Formation of Behavioral Pathology in Female C57BL/6J Mice Exposed to Prolonged Negative Psychoemotional Conditions

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1 Neuroscience and Behavioral Physiology, Vol. 35, No. 9, 2005 Formation of Behavioral Pathology in Female C57BL/6J Mice Exposed to Prolonged Negative Psychoemotional Conditions D. F. Avgustinovich and I. L. Kovalenko Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 90, No. 11, pp , November, Original article submitted July 28, Prolonged exposure of females to negative psychoemotional conditions by permanent keeping with aggressive males on the other side of a perforated partition in a cage with daily witnessing of intermale confrontations had negative influences on the behavior of females, assessed using a variety of ethological tests. The elevated maze test and the open field test provided evidence for the development of marked anxiety in the females, with inhibition of motor and investigative activity. Decreases in the responses of females close to the partition to familiar and unfamiliar males one month after the onset of exposure to negative psychoemotional conditions and before the end of the experiment provided evidence for a decrease in communicativeness. After two months of exposure to negative psychoemotional conditions, intermale confrontations also ceased to interest females, as shown by the partition test. The hotplate test revealed worsening of pain sensitivity in experimental females. More than half of the individuals demonstrated impairment to the cyclicity of sexual cycles with increases in the duration of diestrus. After being kept in negative psychoemotional conditions, females ceased to differentiate littermates by social status and gender, as shown by social/sexual recognition tests. All these data provide grounds for suggesting the development of an anxious-depressive state in females after being kept in negative psychoemotional conditions for prolonged periods. KEY WORDS: C57BL/6J mice, negative psychoemotional conditions, anxiety, social recognition, pain sensitivity, communicativeness, estral cycle, anxious-depressive state. Stress, especially social stress, is known to have significant negative effects both in humans [12] and animals [10, 11]. Experimental modeling of social stress, which has psychoemotional effects on animals to facilitate the development of a pathological state in individuals, is of current relevance. Such models allow complex studies of developing pathological states, as they contain causal mechanisms common to humans and animals. However, there are gender-related characteristics in responses to psychoemotional stress, such that a given treatment does not always have identical consequences in males and females [27]. Thus, chronic social stress in humans elicits the development of anxious-depressive pathology twice as often in women as in men [17, 33]. In experiments, prolonged isolation of animals is traditionally used as a model of the behavioral and Institute of Cytology and Genetics, Siberian Division, Russian Academy of Sciences, 10 Lavrent ev Prospekt, Novosibirsk, Russia. physiological effects of social stress [21], and has different effects on males and females [27]. Our previous studies have demonstrated that modeling of social stress in C57BL/6J mice induces the development of an anxious-depressive state in males [23]. This naturally led to the wish to obtain a model of an analogous pathological state in females of this genotype. As it is virtually impossible to induce confrontations between females by non-pharmacological methods, as females are not aggressive by nature (with the exception of pregnant and lactating individuals), other types of negative psychoemotional conditions were used to induce the development of a pathological state [1 3]. Prolonged restriction stress [2] and the conditions of social instability [3] had no significant pathological effect on the behavior of females. Marked anxiety in females was established after prolonged keeping in isolation [3]. However, analysis of the behavior of females kept for one month in constant sensory contact, through a partition, with aggressive males and witnessing daily intermale /05/ Springer Science+Business Media, Inc.

