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Male aromatase-knockout mice exhibit normal levels of activity, anxiety and “depressive-like” symptomatology
Behavioural Brain Research 163 (2005) 186–193
Research report
Male aromatase-knockout mice exhibit normal levels of activity, anxiety and “depressive-like” symptomatology C. Dalla a,b , K. Antoniou b , Z. Papadopoulou-Daifoti b , J. Balthazart a , J. Bakker a,∗ a
Center for Cellular and Molecular Neurobiology, University of Li`ege, 17 Place Delcour, B-4020 Li`ege, Belgium b Department of Pharmacology, Medical School, University of Athens, Greece Received 24 March 2005; received in revised form 28 April 2005; accepted 29 April 2005 Available online 18 July 2005
Abstract It is well known that estradiol derived from neural aromatization of testosterone plays a crucial role in the development of the male brain and the display of sexual behaviors in adulthood. It was recently found that male aromatase knockout mice (ArKO) deficient in estradiol due to a mutation in the aromatase gene have general deficits in coital behavior and are sexually less motivated. We wondered whether these behavioral deficits of ArKO males could be related to changes in activity, exploration, anxiety and “depressive-like” symptomatology. ArKO and wild type (WT) males were subjected to open field (OF), elevated plus maze (EPM), and forced swim tests (FST), after being exposed or not to chronic mild stress (CMS). CMS was used to evaluate the impact of chronic stressful procedures and to unveil possible differences between genotypes. There was no effect of genotype on OF, EPM and FST behavioral parameters. WT and ArKO mice exposed to CMS or not exhibited the same behavioral profile during these three types of tests. However, all CMS-exposed mice (ArKO and WT) spent less time in the center of the EPM. Additionally, floating duration measured in the FST increased between two tests in both WT and ArKO mice, though that increase was less prominent in mice previously subjected to CMS than in controls. Therefore, both ArKO and WT males displayed the same behavior and had the same response to CMS however CMS exposure slightly modified the behavior displayed by mice of both genotypes in the FST and EPM paradigms. These results show that ArKO males display normal levels of activity, exploration, anxiety and “depressive-like” symptomatology and thus their deficits in sexual behavior are specific in nature and do not result indirectly from other behavioral changes. © 2005 Published by Elsevier B.V. Keywords: Aromatase knockout mice; Estrogen; Open field; Chronic mild stress; Forced swim test; Elevated plus maze test; Anxiety; Depression
1. Introduction It is well established that estradiol derived from neural aromatization of testosterone plays a crucial role in the development of the male brain and the display of sexual behaviors in adulthood [3,6,28,31]. This notion was further supported by the recent finding that male aromatase knockout mice (ArKO), deficient in estradiol due to a targeted mutation in the Cyp19 gene, show impaired sexual behavior and sexual motivation [5]. In particular, male ArKO mice rarely
Abbreviations: ArKO, aromatase knockout mice; CMS, chronic mild stress; EPM, elevated plus maze; FST, forced swim test; OF, open field; WT, wild-type ∗ Corresponding author. Tel.: +32 4 355 5978; fax: +32 4 366 5971. E-mail address: [email protected] (J. Bakker). 0166-4328/$ – see front matter © 2005 Published by Elsevier B.V. doi:10.1016/j.bbr.2005.04.020
displayed coital behavior when paired with an estrous female and showed a reduced interest in investigating olfactory and visual cues from conspecifics in a Y-maze [5]. These behavioral changes suggest that ArKO male mice have general deficits in sexual behavior including sexual preferences and sexual motivation due to the absence of estradiol. It was recently found that female ArKO mice exhibited similar deficits in sexual behavior [4] and that these deficits are associated with a “depressive-like” symptomatology, as suggested by their increased floating behavior in the forced swim test (FST) [16]. The behavioral deficits in the FST were not corrected by adult estradiol treatment indicating an organizational role of estradiol in the female brain. The presence of a “depressive-like” symptomatology in ArKO females opens the possibility that the deficits affecting their sexual behavior might indirectly result from a general
C. Dalla et al. / Behavioural Brain Research 163 (2005) 186–193
decrease in motivation. The present studies were therefore initiated to determine whether male ArKO mice also exhibit a “depressive-like” behavioral profile as previously observed in ArKO females [16]. To obtain a broad characterization of ArKO male mice, their levels of motor activity, exploration, anxiety and “depressive-like” symptomatology were quantified by well established procedures. Taking into consideration that prolonged exposure of rodents to stressors simulates “depressive-like” symptomatology [42,43], we also investigated whether the application of a chronic mild stress (CMS) procedure would differentially affect the behavior of ArKO and wild type (WT) males. Previous chronic exposure to stress could possibly unmask behavioral alterations in ArKO male mice, affecting motor activity, exploration, coping behavior, anxiety-like and “depressive-like” symptomatology.
