Estrogen Increases Locomotor Activity in Mice through Estrogen Receptor : Specificity for the Type of Activity
Abstract2q, http://www.100md.com
Estrogens are known to increase running wheel activity of rodents primarily by acting on the medial preoptic area (mPOA). The mechanisms of this estrogenic regulation of running wheel activity are not completely understood. In particular, little is known about the separate roles of two types of estrogen receptors, ER{alpha} and ERß, both of which are expressed in mPOA neurons. In the present study the effects of continuous estrogen treatment on running wheel activity were examined in male and female mice specifically lacking either the ER (ERKO) or the ERß (ßERKO) gene. Mice were gonadectomized and 1 wk later implanted with either a low dose (16 ng/d) or a high dose (160 ng/d) of estradiol benzoate (EB) or with a placebo control pellet. Home cage running wheel activity was recorded for 9 d starting 10 d after EB implants. The same mice were also tested for open field activity before and after EB implants. In both female and male {alpha} ERKO mice, running wheel activity was not different from that in corresponding wild-type (WT) mice in placebo control groups. In both females and males it was increased by EB only in WT, not ERKO, mice. In ßERKO mice, on the other hand, both doses of EB equally increased running wheel activity in both sexes just as they did in ßWT mice. Absolute numbers of daily revolutions of EB-treated groups, however, were significantly lower in ßERKO females compared with ßWT females. Before EB treatment, gonadectomized ERKO female were significantly less active than WT mice in open field tests, whereas ßERKO females tended to be more active than ßWT mice. In male mice there were no effect of ER or ERß gene knockout on open field activity. Unlike its effect on running wheel activity, EB treatment induced only a small increase in open field activity in female, but not male, mice. These findings indicate that 1) in both sexes estrogenic regulation of running wheel activity is primarily mediated through the ER{alpha} , not the ERß; and 2) hormone/genotype effects are specific to the type of locomotor activity (i.e. home cage running wheel activity and open field activity) measured.
Introductionwdd, 百拇医药
IT IS WELL established that estrogen regulates running wheel activity in female and male rats. Gonadally intact female rats show the highest activity during proestrus, when plasma levels of estrogen are elevated (1). In gonadectomized female and male rats, it has been shown that estrogen treatment increases running wheel activity (2, 3, 4, 5), although estrogen failed to do so in ferrets of both sexes (6). A number of lesion (7), electrophysiological (8), and site-specific steroid implant studies (9) revealed that the medial preoptic area (mPOA) is the primary brain site responsible for this behavioral effect of estrogen, although the anterior hypothalamic area just posterior to mPOA may also be involved. Two types of nuclear estrogen receptors, ER{alpha} and ERß, are both localized in the mPOA as shown by in situ hybridization as well as immunocytochemical studies (10, 11, 12, 13, 14). It is not known, however, which ER is responsible for estrogenic regulation of running wheel activity. In the present study we examined the effects of estrogen treatment on running wheel activity in male and female mice specifically lacking either ER{alpha} ({alpha} ERKO) or ERß (ßERKO) genes.
Previous studies have shown that estrogen also regulates open field activity in female rats (15). It is generally assumed that estrogen may control open field activity and running wheel activity through different mechanisms. Open field activity measured in an unfamiliar testing apparatus represent an animal’s response to a new environment, whereas running wheel activity is the animal’s home cage activity. Brain site-specific estrogen implants effective in increasing running wheel activity in gonadectomized female rats failed to increase open field activity (9). These findings in rats suggest that estrogenic regulation of running wheel activity may be primarily localized in the mPOA, whereas that of open field activity may involve more than one specific brain site. Furthermore, a recent study in C57BL/6J mice showed that systemic estrogen treatments stimulating running wheel activity did not necessarily increase or could even decrease certain measurements of open field activity (16). It is assumed that activity measured in open field tests may be confounded by a number of factors, such as fear and emotionality, depending on testing conditions, e.g. lighting intensity and size of the apparatus (17). Indeed, it is reported that open field activity levels were highly correlated with behavioral responses in other fear-related behavioral test paradigms, such as fear conditioning and dark/light transition tests, rather than with home cage running wheel activity levels (16). Taken together, these findings suggest that two types of activity may be differentially regulated by estrogen via two types of ERs.