2 960 confrontations, as well as placing females on the territory of aggressive interactions between males after confrontations [1], provided evidence supporting the potential of using this experimental approach to model the pathological state in females. In these conditions, the aggressive environment of the psychoemotional conditions had more significant influences, as anxiety in females develops more quickly (in one month) than in conditions of prolonged keeping in isolation (2 3 months) [1, 3]. Although neither case produced marked changes in the behavior of females in the Porsholt test, often used in preclinical experiment for assessing the depressive status of individuals [9], females showed an increase in drift in conditions of an aggressive environment [1]. It was suggested [1] that modification of this approach, with increases in the duration of keeping females in the aggressive environment to 2 3 months, should facilitate the development of marked depressive pathology in females. In addition, more detailed and multilateral analysis of the behavior of females after exposure to the prolonged psychoemotional effects of the aggressive environment were needed to confirm the presence and depth of the developing pathological process. The aim of the present work was therefore to perform complex studies of the behavior of females, assessing this in terms of their investigative and movement activity, social recognition, pain sensitivity, level of anxiety and depressivity after being kept for prolonged (2.5 months) periods in negative psychoemotional conditions imposed by the combination of constant keeping of females in sensory contact with aggressive males on the other side of a partition in the cage and daily witnessing of intermale confrontations. A series of widely used ethological test was used, i.e., the elevated cross maze test, the open field test, the hotplate test, and the Porsholt test, as well as the partition test developed in our laboratory, and tests for sexual and social recognition. METHODS Experiments were performed on mature female mice of the inbred line C57BL/6J aged months. Animals were reared and kept in standard animal-house conditions at the Institute of Cytology and Genetics, Siberian Division, Russian Academy of Sciences in a 12:12 h (light:dark) light regime. Standard granulated feed (from Sel skokhozyaistvennye Tekhnologii, Moscow) and water were available ad libitum. From the age of one month to maturity, females were kept in standard cages of size cm. In accord with the experimental aims, females were transferred to metal cages of size cm, divided into two equal sectors by a transparent perforated partition. One half of the cage was constantly occupied by a female, the other by an aggressive male which at the start of the experiment already had experience of victories in intermale Avgustinovich and Kovalenko confrontations (fights). Every day, during the second half of the day, a victim was placed with the aggressive male, the victim being a male mouse with previous experience of defeats in confrontations with an aggressor. This transfer of victims resulted in sequential intermale fights occurring before the eyes of the female on the other side of the partition. The interaction between the males lasted 10 min, after which the victim was removed from the cage. Aggressors were then transferred to the neighboring cage on litter (sawdust) on which the intermale confrontation had taken place, on the other side of the partition from another female. Females were left in their own territory throughout the experiment, unlike the situation in previous experiments [1], in which females were transferred daily to the litter of the aggressor of the neighboring cage in which fights between males had taken place. The duration of exposure of females to negative psychoemotional conditions, including intermale fights and constant keeping of females with aggressors on the other side of the partition, was 2.5 months. Controls consisted of intact females of the same age, kept in groups of five individuals in standard cages of size cm, initially placed together from the age of one month before the experiment. Groups consisted of 18 experimental and 25 control females. During the period of exposure to negative psychoemotional conditions, measurements were made of the dynamics of the females behavior (every 5 7 days) close to the partition in response to both a familiar aggressive male and an unfamiliar aggressive male (this is the partition test). Observations of females were made after replacement of the metal roof with transparent organic glass. The time (sec) spent by the female close to the transparent partition with openings was measured during the first 5 min, along with the numbers of approaches and/or turns towards the partition on the other side of which was an aggressive male. The familiar aggressive male was then replaced by an unfamiliar aggressive male and the behavior of the female close to the partition was again observed for 5 min. Thus, quantitative assessments of the responses of females to males in the neighboring sector of the cage were obtained. The periods during which the females placed their noses in the openings of the partition to sniff their neighbors were measured, along with the periods of touching the partition with the forepaws and moving along the partition. The day after the partition test, the responses of females to intermale fights were studied. After placing a victim with the aggressive male, recordings were made, as on the previous day, of the female s behavior close to the partition during the 10 min of the intermale confrontations, separating this period in to the first and second 5-min periods. Thus, the dynamics of female responses to intermale confrontations were studied throughout the experimental period. At the end of exposure to negative psychoemotional conditions, all females of both the experimental and control groups were compared in ethological tests (one test per day)