2. Material and methods 2.1. Animals and general design Male ArKO mice were generated by target disruption of exons 1 and 2 of the Cyp19 gene [24]. Heterozygous males and females of the C57Bl6 strain were bred to generate wild type (WT) and homozygous-null (ArKO) offspring. Mice were genotyped by PCR analysis of tail DNA [5]. All breeding and genotyping were performed at the School of Veterinary Medicine, University of Li`ege, Belgium. Mice were 8–10 weeks old at the start of the experiment. All experimental mice were group-housed under a light–dark cycle (12:12 LD, lights off at 20:00). Food and water were always available ad libitum, unless required by the experimental procedure. All experiments were conducted in accordance with the guidelines set forth by the National Institutes of Health Guiding Principles for the Care and Use of Research Animals and were approved by the Ethical Committee for Animal Use of the University of Liege. The order and timing of the behavioral tests was selected in order to avoid, as much as possible, any interference between the tests. Specifically, an open field (OF) test was first used to investigate possible differences in activity and exploration [11] between ArKO and WT male mice. Then, one half of the ArKO and WT mice were subjected to CMS, a well-known animal model of depression [30] while other subjects were kept as controls. All mice were subsequently subjected to another OF test, and then to the FST and elevated plus maze (EPM) test. The FST was used to research behavioral indices that simulate “depressive-like” symptomatology [13,33] and to detect, in ArKO males previously subjected to CMS, possible alterations concerning coping with a novel stressor, such as FST. The EPM test was used to detect possible anxiety-related alterations [18] in ArKO males previously subjected to CMS or not. 2.2. Experimental procedure 2.2.1. Open field test All mice were first subjected to an OF test. Gonadally intact WT males (N = 10) and ArKO males (N = 12) were brought into the test room at least 1 h before the start of behavioral testing and remained in the same room throughout the test. The OF consisted of a Plexiglas arena (51 cm long × 18 cm high × 30 cm wide). At the beginning
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of the test, each male was placed in the middle of the arena and the duration of the following behaviors were recorded for 10 min: standing (sitting on all four feet), moving (walking on all four feet), rearing (body inclined vertically with hindpaws on the floor of the arena and forepaws either on the wall of the arena or in the open) and grooming (washing the face or any other part of its body with the forepaws) [11]. All behaviors displayed in the arena were analyzed continuously using a computerized technique for quantification of duration of each behavioral response during the 10 min observation period as described by Spruijt and Gispen [37] with some minor modifications [2]. Mice were tested individually during the light phase of the LD cycle (between 10:00 and 14:00 h) in a random order. The arena was cleaned with 70% ethanol to eliminate odors after each test. 2.2.2. Chronic mild stress At the end of the OF test, WT and ArKO male mice were subdivided into two subgroups: males of the first group were subjected to CMS whereas those of the second group remained in their home cage and served as controls. The CMS protocol lasted for four weekly cycles that consisted of two different alternating stressors per day [30] (see detailed program applied each week in Table 1). The stressors were: food or water deprivation, stroboscopic illumination (120 flashes/min), overnight illumination (lights on during the dark phase), cage tilting (45◦ ), soiled cage (50 ml of plain water into the sawdust bedding) followed by cage cleaning. Each stressor lasted 8–16 h. 2.2.3. Open field test following CMS In order to determine whether CMS affected general motor activity of WT and ArKO male mice, all subjects, i.e. CMS and control mice, were again tested for OF behavior for a 10 min period. The test was performed 24 h after the final CMS application using the same method as described above. 2.2.4. Forced swim test Two days later, CMS and control mice were subjected to two tests of the FST, with an interval of 24 h in between. The duration Table 1 Detail of the weekly experimental manipulations performed within the chronic mild stress (CMS) protocol Weekly CMS protocol Monday 10:00 Monday 18:00 Tuesday 10:00 Tuesday 20:00 Wednesday 10:00 Wednesday 20:00 Thursday 8:00 Thursday 18:00 Friday 10:00 Friday 20:00 Saturday 8:00 Sunday 8:00
Water deprivation for 8 h Soiled cage (150 ml of water was poured into the sawdust bedding) for 16 h Cage cleaning, followed by food deprivation for 8 h Overnight illumination (lights switched on during the dark phase) Water deprivation for 8 h Overnight illumination (lights switched on during the dark phase) Stroboscopic illumination for 10 h (lights switched off) Tilting of the cages backwards (45◦ ) for 16 h Food deprivation for 8 h Overnight illumination (lights switched on during the dark phase) Stroboscopic illumination for 10 h (lights switched off) Stroboscopic illumination for 10 h (lights switched off)
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of each test was chosen to be 5 min based on the observation that mice from the C57Bl6 strain quickly develop an immobile posture after the first swim test [34]. Mice were gently placed in a large glass cylinder (diameter 12 cm, height 20 cm) containing 1200 ml of water at 24 ± 2 ◦ C. This amount of water created a column of water that was high enough to prevent subjects from reaching the bottom of the cylinder with their tail. The diameter of the cylinder was chosen to be larger [1,27] than traditionally used for FST because it was reported that a larger diameter increases the predictive validity of the FST test in mice [33,38]. Males were tested individually under normal white lighting, during the light phase of the light–dark cycle (between 10:00 and 14:00 h) in a random order. The duration (in seconds) of each of the following behavioral responses was recorded: struggling = attempts at climbing the walls of the cylinder and jumping out of the water, mice were moving all four limbs with the two front limbs breaking the surface of the water or touching the walls; swimming = active swimming around in circles, and floating = total absence of movement or small movements of one of the posterior paws that do not produce displacement (paddling), in order to keep the mouse’s head above the water. After the FST, mice were removed from the cylinder, dried with towels, and placed in another cage to further dry before being returned to their home cages. The water in the cylinder was changed after each test. 2.2.5. Elevated plus maze test One week later, all subjects were tested for their behavior in the EPM. Control and CMS male mice were brought into the test room at least 1 h before the onset of behavioral testing and remained in the same room throughout the test. The EPM consisted of four arms (each arm 30 cm long × 15 cm high × 8 cm wide); two open and two closed arms formed a cross, which was raised 80 cm above the floor. At the beginning of the test, each mouse was placed in the center area and subsequently the time spent in the center, open and closed arms was recorded for 5 min. In addition the number of entries into either the open or closed arms was registered. Behavioral variables were recorded using a computerized method as mentioned before. It was considered that the mouse was in the open (or closed) arm when its four legs were not in the center area. Males were tested individually under normal white lighting, during the light phase of the light–dark cycle (between 10:00 and 14:00 h) in a random order. The maze was cleaned with 70% ethanol to eliminate odors after each test. 2.2.6. Statistical analysis Behavioral data derived from the OF and FST were analyzed by repeated measures ANOVA, with genotype and CMS exposure as independent factors and tests as repeated factor. When appropriate, repeated ANOVAs were followed by Tukey highest significant difference (HSD) post hoc comparisons adapted for repeated-measures ANOVA. Behavioral data derived from EPM were analyzed by twoway ANOVA, with genotype and CMS exposure as independent factors. All experimental data are presented in the figures as mean values ± SEM.