Although the effects of estrogen are not yet known, our previous studies suggested that ER{alpha} gene disruption might modify baseline open field activity in a gender-dependent fashion. In gonadally intact male mice, ER{alpha} , but not ERß, gene disruption significantly increased open field activity (18, 19). In females, on the other hand, ER{alpha} gene disruption tended to decrease both open field activity in gonadally intact mice and chamber transitions in the dark/light transition tests in both gonadally intact and gonadectomized mice (20). In the present study we first pretested gonadectomized mice of both sexes for open field activity to assess sex- and/or gene-dependent effects of gene knockout. We then assigned mice to one of three treatment groups based on activity levels and examined whether estrogen might affect running wheel and open field activities separately by using the same mice in both tests.8d*, 百拇医药
Materials and Methods8d*, 百拇医药
Mice
A total of 172 mice deficient in the ER{alpha} ({alpha} ERKO) or ERß (ßERKO) gene and their respective wild-type (WT) littermates of both sexes were used. They were obtained from the {alpha} ERKO and ßERKO breeding colonies maintained at Rockefeller University by mating heterozygous male and female mice. Original breeding pairs (mixed background of C57BL/6J and 129) were obtained from the NIEHS. Animal rooms were maintained on a 12-h light, 12-h dark cycle at a constant temperature (22 C). Experimental mice were group-housed with the same sex and mixed genotype cage mates from weaning (21 d old) until used for the experiment. Food and water were available ad libitum throughout the studies.%@3, 百拇医药
Procedure%@3, 百拇医药
At the age of 9–11 wk, mice were gonadectomized (d 1; see Table 1). On the sixth day all mice were tested once for open field behavior (see below) and assigned to 1 of 3 treatment groups based on their activity levels (see below) and body weights. The numbers of animals in each treatment group of each genotype are indicated in parentheses in Tables 2 and 3. Two days later the mice were implanted with either a ß-estradiol 3-benzoate (EB) capsule with a dose of 16 or 160 ng/d or a placebo capsule (Innovative Research of America, Toledo, OH). At the same time, they were weighed, single-housed in plastic cages (27 x 16 x 13 cm), and given a phytoestrogen-free diet (AIN76A, Ralston Purina Co., St. Louis, MO). On the 15th day (1 wk after the implant) mice were transferred to running wheel activity cages (Mini Mitter, Bend, OR) and monitored daily for the number of wheel revolutions (d 16–25; data starting on d 17 were included for the analysis). The day after the last recording, they were transferred again to regular single-house cages and tested for open field behavior twice 1 wk later (d 33 and 34).
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Table 1. Experimental procedure], http://www.100md.com
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Table 2. Preimplant body weight], http://www.100md.com
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Table 3. Preimplant open field activity (total distance)], http://www.100md.com
Open field behavior tests], http://www.100md.com
Mice were tested for 5 min in an open field apparatus (40.5 x 40.5 cm, 30-cm high wall), which was illuminated with red light from the top at the center of the apparatus. At the beginning of the test a mouse was placed gently in a corner square with its head facing the corner. Activity was monitored automatically with infrared beams, and data were analyzed and stored using a Digiscan Analyzer and Digiscan software (Digiscan model RXYZCM, Accuscan Instruments, Columbus, OH). The total horizontal and vertical moving distances (total activity), total horizontal moving distance (total distance), cumulative duration of horizontal moving (moving time), moving distance in the center area (center distance), and time spent in the center area (center time) were recorded for each mouse. The first three measurements primarily indicated general exploratory activity levels in a novel environment, whereas the two measurements in the center area were interpreted to also be related to the animals’ anxiety and fear levels. The center area was defined as the area more than 1 in. away from the wall. After completion of the open field test, mice were weighed and assigned to one of three treatment groups balanced in terms of total distance and body weight.