3 Formation of Behavioral Pathology in Female C57BL/6J Mice 961 in the following sequence: elevated maze test, open field test, Porsholt test, sexual preference test, social preference test, and hotplate test. Each test was always applied once to each animal. Elevated Cross Maze Test. This test, in which the animal s behavior is sensitive to the actions of anxiogenic and anxiolytic agents, is one of the most widely used tests for assessing anxiety states in individuals [25, 31]. The cross maze was elevated above the floor to a height of 50 cm and consisted of two open and two closed (enclosed on three sides) arms. The mouse was placed in a darkened room 5 min before the test, and the individual s behavior was tested in the maze for 5 min. The animal was placed in the center of the maze with the nose in a closed arm and measures adequately reflecting the anxiety state were recorded: the time spent in the open arms, the center, and the closed arms of the maze (data presented as the times spent in each part of the maze as percentages of the total test duration); the numbers of excursions into the open arms, center, and closed arms, again expressed as percentages; the total numbers of excursions into the open arms and center and entries into the closed arms of the maze; the numbers of transfers from one closed arm to the other; and the number of glances beneath the maze. The maze was thoroughly washed and dried after testing each individual. Open Field Test. The animals behavior in an open field of size cm, divided into squares of cm, was studied. The field was illuminated with a bright light (100 W) located 1 m above the floor surface. Animals were placed in the center of the field and the latent periods (sec) of the first excursions from the central square were measured, along with the numbers of squares crossed, the duration (sec) and number of grooming episodes, and the number of rearings onto the hindpaws per 5-min test. The surface of the open field was thoroughly washed with water and dried after each animal. Porsholt Test (forced swimming test). This test, sensitive to the actions of a variety of antidepressants [13, 28, 29], is regarded as a test for depressivity in rats and mice [16, 24, 36]. For testing, each mouse was placed in unavoidable conditions in a 1-liter glass flask (diameter 12 cm, height 20 cm) filled with water at 25 ± 1 C to 9 cm. During the 5 min of the test, measurements were made of the duration (sec) and number of episodes of passive swimming by the mouse, this consisting of drift and the state of complete immobility. Mice were regarded as immobile when they floated in the water without moving. In drift, the mouse made only the weak movements with one or two hindpaws to keep the head above the surface of the water. In addition, measurements were made of the latent period of the first period of immobility (sec). Sexual Recognition Test. Females were placed for one day individually in standard cages of size cm, previously fitted with transparent perforated partitions in opposite corners. On the next day, an intact group male was placed behind one of the partitions, with a male being placed behind the other partition. The latent periods (sec) of the approaches to the male and female were measured in experimental and control females, along with the numbers of approaches and durations (sec) of periods spent alongside the partitions during the 10-min observation period. Social Recognition Test. The responses of females of two experimental groups were studied the day after the sexual preference test; in this test, an aggressive male was placed behind one of the partitions and a victim behind the other. During the 10-min observation period, the latent period (sec) of approaches to the aggressive male and victim were measured, along with the numbers of approaches to each of these and the duration (sec) of periods spent close to each partition. Hotplate Test. Animals were placed on a round areas of diameter 17.5 cm connected to a thermostat maintaining the area at a temperature of 55 ± 1 C. The time (sec) from the moment at which individuals were placed on the area to the first licking of the paws was measured. Females were returned to their home cages after measuring this latent period. All behavioral measures were recorded using an Étograf apparatus, which allows the frequency and durations of behavioral acts to be recorded. When experiments were complete, control studies of the phase of the sexual cycle were performed in experimental and control females on five