3. Results 3.1. Open field tests Three-way repeated ANOVAs of the behaviors expressed in the OF with genotype and CMS as independent factors
and tests (before and after CMS) as repeated factor revealed no effect of genotype or CMS (and no significant interaction between them) on standing and moving duration. Nevertheless, this analysis identified an effect of repeated testing on the duration of standing and moving [F(1,18) = 95.173, p < 0.001; F(1,18) = 89.134, p < 0.001, respectively], but no significant interaction of this factor with any of the others. Standing duration increased, while moving duration decreased over repeated testing in all mice (Fig. 1). There was no significant effect of genotype, CMS exposure or repeated testing on the duration of grooming and rearing (Fig. 1). The behaviors displayed in the OF were thus very similar between ArKO and WT males and did not appear to be affected by CMS exposure. In addition, the duration of standing and moving of all groups were affected in the same way by repeated testing. 3.2. Forced swim test Three-way repeated ANOVA of floating duration with genotype and CMS as independent factors and tests as repeated factor revealed a significant effect of repeated testing [F(1,18) = 127.791, p < 0.001], as well as an interaction of repeated testing with CMS [F(1,18) = 5.383, p = 0.032], but no significant effect of genotype, CMS or any other significant interaction. ArKO mice did not differ from WT in either test of FST. Post hoc analysis showed that floating duration increased over repeated testing in all mice, but mice previously subjected to CMS increased their floating duration from test 1 to test 2 less than controls (Fig. 2A). To better illustrate the changes in floating duration during repeated testing and their interaction with previous exposure to the CMS, data were transformed as a percentage of the duration expressed during test 1 and results for both genotypes were pooled since there was no significant effect of genotype nor did it interact with any of the other factors. As can be seen in Fig. 2A (bars), the increase in floating duration between test 1 and test 2 was almost 50% smaller in mice that had been subjected to CMS than in controls. Three-way ANOVAs of the struggling and swimming durations also showed a significant effect of repeated testing [F(1,18) = 5.771, p = 0.027; F(1,18) = 155.347, p < 0.001, respectively], but again no overall effect of genotype or CMS, and no significant interactions. In particular, there was here no interaction between repeated testing and exposure to CMS. Swimming and struggling duration decreased over repeated testing in all mice independently of genotype or previous exposure to CMS (Fig. 2B and C, respectively). 3.3. Elevated plus maze Two-way ANOVAs with genotype and CMS as factors revealed no difference between WT and ArKO males and no effect of previous exposure to CMS on the time spent in either the open arms or closed arms of the EPM (Fig. 3). There was also no significant interaction between these factors. Nevertheless, the same analysis revealed an effect of CMS
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Fig. 1. Means + S.E. for the duration of standing, moving, rearing and grooming in the first (before CMS) and second (after CMS) OF test for male WT and ArKO mice previously exposed to CMS or not. +, ++, +++ = p < 0.05, p < 0.01, p < 0.001, respectively for statistically significant difference between test 1 and 2 in the corresponding group.
on the time spent in the center of the EPM [F(1,21) = 4.821, p = 0.041]: both WT and ArKO mice previously exposed to CMS spent less time in the center of the EPM in comparison with WT and ARKO control mice (Fig. 3). Furthermore, the number of entries in the closed and open arms and in the center did not differ between ArKO and WT males nor was it affected by previous exposure to CMS (Fig. 3).
4. Discussion The present study shows that male ArKO mice exhibited similar levels of motor activity, exploration, anxiety-like and “depressive-like” symptomatology compared to WT males, as implied by their results in the OF, FST and EPM tests. Furthermore, CMS had the same impact on ArKO and WT males, since the aforementioned behavioral variables did not differ between the two genotypes following exposure to CMS. Nevertheless, CMS slightly modified the behavioral performance of both WT and ArKO mice, displayed in the FST and EPM, thus confirming its behavioral efficacy. These findings provide important new information allowing a better characterization of the behavioral phenotype of male ArKO mice. 4.1. The behavioral profile of ArKO males ArKO and WT males exhibited similar levels of motor activity, exploration, anxiety-like and “depressive-like”
symptomatology. In particular, ArKO males did not show any changes in the duration of standing, moving, rearing and grooming in the OF test indicating similar levels of general motor and exploratory activity in a novel environment as WT males. Bakker et al. [5] previously reported a decreased activity in ArKO males compared to heterozygous subjects based on the number of lines crossed during a different form of an OF test (the difference with WT males did not reach significance). The discrepancy between these and the present results is potentially explained by the fact that a more detailed analysis of behavior was used here (measure of actual duration of movements rather than number of lines crossed) providing more accurate information on the behavior of the subjects in an OF. WT and ArKO mice exhibited similar behaviors in the FST indicating that they display the same levels of “depressivelike” symptomatology and/or coping behavior [13,33]. It is important to mention that female ArKO mice, in contrast with ArKO males, exhibited an increased “depressive-like” symptomatology in the FST when compared with WT females, and that remained elevated over four consecutive tests [16]. The increase in floating duration, along with deficits in sexual behavior [4] was not reversed by estrogen treatment in adulthood pointing to important organizational effects of estrogens on the female brain. Similar alterations in “depressive-like” and/or coping behavior do not seem to be present in ArKO males indicating that such general changes in behavior cannot explain the deficits in male copulatory behavior that have
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Finally, in the EPM test, which has been widely used to evaluate anxiety in rodents [18], ArKO males did not spend more time in the open or closed arms as WT males. Thus, ArKO males appeared to exhibit comparable levels of motor activity, exploration and anxiety-like behavior as WT males. These findings are in general agreement with previous results concerning other strains of male knockout mice that are lacking estrogen receptors of the alpha or beta subtype and that were shown to exhibit normal levels of behavior in the OF [32] and EPM [26]. Taken together, these studies indicate that although activational and organizational effects of estrogens are very important for the display of male sexual behavior [28], evidence for a role of this steroid in mediating non-reproductive behaviors in the male is poor. It has however been suggested that these behaviors could be influenced by the action of non-aromatizable androgens such as dihydrotestosterone [9,19,35]. 4.2. Response to CMS is not affected in ArKO male mice CMS had no differential impact on any of the behaviors measured in the OF, FST, and EPM of ArKO or WT mice, suggesting that both genotypes responded in the same way to CMS. Taking into consideration that CMS is a well-known animal model of depression which has been broadly used for studying depression-related behaviors in knockout mice [14,33], the non-response of ArKO males to CMS strongly supports the notion that these mice do not suffer from any “depressive-like” symptomatology. Fig. 2. (A) Means + S.E. for the duration of floating in the two consecutive tests of the FST for male WT and ArKO mice previously exposed to CMS or not. Means + S.E. for the % increase in floating duration in test 2 for control and CMS mice. +, ++, +++ = p < 0.05, p < 0.01, p < 0.001, respectively for statistically significant difference between test 1 and 2. (B) Means + S.E. for the duration of swimming in the two consecutive tests of the FST for male WT and ArKO mice previously exposed to CMS or not. +, ++, +++ = p < 0.05, p < 0.01, p < 0.001, respectively for statistically significant difference between test 1 and 2. (C) Means + S.E. for the duration of struggling in the two consecutive tests of the FST for male WT and ArKO mice previously exposed to CMS or not.
