Social stress in mice: Gender differences and effects

Physiology & Behavior 73 (2001) 411 ± 420

Social stress in mice: Gender differences and effects of estrous cycle and social dominance Paola Palanza*, Laura Gioiosa, Stefano Parmigiani Dipartimento di Biologia Evolutiva e Funzionale, Parco Area delle Scienze 11A, UniversitaÁ di Parma, 43100 Parma, Italy Received 16 October 2000; accepted 22 February 2001

Abstract A large discrepancy in the possibility of inducing social stress in the two genders exists. Since generalizations of findings from one sex to the other appear not to be valid, reliable models of social stress in females are needed. We examined the effects of social context in the housing environment, as a possible source of stress, on exploration and anxiety in male and female mice, taking into account the estrous phase for females and the social status for males as additional variables. Mice housed individually or with siblings were tested in a freeexploratory paradigm of anxiety (where test animals have a choice to stay in their home cage or to explore an open field, OF). Individually housed females did not leave their home cage for long periods, explored less the unfamiliar area and displayed higher risk assessment, a behavioral profile suggestive of lower propensity for exploration and higher level of anxiety compared with group-housed females. Individually housed males tended to show an opposite profile. Proestrus mice were less sensitive to the decrease of exploratory propensity induced by individually housing compared to estrus and diestrus mice. Social dominants and social subordinates in sibling groups did not differ in their exploratory responses to the OF. Different housing procedures, as means to provide different social environment, may differentially induce mild social stress in male and female mice. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Dominance; Subordination; Male; Female; Ovarian hormones; Anxiety; Exploration

1. Introduction Stressful events are thought to participate in the induction of many different pathologies [1]. Cardiovascular disease [2], ulcerative colitis [3], infectious disease [4], neoplastic disease [5] and psychiatric disorders [1,6] have all been related to stress of various nature. In particular, epidemiological data strongly support the view that psychosocial stress contributes significantly to the development and expression of disease concerning the cardiovascular system (hypertension, cardiac dysrhythmicity or sudden cardiac death Ð see Sgoifo et al., this issue), food intake and metabolism (obesity, anorexia and bulimia Ð see Brambilla, this issue) or mood and motivation (depression, phobias, compulsive panic disorders, anxiety, etc. Ð see Troisi, this issue). A number of animal models have been developed to investigate the impact of stress of various natures on development and expression of disease [7]. The conventional

* Corresponding author. Tel.: +39-521-905628; fax: +39-521-905657. E-mail address: [email protected] (P. Palanza).

animal models of stress include restraint stress, uncontrollable electric shocks, forced running, forced swim and sequential exposure to a variety of severe or mild chronic stress (reviewed in Ref. [8]). Although effective and useful, these stressors are physical, potentially painful and they offer little face validity compared to social and psychological stressors that are of particular interest in humans. These conventional animal models of stress thus appear to be quite far from real life events, either for the experimental animal model or the human counterpart [9]. Consequently, animal models that involve a social context seem to be more appropriate they mimic the stressful situations that an animal may meet in its everyday life in a natural habitat, and for which behavioral and neuroendocrine responses had been shaped by evolutionary processes [9,10]. The colony model (Visible Barrow System [11]), the chronic psychosocial stress [12], the sensory contact model [13] and the resident/intruder test [10,14,15], all focus on the behavioral and physiological effects of the fight-induced subordination and social defeat (reviewed in Ref. [9]). Many physiological and behavioral alterations have been described in subordinates, and the concept of subordination

0031-9384/01/$ ± see front matter D 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 3 1 - 9 3 8 4 ( 0 1 ) 0 0 4 9 4 - 2