sequential days. Vaginal smears were collected each day at the same time of day. In addition, smears were collected each day after observation of females in behavioral tests. Smears were stained with azure-eosin by the Giemsa Romanovskii method for microscopic identification of the stage of the estral cycle. Behavioral measures in control females and individuals exposed to negative psychoemotional conditions from each test were compared using the non-parametric Mann Whitney U test. Pairwise comparisons of behavioral measures from the various tests for each experimental group were based on the non-parametric Wilcoxon Matched Pairs T test, as were data on the dynamics of behavior of experimental groups of females in the partition test and tests for responses to intermale confrontations. Statistical analyses of data were run on Statistica 6.0. RESULTS As established by the partition test, prolonged keeping of females in cages with aggressive males fighting every day with other males induced changes in the behavior of the females close to the partition (Fig. 1). While females showed interest in both the familiar and unfamiliar males on the other side of the partition at the start of the experiment, this interest was significantly decreased by one month (measurements 4 5) as compared with the first measure-

4 962 Avgustinovich and Kovalenko Fig. 1. Dynamics of changes in the behavior of females exposed to prolonged negative psychoemotional conditions in responses to familiar (A) and unfamiliar (B) males in the partition test. The vertical axes of the upper plots show numbers of approaches to the partition; the vertical axes of the lower plots show response durations, sec. 1 9) Measurement numbers. *p < 0.05; **p < 0.01; ***p < compared with the first measurement. Fig. 2. Dynamic changes in the behavior of females in response to intermale confrontations. The abscissa shows measurement numbers; the ordinate in A shows the number of approaches to the partition, while the ordinate in B shows response durations, sec. The continuous line shows results obtained after the first 5-min period; the continuous line shows results obtained after the second 5-min period. *p < 0.05; **p < 0.01; ***p < compared with the first measurement. ment. In both cases, females showed decreased numbers of approaches to the partition and decreased durations of time spent close to the partition. The interest of females in the intermale confrontation itself also decreased, though later in time (Fig. 2). At about two months from the start of the experiment (measurements

5 Formation of Behavioral Pathology in Female C57BL/6J Mice 963 TABLE 1. Comparative Analysis of the Behavior of Control Females and Individuals Exposed to Negative Psychoemotional Stress in the Elevated Cross Maze, Open Field, and Hotplate Tests Measure Control females Females exposed to negative psychoemotional conditions Elevated cross maze Number of excursions into open arms, % 10.2 ± ± 1.6* Time spent in open arms, % 4.4 ± ± 0.7* Number of excursions to center 47.6 ± ± 0.9 Time spent in center, % 14.6 ± ± 2.7 Number of entries into closed arms, % 42.2 ± ± 1.9 Time spent in closed arms, % 81.1 ± ± 3.0 Total number of entries/exits 16.2 ± ± 2.1 Number of transfers 2.5 ± ± 0.8 Number of glances 3.5 ± ± 0.7 Open field Latent period, sec 5.2 ± ± 7.3** Number of squares ± ± 10.3*** Number of rearings 15.6 ± ± 2.7 Duration of rearings, sec 22.0 ± ± 3.2** Number of grooming episodes 4.3 ± ± 1.7* Duration of grooming, sec 17.0 ± ± 5.7 Hotplate Latent period to first paw lick, sec 8.6 ± ± 0.9** Notes. *p < 0.05, **p < ***p < compared with controls (Mann Whitney, U test). 9 10), females showed significant decreases in the numbers of approaches to the partition and the durations of time spent close to the partition during intermale confrontations as compared with values obtained at the first measurements. This was seen both in the first 5 min and the second 5 min of the intermale confrontations. Analysis of the females behavior in ethological tests after being kept in negative psychoemotional conditions also demonstrated significant impairments. It should first be noted that vaginal smears taken after each ethological test identified individual cases of females at the stage of proestrus and metestrus. These females were therefore excluded from further statistical analysis. Significant differences in behavior between females at the stage of estrus and females at the stage of diestrus were not seen in the tests used here, except for the Porsholt test. Thus, combined data from females in these two stages of the estral cycle are presented for all tests other than the Porsholt test. For the Porsholt test, comparisons were made for control females and females exposed to negative psychoemotional conditions separately for the estrus and diestrus stages. As established in the elevated cross maze test (Table 1), females kept in negative psychoemotional conditions showed significant decreases in the numbers (U = 61.0, p < 0.029) and durations (U = 64.0, p < 0.040) of periods spent in the open arms of the maze. In addition, there was a tendency for more frequent excursions into the closed arms (U = 70.5, p < 0.093). Even more marked differences between control females and females placed with