been identified in these males. Furthermore, since ArKO males have been deprived of estrogens like ArKO females, these results imply additional gender-dependent factors that interact with estrogens in the display of “depressive-like” symptomatology. The mechanisms underlying this sexually differentiated behavioral response to the deletion of the aromatase gene is unclear at present but may relate to compensatory mechanisms triggered by testosterone in males or to the differential location of the behaviorally relevant aromatase in the two sexes (essentially in the brain for males and in the ovary for the females). Additional studies should be designed to specifically address this topic and determine the behavioral significance, if any, of brain aromatase activity in females.
4.3. The impact of CMS on subsequent behavioral responses CMS application in male mice did not influence any behavioral measurements in the OF which is in agreement with findings previously reported by Duncko et al. [17]. Other studies reported either decreased [15,22,25,36] or increased [7,41] motor activity and/or exploration after exposure of male rodents to CMS, showing that the effects of CMS on OF behavior are not consistent and depend strongly on the animals and methods used in each study. CMS had a slight effect on the behavior expressed in the FST in male mice. As expected, all mice increased their duration of floating during the second test of FST, but this increase was less pronounced in mice previously subjected to CMS than in controls. Increased floating behavior in the second test of the FST has been characterized as an index of “depressive-like” symptomatology in rodents [1,34]. Alternatively, floating duration has been also considered as an index of coping strategy that is essential for adaptation to the stressful procedure [10,40]. It can thus be suggested that mice previously subjected to CMS respond differently to a novel stressful procedure, such as the FST, and exhibit an altered coping behavior in comparison with controls. Studies in male rodents have shown that previous exposure to CMS
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Fig. 3. Means + S.E. of the time spent in the open arms, closed arms or center of the EPM and numbers of entries in the open arms, closed arms and center of the EPM for male WT and ArKO mice previously exposed to CMS or not. # = p < 0.05: statistically significant difference between control and CMS-exposed mice.
increases [8,15,20,29,39] or decreases [21,23] passive behaviors, such as floating, in the FST, suggesting a variable effect of chronic stress on coping strategies and response to novel stressors. Previous exposure of male mice to CMS also resulted in a decrease in the time spent in the center of the EPM. The number of entries and the time spent in the closed arms of the EPM has been associated with anxiety-like behavior, while the number of entries and time spent in open arms is supposed to reflect the absence of anxiety [12,18]. In addition, the time spent in the center of the EPM is also a measure of anxietyrelated behavior, though less specific, since mice are more exposed in the center of the EPM than near the walls and generally do not prefer this place [12]. Therefore, it could be suggested that mice exposed to the CMS were somewhat more anxious since they tended to spend less time in the center of the EPM. Future work will have to address the question of whether these behavioral alterations following CMS are only transient and strongly associated with the sequence of manipulations experienced by the mice or whether they result from more permanent modifications that would have affected
in a more or less irreversible manner the brain and behavior of the subjects. 4.3.1. Concluding remarks In conclusion, our findings show that ArKO male mice exhibit normal levels of motor activity, exploration, anxietylike and “depressive-like” symptomatology, despite their known deficits in sexual behavior [5]. Moreover, the finding that CMS had the same impact in ArKO and WT males further confirms the notion that these mice do not exhibit any behavioral alterations related with response to stress and depression. These findings do not, however, exclude a potential role for estrogens in the control of these behaviors. One must indeed consider that compensatory mechanisms during the development of the brain in male ArKO mice could lead to a normalization of the aforementioned behaviors despite the chronic lack of exposure to estrogens [14]. Interestingly, the present study differentiates the behavioral profile of ArKO males from that of ArKO females which have been shown to exhibit “depressive-like” symptomatology [16] in addition to their deficits in sexual behavior [4]. In
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contrast, no sex differences were observed in general activity and anxiety-like behavior of ArKO mice, since both males (present study) and females [16] displayed normal patterns of behavior in the OF and EPM tests. The comparison of these two studies therefore suggests the existence of a sexual differentiation in the type of behaviors modulated by estrogens and this finding deserves further investigation. The present results thus contribute to the understanding of the role of estrogens in the establishment of sexually differentiated behaviors shown both in a reproductive and non reproductive context. The distinct role in males and females of estrogens on mood and mental state could possibly underline the sex differences in the appearance, course and treatment of affective disorders.
Acknowledgements The authors would like to thank Sandrine Seyen and Christel Dejace for their excellent technical assistance. We also thank Drs. N. Harada and S. Honda for providing us with their strain of ArKO mice. This research was supported by a Marie Curie fellowship to C. Dalla within the EC Programme “Quality of Life and Management of Living Resources” under contract number QLK6-CT-2000-60042 and fellow reference number QLK6-GH-00-60042-03. It was also supported by a grant from the National Institutes of Child Health and Human Development (HD044897) and by a Belgian grant from the Fonds National de la Recherche Scientifique to J. Bakker and by grants from the Government of the French Community of Belgium (ARC 99/04-241) and the National Institutes of Mental Health (MH50388) to J. Balthazart.