412

P. Palanza et al. / Physiology & Behavior 73 (2001) 411±420

stress [16,17] is currently the most studied model of social stress. In addition, isolation (e.g., Ref. [18]) and crowding (e.g., Ref. [19]) have been traditionally used to model behavioral and physiological effects of social stress. However, there is a large discrepancy in the possibility of inducing social stress in the two genders. While there are a number of models of social stress in males, reliable social stress models for females are virtually lacking, and the great majority of studies are conducted only on male rodents' models. The current models of defeat-related psychosocial stress used for male rodents, such as the resident/intruder paradigm or the colony model, are not appropriate for females, as it is generally difficult to induce high levels of aggression in nonreproductive females or to obtain a strong dominance relationship in the laboratory rodents (mice and rats) generally used as experimental models. Models of social stress efficient in one sex have been demonstrated to be inefficient for the other sex, and generalizations of findings from one sex to the other appear to be a tenuous process at best [19,20]. While crowding induces social stress in male rats, females are not strongly affected by this condition but show higher levels of corticosterone, a biochemical index of stress, when individually housed [19]. Haller et al. [20] have reported that male and female rats were differentially affected by two different models of social stress, defeat in aggressive encounters (induced by aggressive male residents and female residents made aggressive by surgical mediobasal hypothalamic lesion) and social instability (obtained by alternating isolation and crowding phases). By measuring physiological biomarker of stress, they concluded that defeat is a major stress for males but not for females, whereas social instability is more stressful for females than for males [20]. On the other hand, epidemiological studies consistently report significant gender differences in the prevalence, etiology and responses to treatments of neuropsychiatric disorders [21]. It is generally acknowledged that alcoholism and other drug abuse, antisocial personality, attention deficit disorders, alcohol use disorders, Tourette's syndrome and completed suicide predominate in men, whereas major depression, anxiety, eating disorders and attempted suicide are more common in women (e.g., Refs. [22 ±24]). Furthermore, gender differences in drug sensitivity have been acknowledged, and hormonal profiles (e.g., ovarian steroids) have been shown to affect activity and effects of psychoactive drugs (reviewed in Ref. [25]). Sex differences in emotional behavior (e.g., Refs. [26 ± 30]) as well as sexlinked differences in neurotransmitter and neuromodulatory systems potentially related to stress- or fear-eliciting behaviors [31,32] have been well documented in a variety of mammals. Significant Drug Gender interactions were consistently obtained in a high proportion of those rare studies in which male and female subjects were compared [33 ± 37], thus indicating differential responses to psychoactive drugs in the two sexes. However, the great majority of basic researches on anxiolytic or antidepressant drugs focus

on male rats or mice as experimental models. Females as subjects in behavioral research are often neglected because they are thought to be too ``variable,'' and it is easier and cheaper to use only males [38]. Ovarian hormone fluctuations may indeed lead to behavioral changes that may be related to emotionality or anxiety [39,40]. In the present study we examined the effects of social context in the housing environment, as a possible source of stress, on exploration and anxiety in male and female mice, taking into account the estrous phase for females and the social status for males as additional variables. We have used a modified free-exploratory paradigm where the animals have the opportunity to choose between a novel and a familiar compartment [41,42]. It is known that the behavior of animals exposed to a novel situation results from a competition between an exploratory tendency (novelty seeking, curiosity) and a withdrawal tendency (fear). By using this paradigm rather than a traditional open field (OF), it is possible to measure not only specific defensive reactions of mice (such as risk assessment), which have been shown to be a reliable index of anxiety in other fear/defense paradigms [43], but also their exploratory propensity (i.e., novelty seeking or ``curiosity''). In most of the exploratory test currently used to measure anxiety responses, such as the OF (reviewed in Ref. [44]) or the elevated plus-maze [45], test animals are ``forced'' into a novel environment and it has been questioned whether the so-called fear reactions might be elicited by such ``forced'' situations rather than by novelty per se [46]. 2. Materials and methods 2.1. Subjects Subjects were CD-1 Swiss albino mice (Mus domesticus) born and reared in our lab from a stock originally purchased from Charles River Italia (Calco, Lecco). After weaning, animals were housed in same-sex sibling groups (4 ± 6 animals/cage) in cages measuring 40 25 15 cm. Room temperature was maintained at 22 ± 24°C on a 12L:12D cycle, with light coming on at 07:00 h. Experimentally, naõÈve mice were used in each experiment. 2.2. Apparatus The apparatus consisted of two sections: (1) a home cage (40 20 20 cm) in which animals were individually housed for 24 h before testing. The floor of the cage was covered by sawdust, and food and water were freely available. The top of the chamber was made of transparent Plexiglas to allow observation and videotape recording. One of the side-walls of the cage was made by wire mesh. (2) An unfamiliar area (OF), a rectangular arena of 73 110 cm bordered along by a 50-cm high polypropylene wall, and divided into 20 squares in which a bright and a dark zone


413

Fig. 1. Exploratory behavior in the open area of the modified free-exploratory paradigm in individually or group-housed male and female mice. The influence of the estrous cycle on female responses is shown. Risk assessment behavior, latency to enter the OF and time-spent exploring the OF are presented as duration time in seconds. * P < .05, + P < .1 vs. same-sex group-housed counterparts; xP < .05 vs. group-housed males.

were created. A light was positioned behind and above the wall so as to cast a shadow along the length of the OF. The home-cage burrow was connected to the OF by means of a small opening (5 cm diameter), which was closed with a removable barrier until testing. 2.3. Procedure Before 24 h of testing, all subjects were moved into the experimental home cages. Group-housed subjects were moved together, but only one animal was introduced into

the home cage; the remaining two siblings were placed in a wire mesh cage adjacent to the wire mesh wall of the home cage in order to maintain tactile and olfactory contact between group-housed siblings. Tests were conducted between 16:00 and 20:00 h in a darkened room illuminated with a white light, which was positioned behind and above the wall of the OF. During observation, the experimenter sat 2.5 m distant from the apparatus. Five minutes before testing, the home cage was placed at one end of the rectangular OF. Once the barrier was removed, behaviors (risk assessment behaviors) were

