exacerbated aggression in groups of mice lab. Animals maintained in the absence of social interaction, described in the literature as a model for hypoactivity or depression-like behavior, were used as a reference [18,19].

Pattern of aggressive behavior (PAB)
We evaluated the daily routine, looking for the appearance of signs of aggression, fi ghts, bites and dominance in each animal group. We also noted other stress signs such as vocalizations,

Open fi eld test
This test consisted of the measurement of behavioral variables in experimental individuals, placed in an arena limited to sixteen spaces of equal size [20]. Behavioral assessment was carried out by evaluating motor activity by measuring the number of spaces crossed (horizontal displacements) by each mouse in a period of fi ve minutes, while the exploratory activity was measured through the number of vertical displacements (rearing) independent of the location in the arena, also in a period of fi ve minutes.

Elevated plus maze test
This test was used to assess the motor ability and exploratory interest of each animal [21].

Statistical analysis
Results were expressed as mean-standard deviation (±SD).
Signifi cance between means was determined using the Student t-test and results were regarded as signifi cant when P ≤ 0.05.

Mice lab observation and PAB and isolation social behavior
in their daily routine we divided them into respective categories. The behavior types was demonstrated graphically ( Figure 1). The fi rst category described is of mice that did not demonstrate aggressive behavior in the group, denominated as normal ( Figure 1A). In relation to PAB groups we distinguished two categories of individual aggressive behavior. Dominant animals were those who showed no signs of fi ghts or bites. Subordinate animals, in turn, showed signs of fi ghts and bites. In these groups, two types of social dominance was suggested: i) «one against all» when an individual fought all components ( Figure 1B) and ii) «all against one» when several components fought only one individual component of the group (Figure C), but we have not yet confi rmed this dynamic of aggressiveness. Moreover, we also evaluated the activity of mice in social isolation ( Figure 1D).
Open fi eld test results (Figure 2) showed that both, dominant and subordinate mice, showed a signifi cant decrease in motor (Figure 2A) and exploratory activity ( Figure 2B) when compared to normal mice. Subordinate animals demonstrated lower activity when compared with the dominant mice and a similar level in relation to the animals in social isolation ( Figure  2A Figure 4A). Concerning exploratory activity, the time remaining in closed arms was higher for the subordinate mice. These values, in number of visits or time in arms, were similar for subordinate and solitary mice ( Figure  4B).

Discussion
Our results, in agreement with data from literature, indicate that groups of male mice in adulthood may present PAB [7]. The aggressive behavior can have various causes. The genetic infl uence may be related to segments of chromosomes with effects on aggressive behavior. This approach is illustrated by the effect of the male-specifi c part of the mouse Y chromosome on aggressive behavior. It´s proposed that a positional candidate for this effect is Sry [22]. Testosterone also increases adrenal corticoid hormone (ADH) levels in the medial amygdala, lateral hypothalamus, and preoptical medial area, which are involved in aggressive behavior [10]. Neurotransmitter expression, as well as excessive aggressive and impulsive traits of neuronal NO synthase knockout (nNOS-/-) mice were shown to be caused by reductions in serotonin (5-HT) turnover and defi cient 5-HT1A and 5-HT1B receptor function in brain regions regulating emotion [23]. We believe that the presence of aggressive behavior and social dominance in adult male mice may be the result of these factors together.   However, one question still needs to be clarifi ed. Why would the PAB appear in some groups and not others, under the same environmental conditions? Issues such as sexual competition [24] and the inclusion of intruders [25] was described in the literature, but this did not happen in our study. Several studies have described social dominance in male mice and related the description of dominant and submissive categories [26,27].
The motor and exploration activity of the mice is born of the requirement for information related to the new environment. The animal acquires information in two ways, evaluated by testing in the open fi eld and the elevated plus-maze. Horizontal displacement, measured by the number of spaces crossed (motor activity) or displacement in arms (open and closed) in the elevated plus-maze, respectively. Exploratory activity was measured by the number of vertical displacements (rearing) while the animal is anywhere in the arena and time spent in the arms. The results for the two tests were similar. Our results showed that dominant and subordinated mice showed decreased motor and exploratory activity in relation to animals without the PBA, that is, despite presenting different categories, the stress promoted by aggressiveness affects individuals of both categories. So, decrease in activity may be related to the chronic stress caused by the aggressive behavior. Furthermore, subordinate animals showed serious decreased in the activity compared with the dominant mice and similar animals in social isolation, what can we characterize as a state of depression (depression-like) in booth categories [28].
Individual housing has also frequently been reported to be uncomfortable for mice and has even been used as a model for social deprivation in man [29]. It may cause both physiological and behavioral abnormalities, referred to as 'isolation syndrome' [30]. The clinical depression, understanding of the etiology behind these disorders is far from complete, though theories involving various neurotransmitter systems have been advocated [31]. The symptoms -reduced activity of the submissive and isolated animals -suggest a connection to neurotransmitter imbalance similar to the clinical signs of depression in humans [32]. In consequence, these animals show reduced motor and exploratory activity.

Conclusion
We still do not know the cause of the appearance of PAB in some groups of male mice. The high and/or low level of (mainly motor) activity of the animals comes after the process of social dominance or as consequence of this. Are there two types of social dominance that could arise from the activity level of individual mice? Our goal is to propose a model of maintenance of male mice in the laboratory based on their motor and exploratory activity designed to minimize aggressive behavior. We can conclude that animals which have aggressive disorders in their group show alterations in their motor and exploratory activity and similar with isolation social behavior in suggest depression-like condition.