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Research report
Male aromatase-knockout mice exhibit normal levels of activity, anxiety and “depressive-like” symptomatology C. Dalla a,b , K. Antoniou b , Z. Papadopoulou-Daifoti b , J. Balthazart a , J. Bakker a,∗ a
Center for Cellular and Molecular Neurobiology, University of Li`ege, 17 Place Delcour, B-4020 Li`ege, Belgium b Department of Pharmacology, Medical School, University of Athens, Greece Received 24 March 2005; received in revised form 28 April 2005; accepted 29 April 2005 Available online 18 July 2005
Abstract It is well known that estradiol derived from neural aromatization of testosterone plays a crucial role in the development of the male brain and the display of sexual behaviors in adulthood. It was recently found that male aromatase knockout mice (ArKO) deficient in estradiol due to a mutation in the aromatase gene have general deficits in coital behavior and are sexually less motivated. We wondered whether these behavioral deficits of ArKO males could be related to changes in activity, exploration, anxiety and “depressive-like” symptomatology. ArKO and wild type (WT) males were subjected to open field (OF), elevated plus maze (EPM), and forced swim tests (FST), after being exposed or not to chronic mild stress (CMS). CMS was used to evaluate the impact of chronic stressful procedures and to unveil possible differences between genotypes. There was no effect of genotype on OF, EPM and FST behavioral parameters. WT and ArKO mice exposed to CMS or not exhibited the same behavioral profile during these three types of tests. However, all CMS-exposed mice (ArKO and WT) spent less time in the center of the EPM. Additionally, floating duration measured in the FST increased between two tests in both WT and ArKO mice, though that increase was less prominent in mice previously subjected to CMS than in controls. Therefore, both ArKO and WT males displayed the same behavior and had the same response to CMS however CMS exposure slightly modified the behavior displayed by mice of both genotypes in the FST and EPM paradigms. These results show that ArKO males display normal levels of activity, exploration, anxiety and “depressive-like” symptomatology and thus their deficits in sexual behavior are specific in nature and do not result indirectly from other behavioral changes. © 2005 Published by Elsevier B.V. Keywords: Aromatase knockout mice; Estrogen; Open field; Chronic mild stress; Forced swim test; Elevated plus maze test; Anxiety; Depression
1. Introduction It is well established that estradiol derived from neural aromatization of testosterone plays a crucial role in the development of the male brain and the display of sexual behaviors in adulthood [3,6,28,31]. This notion was further supported by the recent finding that male aromatase knockout mice (ArKO), deficient in estradiol due to a targeted mutation in the Cyp19 gene, show impaired sexual behavior and sexual motivation [5]. In particular, male ArKO mice rarely
Abbreviations: ArKO, aromatase knockout mice; CMS, chronic mild stress; EPM, elevated plus maze; FST, forced swim test; OF, open field; WT, wild-type ∗ Corresponding author. Tel.: +32 4 355 5978; fax: +32 4 366 5971. E-mail address: [email protected] (J. Bakker). 0166-4328/$ – see front matter © 2005 Published by Elsevier B.V. doi:10.1016/j.bbr.2005.04.020
displayed coital behavior when paired with an estrous female and showed a reduced interest in investigating olfactory and visual cues from conspecifics in a Y-maze [5]. These behavioral changes suggest that ArKO male mice have general deficits in sexual behavior including sexual preferences and sexual motivation due to the absence of estradiol. It was recently found that female ArKO mice exhibited similar deficits in sexual behavior [4] and that these deficits are associated with a “depressive-like” symptomatology, as suggested by their increased floating behavior in the forced swim test (FST) [16]. The behavioral deficits in the FST were not corrected by adult estradiol treatment indicating an organizational role of estradiol in the female brain. The presence of a “depressive-like” symptomatology in ArKO females opens the possibility that the deficits affecting their sexual behavior might indirectly result from a general
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decrease in motivation. The present studies were therefore initiated to determine whether male ArKO mice also exhibit a “depressive-like” behavioral profile as previously observed in ArKO females [16]. To obtain a broad characterization of ArKO male mice, their levels of motor activity, exploration, anxiety and “depressive-like” symptomatology were quantified by well established procedures. Taking into consideration that prolonged exposure of rodents to stressors simulates “depressive-like” symptomatology [42,43], we also investigated whether the application of a chronic mild stress (CMS) procedure would differentially affect the behavior of ArKO and wild type (WT) males. Previous chronic exposure to stress could possibly unmask behavioral alterations in ArKO male mice, affecting motor activity, exploration, coping behavior, anxiety-like and “depressive-like” symptomatology.