414


recorded from the first approach to the entrance and, a cutoff of 10 min was used for animals that did not emerge on the surface of the OF. These animals were included in the statistical analysis with a latency to enter the OF of 10 min and 0 time spent exploring the OF. The test was considered to have started after the first entry into the unfamiliar OF (with the four paws) and lasted 5 min. To reduce any lingering olfactory cues, the apparatus was wiped with clean damp cloth and a solution of 2% ethyl alcohol between successive tests. Sessions were recorded on videotape by a VHS video camera situated 2 m above the apparatus. Following the OF test, the estrus phase of females was checked by taking vaginal smears. 2.3.1. Experiment 1 When 90-day-old, sexually naive males and virgin females were randomly allocated to two experimental groups: (a) individually housed for a week in Plexiglas cages measuring 20 25 15 cm (N = 44 females, 16 males); (b) group-housed with same-sex siblings (littermates that were housed together since weaning; 3 animals/cage measuring 45 25 15 cm) (N = 36 female groups, 12 male groups). Only one female per group was tested. Male groups were observed for 1 h when placed in the clean cage and once a day in the following days in order to determine social dominance. Only subordinate males were used for testing. All the animals were gently handled once a day, and females were checked for estrous phase by taking vaginal smears. 2.3.2. Experiment 2 Sixty 90-day-old male mice were group-housed with same-sex siblings (three animals/cage measuring 40 25 20 cm), which were littermates housed together since weaning (N = 24 groups). Male groups were observed for 1 h when placed in the clean cage and twice a day in the following days in order to determine social dominance. On the basis of the agonistic interactions displayed during the observation sessions, animals were assigned to two categories: dominant and subordinate. Two groups were not used for testing, as it was not possible to detect a clear and stable hierarchy. For half of the groups (N = 10) only the dominant was tested. For the remaining 11 groups only a subordinate male was tested. 2.4. Behavioral analysis Behaviors were scored off videotape by a trained observer who remained blind to treatment conditions until data analysis was complete. Data were logged by a series of electronic counters and timers. With the exception of the number of transitions and of crossed squares, all measures were scored as duration (s). The following patterns of behavior have been recorded in the ethological analysis: latency to enter the OF (10 min maximum time), i.e., the time from first approach to the opening of the home cage to

actual entrance into the OF with all four paws; risk assessment, defined as forward elongation of the head and shoulders with scanning the unfamiliar OF occurring from the home cage (this posture was recorded as total time spent before the first exit into the OF); total time spent in the OF; time spent in the bright area, i.e., percentage of time spent in the bright area of the OF (as calculated on the total time spent in the OF); number of returns between the home cage and the OF; rearing, i.e., standing with the forepaws touching the walls of the OF (percent time); locomotor activity was evaluated on the basis of the number of floor squares crossed by the test animal while in the OF. 2.5. Statistical analysis Females were examined for the effect of estrous stage in relation to social context by two-way ANOVA (independent factors of estrous phase and social context). The effects of social status on grouped males were analyzed by oneway ANOVA. Sex differences were analyzed by two-way analysis of variance (ANOVA; independent factors of sex and social context), and only group-housed subordinate males were used for this comparison. Duration data expressed as percent time (i.e., rearing and time spent in the bright area) were initially arcsin-transformed to give normal distributions. Unplanned comparisons were used for binary contrasts.

3. Results Data on the effects of social context on males and females in different estrous phases are summarized in Figs. 1± 3. 3.1. Effects of estrous stage in females Only three metaestrous females were found (two individually housed females and one group-housed females). As the paucity of the sample did not allow statistical analysis, only proestrous, estrous and diestrous females were compared. A significant effect of social context [ F(1,71) = 5.77, P < .02], but not of estrous stage, and no interaction effect were recorded on the latency to enter the OF. Comparisons between groups indicated that individually housed females displayed significantly higher latencies than group-housed females during the stages of estrous ( P < .01) and diestrous ( P < .1), but not of proestrous. Estrous stage did not affect risk assessment behavior, while a significant effect of social context was found [ F(1,71) = 15.94, P < .001]. Comparisons between groups indicated that individually housed females displayed significantly more risk assessment than group-housed females during the stages of estrous and diestrous ( P < .01).


415

Fig. 2. Rearing behavior and time spent in the bright area of the OF by male and female mice housed individually or with same-sex siblings. The influence of the estrous cycle on female responses is shown. Data are expressed as percent time upon total time spent in the OF. * P < .05, + P < .1 vs. same-sex grouphoused counterparts.