aggressors were seen in the open field test (Table 1). Females kept in negative psychoemotional conditions showed significant increases in the latent period of excursions from the center of the field (U = 83, p < 0.006), decreases in the number of squares crossed (U = 39.5, p < 0.000), increases in the number of grooming sessions (U = 97.5, p < 0.019), and decreases in the duration of rearings onto the hindpaws (U = 73.5, p < 0.002). Prolonged keeping in negative psychoemotional conditions also decreased pain sensitivity in females, as demonstrated by the hotplate test (Table 1). Unlike control females, experimental females showed a significantly longer latent period (U = 99.5, p < 0.007) to the first licking of the paw when placed on the area at a temperature of 55 ± 1 C. Prolonged keeping in negative psychoemotional conditions produced impairments in the behavior of females close to the partition in conditions of simultaneous presentation of an intact group male and female (Fig. 3). Females kept for prolonged periods with an aggressive male showed significant increases in the latent period of the approach to the intact female (U = 121.0, p < 0.050), decreases in the

6 964 Avgustinovich and Kovalenko Fig. 3. Behavioral responses of control females (white columns) and females exposed to negative psychoemotional conditions (black columns) to an intact male or female in the sexual recognition test and to aggressors and victims in the social recognition test. A) Latent period of approach; B) number of approaches; C) duration spent close to partition. The vertical axes in A and C show time, sec. In A and B: 1) to male; 2) to female; 3) to aggressor; 4) to victim; in C: 1) close to male; 2) close to female; 3) close to aggressor; 4) close to victim. *p < 0.05, **p < 0.01 compared with the corresponding parameters in control females; # p < 0.05, ### p < compared with responses to intact male (only for control females); ++ p < 0.01 compared with responses to aggressor (only for control females). Fig. 4. Behavioral measures in control females (white columns) and females exposed to negative psychoemotional conditions (black columns) in the Porsholt test. A) Latent period; B) duration of passive swimming. The vertical axes show time, sec. 1) Estrus, 2) diestrus. **p < 0.01 compared with control females. numbers of approaches to both the intact male (U = 87.0, p < 0.004) and the intact female (U = 98.5, p < 0.010) and in the times spent close to the male (U = 114.5, p < 0.032) and female (U = 124.0, p < 0.060). It should be noted that control females showed a rather greater frequency of approaches to females than to males (T = 49.0, p < 0.012), though there was no difference in the duration of times spent close to males and females. Differences in behavior between control females and females kept in negative psychoemotional conditions on simultaneous presentation of an aggressive male and a victim were less marked (Fig. 3). There were tendencies to increases in the latent periods of approaches to the aggressive male (U = 125.5, p < 0.066) and victim (U = , p < 0.079) in females kept in negative psychoemotional conditions; these, like control females, approached and stayed close to aggressive males and victims to the same extent. However, there were other interesting characteristics in the behavior of females in the two study groups. While control females made more frequent approaches (T = 29.0, p < 0.008) and spent longer (T = 46.5, p < 0.005) periods close to victims than aggressive males, females kept in negative psychoemotional conditions lost this preference. In addition, comparison of the behavior of control females in relation to males of three social groups intact, aggressors, and victims showed that control individuals showed a significant preference for intact males. Control females made significantly more frequent (T = 15.0, p < 0.000) approaches to intact male than to aggressors, and spent longer close to them than to aggressors (T = 3.5, p < 0.000) or victims (T = 60.0, p < 0.018). At the same time, females kept in negative psychoemotional conditions, like control females, lacked this differential behavior: they spent essentially identical periods of time close to males of all three social groups and intact females. The Porsholt test (Fig. 4) showed that there were no significant differences between control females and individuals kept in negative psychoemotional conditions in terms of the latent period of the first episode of immobility, though there was a tendency (U = 44.5, p < 0.061) for this