2. Material and methods 2.1. Animals and general design Male ArKO mice were generated by target disruption of exons 1 and 2 of the Cyp19 gene [24]. Heterozygous males and females of the C57Bl6 strain were bred to generate wild type (WT) and homozygous-null (ArKO) offspring. Mice were genotyped by PCR analysis of tail DNA [5]. All breeding and genotyping were performed at the School of Veterinary Medicine, University of Li`ege, Belgium. Mice were 8–10 weeks old at the start of the experiment. All experimental mice were group-housed under a light–dark cycle (12:12 LD, lights off at 20:00). Food and water were always available ad libitum, unless required by the experimental procedure. All experiments were conducted in accordance with the guidelines set forth by the National Institutes of Health Guiding Principles for the Care and Use of Research Animals and were approved by the Ethical Committee for Animal Use of the University of Liege. The order and timing of the behavioral tests was selected in order to avoid, as much as possible, any interference between the tests. Specifically, an open field (OF) test was first used to investigate possible differences in activity and exploration [11] between ArKO and WT male mice. Then, one half of the ArKO and WT mice were subjected to CMS, a well-known animal model of depression [30] while other subjects were kept as controls. All mice were subsequently subjected to another OF test, and then to the FST and elevated plus maze (EPM) test. The FST was used to research behavioral indices that simulate “depressive-like” symptomatology [13,33] and to detect, in ArKO males previously subjected to CMS, possible alterations concerning coping with a novel stressor, such as FST. The EPM test was used to detect possible anxiety-related alterations [18] in ArKO males previously subjected to CMS or not. 2.2. Experimental procedure 2.2.1. Open field test All mice were first subjected to an OF test. Gonadally intact WT males (N = 10) and ArKO males (N = 12) were brought into the test room at least 1 h before the start of behavioral testing and remained in the same room throughout the test. The OF consisted of a Plexiglas arena (51 cm long × 18 cm high × 30 cm wide). At the beginning
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of the test, each male was placed in the middle of the arena and the duration of the following behaviors were recorded for 10 min: standing (sitting on all four feet), moving (walking on all four feet), rearing (body inclined vertically with hindpaws on the floor of the arena and forepaws either on the wall of the arena or in the open) and grooming (washing the face or any other part of its body with the forepaws) [11]. All behaviors displayed in the arena were analyzed continuously using a computerized technique for quantification of duration of each behavioral response during the 10 min observation period as described by Spruijt and Gispen [37] with some minor modifications [2]. Mice were tested individually during the light phase of the LD cycle (between 10:00 and 14:00 h) in a random order. The arena was cleaned with 70% ethanol to eliminate odors after each test. 2.2.2. Chronic mild stress At the end of the OF test, WT and ArKO male mice were subdivided into two subgroups: males of the first group were subjected to CMS whereas those of the second group remained in their home cage and served as controls. The CMS protocol lasted for four weekly cycles that consisted of two different alternating stressors per day [30] (see detailed program applied each week in Table 1). The stressors were: food or water deprivation, stroboscopic illumination (120 flashes/min), overnight illumination (lights on during the dark phase), cage tilting (45◦ ), soiled cage (50 ml of plain water into the sawdust bedding) followed by cage cleaning. Each stressor lasted 8–16 h. 2.2.3. Open field test following CMS In order to determine whether CMS affected general motor activity of WT and ArKO male mice, all subjects, i.e. CMS and control mice, were again tested for OF behavior for a 10 min period. The test was performed 24 h after the final CMS application using the same method as described above. 2.2.4. Forced swim test Two days later, CMS and control mice were subjected to two tests of the FST, with an interval of 24 h in between. The duration Table 1 Detail of the weekly experimental manipulations performed within the chronic mild stress (CMS) protocol Weekly CMS protocol Monday 10:00 Monday 18:00 Tuesday 10:00 Tuesday 20:00 Wednesday 10:00 Wednesday 20:00 Thursday 8:00 Thursday 18:00 Friday 10:00 Friday 20:00 Saturday 8:00 Sunday 8:00
Water deprivation for 8 h Soiled cage (150 ml of water was poured into the sawdust bedding) for 16 h Cage cleaning, followed by food deprivation for 8 h Overnight illumination (lights switched on during the dark phase) Water deprivation for 8 h Overnight illumination (lights switched on during the dark phase) Stroboscopic illumination for 10 h (lights switched off) Tilting of the cages backwards (45◦ ) for 16 h Food deprivation for 8 h Overnight illumination (lights switched on during the dark phase) Stroboscopic illumination for 10 h (lights switched off) Stroboscopic illumination for 10 h (lights switched off)
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of each test was chosen to be 5 min based on the observation that mice from the C57Bl6 strain quickly develop an immobile posture after the first swim test [34]. Mice were gently placed in a large glass cylinder (diameter 12 cm, height 20 cm) containing 1200 ml of water at 24 ± 2 ◦ C. This amount of water created a column of water that was high enough to prevent subjects from reaching the bottom of the cylinder with their tail. The diameter of the cylinder was chosen to be larger [1,27] than traditionally used for FST because it was reported that a larger diameter increases the predictive validity of the FST test in mice [33,38]. Males were tested individually under normal white lighting, during the light phase of the light–dark cycle (between 10:00 and 14:00 h) in a random order. The duration (in seconds) of each of the following behavioral responses was recorded: struggling = attempts at climbing the walls of the cylinder and jumping out of the water, mice were moving all four limbs with the two front limbs breaking the surface of the water or touching the walls; swimming = active swimming around in circles, and floating = total absence of movement or small movements of one of the posterior paws that do not produce displacement (paddling), in order to keep the mouse’s head above the water. After the FST, mice were removed from the cylinder, dried with towels, and placed in another cage to further dry before being returned to their home cages. The water in the cylinder was changed after each test. 2.2.5. Elevated plus maze test One week later, all subjects were tested for their behavior in the EPM. Control and CMS male mice were brought into the test room at least 1 h before the onset of behavioral testing and remained in the same room throughout the test. The EPM consisted of four arms (each arm 30 cm long × 15 cm high × 8 cm wide); two open and two closed arms formed a cross, which was raised 80 cm above the floor. At the beginning of the test, each mouse was placed in the center area and subsequently the time spent in the center, open and closed arms was recorded for 5 min. In addition the number of entries into either the open or closed arms was registered. Behavioral variables were recorded using a computerized method as mentioned before. It was considered that the mouse was in the open (or closed) arm when its four legs were not in the center area. Males were tested individually under normal white lighting, during the light phase of the light–dark cycle (between 10:00 and 14:00 h) in a random order. The maze was cleaned with 70% ethanol to eliminate odors after each test. 2.2.6. Statistical analysis Behavioral data derived from the OF and FST were analyzed by repeated measures ANOVA, with genotype and CMS exposure as independent factors and tests as repeated factor. When appropriate, repeated ANOVAs were followed by Tukey highest significant difference (HSD) post hoc comparisons adapted for repeated-measures ANOVA. Behavioral data derived from EPM were analyzed by twoway ANOVA, with genotype and CMS exposure as independent factors. All experimental data are presented in the figures as mean values ± SEM.