Time spent exploring the OF was significantly affected by social context [ F(1,71) = 7.94, P < .01]. Individually housed females spent lower amount of time exploring the OF than group-housed subjects when in estrous ( P < .01) and diestrous ( P < .05), but not when in proestrous. Social context influenced the percent of time spent in the bright area of the OF [ F(1,52) = 9.21, P < .005]. No main effect of estrous was found. Individually housed females spent more time in the bright area than grouped females during the stages of proestrus ( P < .01) and, marginally, of estrous ( P < .07), but not when in diestrous. Rearing behavior, as calculated as time percentage upon the time spent in the OF, was affected by social context [ F(1,52) = 7.07, P < .01]. Individually housed females displayed lower rearing than group-housed females when in estrous ( P < .01) and diestrous ( P < .05). Although it did not reach significance, a main effect of estrous stage was found [ F(2,52) = 2,30, P=.11] with estrous females showing significantly lower levels of rearing than diestrous females ( P < .05). Locomotor activity was assessed on the basis of the number of crossed squares in the OF (calculated only on the females entering the OF). Social context significantly

affected activity [ F(1,52) = 5.42, P < .05] with individually housed females less active than group-housed females, but only when in estrous and diestrous ( P < .05). Number of returns between the OF and the home cage (calculated only for females entering the OF) was significantly affected by social context [ F(1,74) = 4.99, P < .03]. Proestrous and estrous, but not diestrous, females showed higher number of returns when individually housed compared to group-housed counterparts ( P < .05). 3.2. Sex differences Risk assessment behaviors were significantly affected by social context [ F(1,104) = 9.78, P < .005] but not by sex; Sex Social context interaction just missed significance [ F(1,104) = 3.17, P < .08]. While social context did not influence risk assessment by males, individually housed females showed significantly higher levels of risk assessment relative to group-housed females ( P < .001). A significant interaction effect of sex and social context on the latency to enter the OF was recorded [ F(1,104) = 6.64, P < .02]. Females that were housed with siblings entered the OF more quickly than individually housed females ( P < .05),

416


Fig. 3. Locomotor activity as calculated on number of crossed squares and number of returns between the home cage and the OF by male and female mice housed individually or with same-sex groups. The effects of the stage of the estrous cycle are shown. Data are calculated on those animals that did enter the OF. * P < .05 vs. same-sex group-housed counterparts.

while a tendency for an opposite profile was found in males ( P =.1). Although males and females did not differ for the latency to enter the OF when individually housed, grouphoused females showed significantly lower latencies than group-housed males ( P < .01). ANOVA showed a significant interaction effect on the time spent exploring the OF [ F(1,104) = 14.52, P < .001]. When group-housed, females spent higher amount of time in the OF as compared to individually housed females ( P < .005) and group-housed males ( P < .005). On the contrary, individually housed males spent more time exploring the OF than group-housed males ( P < .01) and individually housed females ( P < .05). The proportion of time spent in the bright area of the OF was marginally influenced by social context [ F(1,74) = 3.47, P < .07], with group-housed females spending more time in the bright area than individually housed females ( P < .05). ANOVA on rearing behavior revealed a significant interaction effect between sex and social context [ F(1,74) = 5.71, P < .02]. Group-housed females showed more rearing behavior than individually housed females ( P < .005), while group-housed males tended to show lower levels of rearing than individually housed males ( P < .1). Individually housed

males displayed higher amount of rearing as compared to individually housed females ( P < .05). The number of crossed squares and the number of returns between the home cage and the OF were analyzed considering only those animals that entered the OF. The number of crossed squares (an index of locomotor activity) by an animal in the OF was partially affected by sex [ F(1,74) = 3.7, P < .06]. Females displayed higher level of locomotor activity than males, but only when group-housed ( P < .05). Group-housed females showed higher locomotor activity than individually housed females ( P < .05). Number of returns between the home cage and the OF was not significantly affected by sex nor social context. Table 1 Effects of social status on exploration of the OF in group-housed male mice Behavior

Dominants

Subordinates

Risk assessment Latency Time in OF % Bright % Rearing No. of squares No. of returns