7 Formation of Behavioral Pathology in Female C57BL/6J Mice 965 to increase in females at the stage of diestrus. At this same stage of the sexual cycle, there was a significant decrease in the passive swimming time (U = 32.0, p < 0.011) in females kept in negative psychoemotional conditions. Control analysis of vaginal smears in females kept in negative psychoemotional conditions on five sequential days revealed abnormalities of the sexual cycle in 10 of 18 individuals, with increases in the duration of the diestrus phase (4 5 days). Similar abnormalities were seen in the control group in five of the 25 mice studied. DISCUSSION As demonstrated in these studies, many behavioral measures were altered after prolonged keeping in negative psychoemotional conditions. Keeping of females in a single cage on the other side of a partition from an aggressive, constantly fighting male had negative influences primarily on the communicativeness of females. While at the beginning of the experiment females had high levels of interest in both familiar and unfamiliar males, as shown in the partition test, this interest started to decline at one month, though reacting to intermale confrontations remained quite high. However, at months, females also lost interest in fights between males. It is natural to suggest that females adapt to daily intermale fights and permanent living in cages with an aggressive male on the other side of the partition such that the females ceased to respond to these long-repeated events. However, experiments addressing sexual and social recognition by females showed that the females developed a behavioral pathology rather than that they adapted. Thus, presentation of an intact group male and an intact group female to an experimental female did not produce the interest in their littermates that was seen in controls. They avoided making approaches. They failed to differentiate intact individuals kept on the other side of the partition by gender and equally avoided socializing with both the male and the female, while control females discriminated the gender of their littermates and preferred to spend longer times with females. In addition, experimental females kept for prolonged periods with aggressive males stopped discriminating the aggressive males from victims, while control individuals discriminated males in terms of social status, making more frequent approaches to the partition when there was a victim behind it and spending longer periods of time close to victims. Social recognition in animals is known to be a key factor for the recognition of social types of behavior [15]. It allows relationships to be established between individuals during formation of hierarchical structures, which limit aggressive conflicts and facilitate survival of the group as a whole. In addition, correct social recognition is known to be a necessary and important condition for avoiding interactions with infections and parasites. Thus, animals using social information, including chemical signals, have been shown to recognize and avoid interactions with littermates infected with ecto- and endoparasites [15]. In conditions of free selection, females prefer the odors of males which are not infected with parasites [18]. This evolutionarily important factor is also directed to improving the survival of the species as a whole. Disturbances to social recognition in females after chronic exposure to negative psychoemotional conditions can evidently have significant negative consequences for the whole population. Disruption of communicative behavior is evidently associated with increased levels of anxiety in females after prolonged keeping in negative psychoemotional conditions. This is shown by data obtained in the elevated cross maze test and the open field test. These tests are often used for assessing anxiety in rats and mice [25, 30]. In addition, our experiments on male mice of the same line C57BL/6J previously demonstrated decreases in communicativeness with simultaneous increases in anxiety levels in individuals subjected to prolonged social stress [6, 23]. In the present study, females kept in negative psychoemotional conditions, like males, feared excursions into the open arms of the maze, which is evidence of an increase in anxiety in these animals. In the open field test, experimental females showed an almost 2.5-fold decrease in movement activity assessed in terms of the number of squares crossed by the females. Investigative activity was also impaired, as demonstrated by the decreased duration of rearing onto the hindpaws. The latent period of excursions by females from the central square increased. All these points may be evidence for the appearance of anxious behavior in females in new unfamiliar conditions. Simultaneously with the increase in anxiety, females exposed to prolonged negative psychoemotional conditions showed decreases in pain sensitivity, as shown by data obtained in the hotplate test. Experimental females after exposure to negative psychosocial conditions showed increases in the latent period of the first licking of the paw as compared with control individuals, indicating the development of analgesia. The phenomenon of stress-induced analgesia, in which the responses of animals to pain-inducing stimuli decrease after exposure to any stressor, is well known in the literature [34]. This suggests that application of prolonged negative psychoemotional conditions to females, as used in the present experiments, is stressful and promotes the formation of analgesia. In any case, this effect, as shown by our results, facilitates the development of an anxiety state in the individuals. Anxiety, as is known [22, 35], is a consequence of stress applied to the body. Another suggestion is also possible, that the development of anxiety in these conditions facilitates a decrease in pain sensitivity in females. In any case, data have been obtained in mice with genetic knockout of the gene encoding neuropeptide Y, in which there was a parallel between the anx-