3. Results 3.1. Open field tests Three-way repeated ANOVAs of the behaviors expressed in the OF with genotype and CMS as independent factors
and tests (before and after CMS) as repeated factor revealed no effect of genotype or CMS (and no significant interaction between them) on standing and moving duration. Nevertheless, this analysis identified an effect of repeated testing on the duration of standing and moving [F(1,18) = 95.173, p < 0.001; F(1,18) = 89.134, p < 0.001, respectively], but no significant interaction of this factor with any of the others. Standing duration increased, while moving duration decreased over repeated testing in all mice (Fig. 1). There was no significant effect of genotype, CMS exposure or repeated testing on the duration of grooming and rearing (Fig. 1). The behaviors displayed in the OF were thus very similar between ArKO and WT males and did not appear to be affected by CMS exposure. In addition, the duration of standing and moving of all groups were affected in the same way by repeated testing. 3.2. Forced swim test Three-way repeated ANOVA of floating duration with genotype and CMS as independent factors and tests as repeated factor revealed a significant effect of repeated testing [F(1,18) = 127.791, p < 0.001], as well as an interaction of repeated testing with CMS [F(1,18) = 5.383, p = 0.032], but no significant effect of genotype, CMS or any other significant interaction. ArKO mice did not differ from WT in either test of FST. Post hoc analysis showed that floating duration increased over repeated testing in all mice, but mice previously subjected to CMS increased their floating duration from test 1 to test 2 less than controls (Fig. 2A). To better illustrate the changes in floating duration during repeated testing and their interaction with previous exposure to the CMS, data were transformed as a percentage of the duration expressed during test 1 and results for both genotypes were pooled since there was no significant effect of genotype nor did it interact with any of the other factors. As can be seen in Fig. 2A (bars), the increase in floating duration between test 1 and test 2 was almost 50% smaller in mice that had been subjected to CMS than in controls. Three-way ANOVAs of the struggling and swimming durations also showed a significant effect of repeated testing [F(1,18) = 5.771, p = 0.027; F(1,18) = 155.347, p < 0.001, respectively], but again no overall effect of genotype or CMS, and no significant interactions. In particular, there was here no interaction between repeated testing and exposure to CMS. Swimming and struggling duration decreased over repeated testing in all mice independently of genotype or previous exposure to CMS (Fig. 2B and C, respectively). 3.3. Elevated plus maze Two-way ANOVAs with genotype and CMS as factors revealed no difference between WT and ArKO males and no effect of previous exposure to CMS on the time spent in either the open arms or closed arms of the EPM (Fig. 3). There was also no significant interaction between these factors. Nevertheless, the same analysis revealed an effect of CMS
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Fig. 1. Means + S.E. for the duration of standing, moving, rearing and grooming in the first (before CMS) and second (after CMS) OF test for male WT and ArKO mice previously exposed to CMS or not. +, ++, +++ = p < 0.05, p < 0.01, p < 0.001, respectively for statistically significant difference between test 1 and 2 in the corresponding group.
on the time spent in the center of the EPM [F(1,21) = 4.821, p = 0.041]: both WT and ArKO mice previously exposed to CMS spent less time in the center of the EPM in comparison with WT and ARKO control mice (Fig. 3). Furthermore, the number of entries in the closed and open arms and in the center did not differ between ArKO and WT males nor was it affected by previous exposure to CMS (Fig. 3).
4. Discussion The present study shows that male ArKO mice exhibited similar levels of motor activity, exploration, anxiety-like and “depressive-like” symptomatology compared to WT males, as implied by their results in the OF, FST and EPM tests. Furthermore, CMS had the same impact on ArKO and WT males, since the aforementioned behavioral variables did not differ between the two genotypes following exposure to CMS. Nevertheless, CMS slightly modified the behavioral performance of both WT and ArKO mice, displayed in the FST and EPM, thus confirming its behavioral efficacy. These findings provide important new information allowing a better characterization of the behavioral phenotype of male ArKO mice. 4.1. The behavioral profile of ArKO males ArKO and WT males exhibited similar levels of motor activity, exploration, anxiety-like and “depressive-like”
symptomatology. In particular, ArKO males did not show any changes in the duration of standing, moving, rearing and grooming in the OF test indicating similar levels of general motor and exploratory activity in a novel environment as WT males. Bakker et al. [5] previously reported a decreased activity in ArKO males compared to heterozygous subjects based on the number of lines crossed during a different form of an OF test (the difference with WT males did not reach significance). The discrepancy between these and the present results is potentially explained by the fact that a more detailed analysis of behavior was used here (measure of actual duration of movements rather than number of lines crossed) providing more accurate information on the behavior of the subjects in an OF. WT and ArKO mice exhibited similar behaviors in the FST indicating that they display the same levels of “depressivelike” symptomatology and/or coping behavior [13,33]. It is important to mention that female ArKO mice, in contrast with ArKO males, exhibited an increased “depressive-like” symptomatology in the FST when compared with WT females, and that remained elevated over four consecutive tests [16]. The increase in floating duration, along with deficits in sexual behavior [4] was not reversed by estrogen treatment in adulthood pointing to important organizational effects of estrogens on the female brain. Similar alterations in “depressive-like” and/or coping behavior do not seem to be present in ArKO males indicating that such general changes in behavior cannot explain the deficits in male copulatory behavior that have
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Finally, in the EPM test, which has been widely used to evaluate anxiety in rodents [18], ArKO males did not spend more time in the open or closed arms as WT males. Thus, ArKO males appeared to exhibit comparable levels of motor activity, exploration and anxiety-like behavior as WT males. These findings are in general agreement with previous results concerning other strains of male knockout mice that are lacking estrogen receptors of the alpha or beta subtype and that were shown to exhibit normal levels of behavior in the OF [32] and EPM [26]. Taken together, these studies indicate that although activational and organizational effects of estrogens are very important for the display of male sexual behavior [28], evidence for a role of this steroid in mediating non-reproductive behaviors in the male is poor. It has however been suggested that these behaviors could be influenced by the action of non-aromatizable androgens such as dihydrotestosterone [9,19,35]. 4.2. Response to CMS is not affected in ArKO male mice CMS had no differential impact on any of the behaviors measured in the OF, FST, and EPM of ArKO or WT mice, suggesting that both genotypes responded in the same way to CMS. Taking into consideration that CMS is a well-known animal model of depression which has been broadly used for studying depression-related behaviors in knockout mice [14,33], the non-response of ArKO males to CMS strongly supports the notion that these mice do not suffer from any “depressive-like” symptomatology. Fig. 2. (A) Means + S.E. for the duration of floating in the two consecutive tests of the FST for male WT and ArKO mice previously exposed to CMS or not. Means + S.E. for the % increase in floating duration in test 2 for control and CMS mice. +, ++, +++ = p < 0.05, p < 0.01, p < 0.001, respectively for statistically significant difference between test 1 and 2. (B) Means + S.E. for the duration of swimming in the two consecutive tests of the FST for male WT and ArKO mice previously exposed to CMS or not. +, ++, +++ = p < 0.05, p < 0.01, p < 0.001, respectively for statistically significant difference between test 1 and 2. (C) Means + S.E. for the duration of struggling in the two consecutive tests of the FST for male WT and ArKO mice previously exposed to CMS or not.