16.30 4.69 299.20 71.08 105.70 34.63 46.63 7.51 6.12 1.73 54.42 16.97 16.28 4.75

21.61 4.34 355.38 62.57 89.77 25.52 31.17 8.05 6.26 1.64 54.66 13.98 12.44 2.20


3.2.1. Experiment 2 Data are summarized in Table 1. ANOVA did not reveal any significant behavioral differences between socially dominant and socially subordinate mice. 4. Discussion Social factors differentially affected the propensity to explore an unfamiliar open area in the two sexes. When given the opportunity to explore an unfamiliar area, females housed individually for a week did not leave their home cage for longer periods, displayed higher level of risk assessment, explored less the unfamiliar area, spent less time in the bright section, were less active, and showed lower level of rearing, relative to females that were housed with siblings. Risk assessment behavior have been shown to respond to treatment with anxiety reducing agents [41,43], and increased levels of this response may thus be indicative of higher anxiety. The impact of social context appears to be less pronounced on male behavior. However, male behavioral profiles tended to show an opposite trend compared to females, with individually housed males showing higher propensity to explore the unfamiliar area than males housed with siblings. Living alone for a short period or with same-sex siblings may thus have a different psychosocial relevance for the two genders. When challenged to explore an unfamiliar area, female mice housed individually showed decreased exploration (curiosity) and increased anxiety relative to females housed in groups. In contrast, for males, being individually housed for a 7-day period induced higher exploration of the unfamiliar area compared to grouped males. Psychosocial effects of isolation and/or grouping may be interpreted only by taking into account the ``natural'' social behavior of the species examined and interindividual variability, such as sex- and/or age-related differences and individual experiences. Although strain differences exist, male and female house mice show clear differences in their social behavior [41,47]. Male mice are territorial (i.e., aggressively defend their home cage against conspecific intruders) and aggressive to other males; when grouped together, they develop a social structure with a single male being dominant on the others. Conversely, female mice show very low or no aggression towards other females, and do not develop a detectable social hierarchy except when reproductively active (e.g., when cohabiting with a male or during lactation) [48]. According to the Sexual Selection Theory [49], the behavioral strategies in coping with social and environment challenges would differ in males and females when a discrepancy in parental investment exists, as it is in mammalian species. Social status is less important in females, which have higher parental investment and, therefore, lower reproductive competition [50]. Gender-specific response profiles to threatening stimuli may be related to variations in adaptive outcome of

417

specific defensive behaviors for males and females, given their different social and reproductive role. Based on their behavioral profiles when challenged to explore a novel environment and taking into consideration sex differences in social behavior, the present findings suggest that for female mice, being housed alone for a short period may be a mild stressor, while this is not true for males. Males were in fact more affected by being housed with other males, in terms of a reduction of active behaviors in response to the unfamiliar environment. Group housing in male mice and rats is commonly regarded as a stressful social context, mainly for subordinate animals [11,51,52]. As we used only subordinate males for comparison to females and individually housed males in Experiment 1, the finding of reduced exploration of grouphoused males was initially hypothesized as resulting from a mild ``subordination stress'' due to social defeat. However, Experiment 2 found no behavioral differences between group-housed dominants and subordinates in the freeexploratory paradigm. In apparent contrast with these findings, previous studies in both rats and mice have found that social defeat decreased exploration and increased anxiety in different exploratory tests [11,53± 55]. However, in all of these studies a group of animals was formed with adult unknown mice or rats, while in our study mice housed in groups were siblings that had been living together since birth. This methodological discrepancy can explain the different results obtained, as it is well known that male mice show lower rate of aggressive behavior and higher levels of amicable behavior towards kin as compared to unfamiliar unrelated males [56,57]. In addition to similarities in behavioral responses to the OF test, a follow-up study has shown no status-related differences in basal corticosterone level and in a series of parameters of immune function between dominant and subordinate males housed in sibling groups (see Bartolomucci et al., this issue). In light of this result, the observed differences in exploration propensity between group-housed and individually housed males could be related to a general enhanced reactivity of individually housed males to environmental stimuli. A modest anxiolytic-like effect of individual housing in male mice has been reported in different experimental paradigms [53,55], although contrasting reports on the possible stressfulness of isolation in rodents exist. Although isolation has been shown to produce mood disorder-like behavioral deficits in rodents [58], there is little evidence of stressfulness of isolation per se, as many studies showed no endocrine changes in isolated animals as compared with group-housed ones [58,59]. Differences among different studies in the species, the laboratory strain and/or the sex utilized, and particularly the duration and timing of isolation, may explain these discrepancies (e.g., Refs. [19,58 ± 60]). In highly social species and/or strain and/or sex, social deprivation due to prolonged isolation or to rearing in isolation is likely to produce severe endocrine and behavioral disorders (e.g., Ref. [61]). In contrast, for a male mouse