8 966 iogen-like phenotype of these animals and hypoalgesia, assessed using the hotplate test [8]. It is interesting to note that in the Porsholt test, females exposed to prolonged negative psychoemotional conditions and in the diestrus stage showed reductions in the duration of passive swimming. Immobility of animals in this test has been shown to decrease on the background of treatment with many antidepressants [28], so this test is often used by investigators using animal models of depression [19, 26, 32, 38]. The good correlation between effective doses for the antiimmobility effect of various antidepressants and their effective doses for the agents in clinical practice allows the forced swimming test to be used reliably as a preclinical test [9]. Stressors, such as social isolation in mice [39] and uncontrollable electric shock in rats [37], increase the duration of immobility. We have previously demonstrated that chronic social stress in male C57BL/6J mice, due to daily defeats in intermale confrontations and permanent living in sensory contact with an aggressive male, induces increases in immobility in the Porsholt test, this decreasing after two weeks of treatment with antidepressants [23]. However, conversely to the situation in males, various forms of negative psychoemotional conditions, including prolonged restriction [2] and prolonged keeping in solitude and social instability [3], decreased immobility in the Porsholt test in females of the same mouse line, as in the present experiments. It would appear that different psychoemotional stressors improve the state of females, having, like antidepressants, an anti-immobility effect in conditions of forced swimming. However, the development of a pathological state in females is indicated by all the other results obtained here decreased communicativeness, increased anxiety, decreased pain sensitivity, deficiencies in social recognition, and decreases in movement and investigative activity. These data are more likely to provide evidence supporting the development of a depressive-like state, analogous to the state developing in males of this mouse genotype in conditions of chronic social stress [23]. It is possible that there is a genotypic characteristic in the responding of C57BL/6J mice to the more aversive external stimulus in this test (the Porsholt test) in the behavior of the active avoidance of the situation, as noted previously in males [7]. This characteristic is more marked in females as it also appears in the pathological state, which does not occur in males. It is also possible that the Porsholt test is not as indicative in demonstrating a pathological depressive-like state in females as in males. In any case, it is well known that gender differences in behavior are seen in the forced swimming test in both intact animals [5] and on the background of acute and chronic stress [20], as well as on the background of treatment with antidepressants [9, 14]. This is the cause of debate among investigators, as there is a divergence between the fact that depression in human society is more frequent in women, while among animals females are less immobile than males [9]. Despite the results obtained in the Porsholt test, we tend to the view that a pathological depressive-like state with marked anxiety develops in females after prolonged exposure to negative psychoemotional conditions consisting of permanent living with an aggressive male on the other side of the partition in the cage with daily witnessing of intermale confrontations. Strong support for this view comes from the observation of a disturbance to the cyclicity of the phases of the estral cycle in 10 of the 18 experimental individuals. In humans, the incidence of menstrual cycle disturbances is known to reach 70% or more in patients with various types of emotional-affective disorders, including depression, and these disturbances are obligately accompanied by alterations to sexual attraction and frigidity in women [4]. By analogy with humans, it might be suggested that decreased sexual motivation in experimental mice, evidenced, as noted above, by impaired sexual recognition, has the result that females exposed to negative psychoemotional conditions stop distinguishing intact males and females. Thus, abnormalities identified in the behavior of females after prolonged exposure to negative psychosocial conditions suggests the development of a pathological depressive-like state in individuals, similar to the state previously described in males of the same inbred mouse line, C57BL/6J [23]. Just as in males, females showed decreases in investigative and movement activity, communicativeness, and pain sensitivity. Impairments in the cyclicity of the estral cycle were accompanied by negative changes in sexual/social recognition. Females showed marked anxiety, pointing to the presence of an experimental anxious depression as noted previously in males of the same genotype after chronic social stress [23]. All these points provide evidence that the long-term negative psychoemotional conditions used in this study induces pathological behavior in females, reflecting the development of an anxious-depressive state. 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