been identified in these males. Furthermore, since ArKO males have been deprived of estrogens like ArKO females, these results imply additional gender-dependent factors that interact with estrogens in the display of “depressive-like” symptomatology. The mechanisms underlying this sexually differentiated behavioral response to the deletion of the aromatase gene is unclear at present but may relate to compensatory mechanisms triggered by testosterone in males or to the differential location of the behaviorally relevant aromatase in the two sexes (essentially in the brain for males and in the ovary for the females). Additional studies should be designed to specifically address this topic and determine the behavioral significance, if any, of brain aromatase activity in females.
4.3. The impact of CMS on subsequent behavioral responses CMS application in male mice did not influence any behavioral measurements in the OF which is in agreement with findings previously reported by Duncko et al. [17]. Other studies reported either decreased [15,22,25,36] or increased [7,41] motor activity and/or exploration after exposure of male rodents to CMS, showing that the effects of CMS on OF behavior are not consistent and depend strongly on the animals and methods used in each study. CMS had a slight effect on the behavior expressed in the FST in male mice. As expected, all mice increased their duration of floating during the second test of FST, but this increase was less pronounced in mice previously subjected to CMS than in controls. Increased floating behavior in the second test of the FST has been characterized as an index of “depressive-like” symptomatology in rodents [1,34]. Alternatively, floating duration has been also considered as an index of coping strategy that is essential for adaptation to the stressful procedure [10,40]. It can thus be suggested that mice previously subjected to CMS respond differently to a novel stressful procedure, such as the FST, and exhibit an altered coping behavior in comparison with controls. Studies in male rodents have shown that previous exposure to CMS
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Fig. 3. Means + S.E. of the time spent in the open arms, closed arms or center of the EPM and numbers of entries in the open arms, closed arms and center of the EPM for male WT and ArKO mice previously exposed to CMS or not. # = p < 0.05: statistically significant difference between control and CMS-exposed mice.
increases [8,15,20,29,39] or decreases [21,23] passive behaviors, such as floating, in the FST, suggesting a variable effect of chronic stress on coping strategies and response to novel stressors. Previous exposure of male mice to CMS also resulted in a decrease in the time spent in the center of the EPM. The number of entries and the time spent in the closed arms of the EPM has been associated with anxiety-like behavior, while the number of entries and time spent in open arms is supposed to reflect the absence of anxiety [12,18]. In addition, the time spent in the center of the EPM is also a measure of anxietyrelated behavior, though less specific, since mice are more exposed in the center of the EPM than near the walls and generally do not prefer this place [12]. Therefore, it could be suggested that mice exposed to the CMS were somewhat more anxious since they tended to spend less time in the center of the EPM. Future work will have to address the question of whether these behavioral alterations following CMS are only transient and strongly associated with the sequence of manipulations experienced by the mice or whether they result from more permanent modifications that would have affected
in a more or less irreversible manner the brain and behavior of the subjects. 4.3.1. Concluding remarks In conclusion, our findings show that ArKO male mice exhibit normal levels of motor activity, exploration, anxietylike and “depressive-like” symptomatology, despite their known deficits in sexual behavior [5]. Moreover, the finding that CMS had the same impact in ArKO and WT males further confirms the notion that these mice do not exhibit any behavioral alterations related with response to stress and depression. These findings do not, however, exclude a potential role for estrogens in the control of these behaviors. One must indeed consider that compensatory mechanisms during the development of the brain in male ArKO mice could lead to a normalization of the aforementioned behaviors despite the chronic lack of exposure to estrogens [14]. Interestingly, the present study differentiates the behavioral profile of ArKO males from that of ArKO females which have been shown to exhibit “depressive-like” symptomatology [16] in addition to their deficits in sexual behavior [4]. In
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contrast, no sex differences were observed in general activity and anxiety-like behavior of ArKO mice, since both males (present study) and females [16] displayed normal patterns of behavior in the OF and EPM tests. The comparison of these two studies therefore suggests the existence of a sexual differentiation in the type of behaviors modulated by estrogens and this finding deserves further investigation. The present results thus contribute to the understanding of the role of estrogens in the establishment of sexually differentiated behaviors shown both in a reproductive and non reproductive context. The distinct role in males and females of estrogens on mood and mental state could possibly underline the sex differences in the appearance, course and treatment of affective disorders.
Acknowledgements The authors would like to thank Sandrine Seyen and Christel Dejace for their excellent technical assistance. We also thank Drs. N. Harada and S. Honda for providing us with their strain of ArKO mice. This research was supported by a Marie Curie fellowship to C. Dalla within the EC Programme “Quality of Life and Management of Living Resources” under contract number QLK6-CT-2000-60042 and fellow reference number QLK6-GH-00-60042-03. It was also supported by a grant from the National Institutes of Child Health and Human Development (HD044897) and by a Belgian grant from the Fonds National de la Recherche Scientifique to J. Bakker and by grants from the Government of the French Community of Belgium (ARC 99/04-241) and the National Institutes of Mental Health (MH50388) to J. Balthazart.
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