418


a short period of isolation as adult can mimic the establishment of a territory [62]. Behavioral variability may be expected in relation to estrous cycle in female mice. In our experimental paradigm, no main effect of estrous cycle on female behavioral responses was observed. However, a consistent different response observed between females in the various stages of the estrous cycle was that proestrus mice appear to be less sensitive to the decrease of exploratory propensity induced by individually housing compared to estrus and diestrus mice. During proestrous stage, latency to enter the OF, risk assessment, exploration of the OF and rearing levels displayed by individually housed mice were similar to those expressed by group-housed females. Reductions in behavioral indices of anxiety and in sensitivity to experimental procedures across the estrous cycle have been reported in other studies in rats [33,40,63]. It is well known that the peak of ovarian steroid hormones occurs during proestrus [64], and experimental evidence has shown that the ovarian hormones estradiol and progesterone and its metabolites (i.e., active neurosteroids) exert an antianxiety effect in different experimental paradigms of anxiety in both rats and mice [34,40,65,66]. Different housing conditions, as means to provide different social environment, may be models of mild social stressors in male and female mice. In particular, individually housing may induce depression-like symptoms (reduced reactivity and exploration, higher anxiety) in female but not in male mice. The examination of the endocrine effects of these different housing procedures on male and female mice, as well as ethopharmacological tests with selective anxiolytic and antidepressant drugs, are necessary to assess this hypothesis in further studies. It has also to be considered that our subjects were gently handled daily for 6 days prior to testing, and moved into the experimental apparatus 24 h prior to testing. It is possible that these procedures resulted stressful per se and influenced the animal responses to the novel arena differentially in relation to sex and social context. Differential effects of handling on exploration in male and female rats have been reported [67]. However, our behavioral findings of differential effects of isolation and grouping in the two sexes are consistent with reports that female rats had higher levels of corticosterone when they were housed individually than when housed with other females, while males had higher levels of corticosterone when housed with other males compared with their individually housed counterparts [19]. In view of normal housing procedures, this potential source of behavioral variability may be particularly relevant in studies involving mice. Housing animals under particular social conditions may in fact differentially alter males' and females' behavioral responses, as well as physiology and biochemical status (see also Ref. [19]). These possible differences should be identified and accounted for to avoid reaching false conclusions regarding sex differences in response to other variables, such as the effects of pharmacological treatments.

Studies of possible mechanisms underlying gender discrepancy in physiological and behavioral mechanisms of response to stress or threat may help to understand differential vulnerability to psychiatric disorders and other stressrelated diseases.

Acknowledgments This work was supported by grants from the Italian Ministry of University and Scientific and Technological Research (MURST) and the National Council for Research (CNR).

References [1] Brown GW, Harris TO. Life events and illness. London: Unwin Hyman, 1989. [2] Herd JA. Cardiovascular response to stress. Physiol Rev 1991;71: 305 ± 30. [3] Weiss JM. Behavioral and psychological influences on gastrointestinal pathology; experimental techniques and findings. In: Gentry D, editor. Handbook of behavioral medicine. New York: Guilford Press, 1984. pp. 174 ± 221. [4] Sheridan JF. Stress-induced modulation of anti-viral immunity. Brain Behav Immun 1998;12:1 ± 6. [5] Sklar LS, Anisman H. Stress and cancer. Physiol Bull 1981;89(3): 369 ± 403. [6] Brown GW. The role of life events in the aetiology of depressive and anxiety disorders. In: Stannford S, Samon P, editors. Stress: from synapse to syndrome. London: Academic Press, 1993. pp. 23 ± 50. [7] McCabe PM, Sheridan JF, Weiss JM, Kaplan JP, Natelson BH, Pare WP. Animal models of disease. Physiol Behav 2000;68:501 ± 7. [8] Willner P. Animal models as simulations of depression. Trends Pharmacol Sci 1991;12:131 ± 6. [9] Martinez M, Calvo-Torrente A, Pico-Alfonso MA. Social defeat and subordination as a model of social stress in laboratory rodents: a review. Aggressive Behav 1998;24:241 ± 56. [10] Koolhaas JM, Meerlo P, De Boer SF, Strubbe JH, Bolhus B. Social stress in rats: an animal model of depression? Acta Neuropsychiatr 1995;7:27 ± 9. [11] Blanchard DC, Spencer RL, Weiss SM, Blanchard RJ, McEwen B, Sakai RR. Visible barrow system as a model of chronic social stress: behavioral and neuroendocrine correlates. Psychoneuroendocrinology 1995;20:117 ± 34. [12] Fuchs E, Kramer M, Hermes B, Netter P, Hiemke C. Psychosocial stress in tree shrews: clomipramine counteract behavioral and endocrine changes. Pharmacol, Biochem Behav 1996;54:219 ± 28. [13] Kundryavsteva NN. A sensory contact model for the study of aggressive and submissive behavior in male mice. Aggressive Behav 1991;17:285 ± 91. [14] Bohus B, Koolhaas JM, Korte SM. Psychosocial stress, anxiety and depression: physiological and neuroendocrine correlated in animal models. In: Genazzani AR, Nappi C, Petraglia F, Martignoni E, editors. Stress and related disorders from adaptation to dysfunction. Pearl City, NY: Parthenon Publishing, 1990. [15] Albonetti ME, Farabollini F. Social stress by repeated defeat: effects on social behavior and emotionality. Behav Brain Res 1991;430:187 ± 93. [16] Blanchard DC, Sakai RR, McEwen B, Weiss SM, Blanchard RJ. Subordination stress: behavioral, brain and neuroendocrine correlates. Behav Brain Res 1993;58:113 ± 21. [17] Miczek KA, Thompson ML, Tornatzky W. Subordinate animals. Be-


[18] [19] [20]

[21] [22] [23] [24] [25] [26] [27] [28] [29] [30]

[31] [32] [33] [34] [35] [36] [37] [38] [39] [40]

havioral and physiological adaptation and opioid tolerance. In: Brown MR, Koob GF, Rivier C, editors. Stress neurobiology and neuroendocrinology. New York: Marcel Dekker, 1991. pp. 323 ± 57. Kim JW, Kirkpatrick B. Social isolation in animal models of relevance to neuropsychiatric disorders. Biol Psychiatry 1996;40:918 ± 22. Brown KJ, Grunberg NE. Effects of housing on male and female rats: crowding stresses males but calms females. Physiol Behav 1995;58:1085 ± 9. Haller J, Fuchs E, Halasz J, Makara GB. Defeat is a major stressor in males while social instability is stressful mainly in females: towards the development of a social stress model in female rats. Brain Res Bull 1999;50(1):33 ± 9. Earls F. Sex differences in psychiatric disorders: origins and developmental influences. Psychiatr Dev 1987;5:1 ± 23. Rutter M, Smith D, editors. Psychosocial disorders in young people. Chichester: Wiley, 1995. Blehar MC. Gender differences in risk factors for mood and anxiety disorders: implications for clinical treatment research. Psychopharmacol Bull 1995;31:4687 ± 91. Hanna EZ, Grant BF. Gender differences in DSM-IV alcohol use disorders and major depression as distributed in the general population: clinical implications. Compr Psychiatry 1997;38(4):201 ± 12. Godfroid IO. Sex differences relating to psychiatric treatment. Can J Psychiatry 1999;44:362 ± 7. Gray JA. Sex differences in emotional behavior in mammals including man: endocrine bases. Acta Psychol 1971;35:29 ± 46. Archer J. Rodent sex differences in emotional and related behavior. Behav Biol 1975;14:4451 ± 79. Johnston AL, File SE. Sex differences in animal tests of anxiety. Physiol Behav 1991;49:245 ± 50. Blanchard DC, Shepherd JK, De Padua Carobrez A, Blanchard RJ. Sex effects in defensive behavior: baseline differences and drug interactions. Neurosci Biobehav Rev 1991;15:461 ± 8. Perrot-Sinal T, Ossenkopp KP, Kavaliers M. Influence of a natural stressor (predator odor) on locomotor activity in the meadow vole (Microtus pennsylvanicus): modulation by sex, reproductive condition and gonadal hormones. Psychoneuroendocrinology 2000;25: 259 ± 76. Carlsson M, Carlsson A. A regional study of sex differences in rat brain serotonin. Prog Neuropsychopharmacol Biol Psychiatry 1988; 12:53 ± 61. Wilson MA, Biscardi R. Sex differences in GABA/benzodiazepine receptor changes and corticosterone release after acute stress in rats. Exp Brain Res 1994;101:297 ± 306. Fernandez-Guasti A, Picazo O. The actions of diazepam and serotonergic anxiolytics vary according to the gender and the estrus cycle phase. Pharmacol, Biochem Behav 1990;37:77 ± 81. Fernandez-Guasti A, Picazo O. Anxiolytic actions of diazepam, but not buspirone, are influenced by gender and the endocrine stage. Behav Brain Res 1997;88:213 ± 8. Alonso SJ, Castellano MA, Alfonso D, Rodriguez M. Sex differences in behavioral despair: relationships between behavioral despair and open field activity. Physiol Behav 1991;49:69 ± 72. Meng ID, Drugan RC. Sex differences in open field behavior in response to the beta-carboline FG7142 in rats. Physiol Behav 1993;54:701 ± 5. Barros HMT, Ferigolo M. Ethopharmacology of imipramine in the forced-swim test: gender differences. Neurosci Biobehav Rev 1998;23:279 ± 86. Blanchard DC, Griebel G, Blanchard RJ. Gender bias in the preclinical psychopharmacology of anxiety: male models for (predominantly) female disorders. J Psychopharmacol 1995;9(2):79 ± 82. Gray JA, Levine S. Effects of induced estrus on emotional behavior of in selected strain of rats. Nature 1964;201:1198 ± 200. Mora S, Dussaubat N, Diaz-Veliz G. Effects of the estrous cycle and ovarian hormones on behavioral indices of anxiety in female rats. Psychoneuroendocrinology 1997;21(7):609 ± 20.

419

[41] Parmigiani S, Palanza P, Rodgers J, Ferrari PF. Selection, evolution of behavior and animal models in behavioral neuroscience. Neurosci Biobehav Rev 1999;23:957 ± 70. [42] Palanza P. Animal models of anxiety and depression: how are females different? Neurosci Biobehav Rev 2001;25:219 ± 233. [43] Blanchard DC, Blanchard RJ, Rodgers RJ. Risk assessment and animal models of anxiety. In: Olivier B, Mos J, Slangen JL, editors. Animal models in psychopharmacology. Advances in pharmacological sciences. Boston: Birkhauser Verlag Basel, 1991. pp. 117 ± 34. [44] Choleris E, Thomas AW, Kavaliers M, Prato FS. A detailed ethological analysis of the mouse open field test: effect of diazepam, chlorodiazepoxide and an extremely low frequency pulsed magnetic field. Neurosci Biobehav Rev 2001;25. [45] Rodgers RJ, Cole JC. The elevated plus-maze: pharmacology, methodology and ethology. In: Cooper SJ, Hendrie CA, editors. Ethology and psychopharmacology. Chichester: Wiley, 1994. pp. 9 ± 44. [46] Griebel G, Belzung C, Misslin R, Vogel E. The free-exploratory paradigm: an effective method for measuring neophobic behaviour in mice and testing potential neophobia-reducing drugs. Behav Pharmacol 1993;4:637 ± 44. [47] Berry RJ, Bronson FH. Life history and bioeconomy of the house mouse. Biol Rev 1992;67:519 ± 50. [48] Palanza P, Brain PF, Parmigiani S. Intraspecific aggression in mice (Mus domesticus): male and female strategies. In: Haug M, editor. The development of sex differences and similarities in behavior. The Netherlands: Kluwer Academic Publishing, 1993. pp. 191 ± 203. [49] Darwin CH. The descent of man and selection in relation to sex. London: Murray, 1871. [50] Trivers RL. Parental investment and sexual selection. In: Campbell B, editor. Sexual selection and the descent of man 1871 ± 1971. Chicago: Aldine, 1972. pp. 136 ± 179. [51] Bohus B, Koolhaas JM, de Ruiter AJH, Heinen CJ. Stress and differential alterations in immune functions: conclusions from social stress studies in animals. Ned J Med 1991;39:306 ± 15. [52] Hurst JL, Barnard CJ, Hare R, Wheeldon EB, West CD. Housing and welfare in laboratory rats: time-budgeting and pathophysiology in single-sex groups. Anim Behav 1996;52:335 ± 60. [53] Hilakivi LA, Ota M, Lister RG. Effect of isolation on brain monoamines and the behavior of mice in tests of exploration. Locomotion, anxiety and behavioral ``despair''. Pharmacol, Biochem Behav 1989;33:371 ± 4. [54] Parmigiani S, Pasquali A. Exploratory and aggressive behaviour of isolated, dominant and subordinated mice. Accad Sci Lett 1980;114: 3 ± 9. [55] Rodgers RJ, Cole JC. Influence of social isolation, gender, strain and prior novelty on plus-maze behaviour in mice. Physiol Behav 1993;54:729 ± 36. [56] Rowe FP, Redfern R. Aggressive behaviour in related and unrelated wild house mice (Mus musculus). Ann Appl Biol 1969;64:425 ± 31. [57] Kareem AM, Barnard CJ. The importance of kinship and familiarity in social interactions between mice. Anim Behav 1982;30:594 ± 601. [58] Holson RR, Scallet AC, Ali SF, Turner BB. ``Isolation stress'' revisited: isolation rearing effects depend on animal care methods. Physiol Behav 1991;49:1107 ± 18. [59] Misslin R, Herzog F, Koch B, Ropartz P. Effects of isolation, handling and novelty on the pituitary ± adrenal response in the mouse. Psychoneuroendocrinology 1982;7:217 ± 21. [60] Brain PF, Benton D. The interpretation of physiological correlates of differential housing in laboratory rats. Life Sci 1979;24:99 ± 116. [61] Jesberger JA, Richardson JS. Animal models of depression: parallels and correlates to severe depression in humans. Biol Psychiatry 1985;20:764 ± 84. [62] Brain PF, Benton D. What does individual housing mean to a research worker? IRCS J Med Sci 1977;5:459 ± 63. [63] Zimmerberg B, Farley MJ. Sex differences in anxiety behavior in rats: role of gonadal hormones. Physiol Behav 1993;54:1119 ± 24. [64] Butcher RL, Collins WE, Fugo NW. Plasma concentration of LH,

420


FSH, prolactin, progesterone and estradiol-17beta throughout the 4 day estrous cycle of the rat. Endocrinology 1974;94:1704 ± 8. [65] Rodriguez-Sierra JF, Howard JL, Pollard GT, Heindricks S. Effect of ovarian hormones on conflict behavior. Psychoneuroendocrinology 1984;9:293 ± 300.

[66] Rodgers RJ, Johnson NJT. Behaviorally selective effects of neuroactive steroids on plus-maze anxiety in mice. Pharmacol, Biochem Behav 1998;59(1):221 ± 32. [67] Weinberg J, Krahn EA, Levine S. Differential effects of handling on exploration in male and female rats. Dev Psychobiol 1978;11:3251 ± 9.