Running wheel activity[]am, http://www.100md.com
Mice were housed individually in plastic cages (32 x 17 x 14 cm) equipped with a running wheel (25-cm diameter; Mini Mitter). Each wheel revolution was registered by a magnetic switch, which was connected to a counter. The number of revolutions was recorded daily for 9 d as described above.[]am, http://www.100md.com
Statistics[]am, http://www.100md.com
Data from pretreatment behavioral tests were analyzed in each sex with two-way ANOVAs for the main effects of gene (ER{alpha} vs. ERß) and gene knockout (KO vs. WT) and their interactions. Data from posttreatment behavioral tests were analyzed in each sex by three-way ANOVAs for the main effects of gene, knockout, treatment group, and their interactions. Repeated measurement data were analyzed in each sex and gene with three-way ANOVA for the main effects of gene knockout (KO vs. WT) treatment group, test block (as a repeated measure), and their interactions. If applicable, post hoc one- or two-way ANOVAs were performed. Turkey’s test was used for post hoc pairwise comparisons.
Resultsu, 百拇医药
Preestrogen treatment levels of open-field activity and body weightsu, 百拇医药
Gene disruption of ER{alpha} and ERß had opposite effects on open field activity in gonadectomized female, but not male, mice (Fig. 1). In four open field activity measurements, but not in the measurement of center time, there were significant interactions of gene and knockout [total activity: F(1,84) = 9.05; P = 0.004; Fig. 1A; total distance: F(1,84) = 6.31; P = 0.014; Fig. 1B; moving time: F(1,84) = 6.61; P = 0.012; Fig. 1C center distance: F(1,84) = 8.11; P = 0.006; Fig. 1D]. ERKO females were less active than {alpha} WT females, whereas ßERKO female mice tended to be more active than ßWT females.u, 百拇医药
fig.ommitteedu, 百拇医药
Figure 1. Effects of ER{alpha} and ERß gene disruption on open field activity in gonadectomized mice of both sexes. A, Total activity; B, total moving distance; C, cumulative moving time in seconds; D, moving distance in the center area; E, cumulative time spent in the center area; F, body weight. *, P < 0.05 vs. respective WT; {dagger} , P < 0.10 vs. respective WT.
In males, on the other hand, there were no significant effects of gene knockout in either ER{alpha} or ERß genes, except that ßERKO mice tended to show shorter moving times than ßWT. Instead, there were significant main effects of genetic background [total activity: F(1,81) = 5.21; P = 0.025; Fig. 1A; total distance: F(1,81) = 2.97; P = 0.089; Fig. 1B; center distance: F(1,81) = 5.05; P = 0.027 (Fig. 1D); center time: F(1,81) = 11.43; P = 0.001; Fig. 1E]. ßWT and ßERKO were less active than {alpha} WT and {alpha} ERKO mice.{x, 百拇医药
Body weights of female mice were not different among the four genotype groups. In males, there were significant main effects of genetic background [F(1,81) = 14.60; P = 0.0003; Fig. 1F]. Overall, ßWT and ßERKO were heavier than {alpha} WT and {alpha} ERKO mice.{x, 百拇医药
Based on the results of open field tests, mice were assigned to one of three treatment groups, balanced in terms of total moving distance and body weight (Tables 2 and 3). Tables 2 and 3 also indicate the numbers of animals in each treatment group of each genotype.
Effects of estrogen on overall (running wheel, d 1–9) running wheel activity|vnv5, http://www.100md.com
EB treatment significantly increased running wheel activity in both {alpha} WT [F(2,17) = 5.54; P = 0.014] and ßWT [F(2,20) = 6.14; P = 0.009] female mice, although in the former group only the higher dose of EB (160 ng/d) was significantly different from the placebo control (Fig. 2A). Deletion of ER{alpha} completely abolished the facilitatory action of estrogen on running wheel activity. For {alpha} ERKO female mice, there were no differences between the EB-treated groups and the placebo control group.|vnv5, http://www.100md.com
fig.ommitteed|vnv5, http://www.100md.com
Figure 2. Effects of ER and ERß gene disruption on estrogenic regulation of running wheel activity. Mean daily revolutions during the 9-d test period were shown for females (A) and males (B). , Placebo control group; , low dose of EB (16 ng/d) treatment group; , high dose of EB (160 ng/d) treatment group. *, P < 0.05 vs. the respective placebo control group; a, P < 0.05 vs. WT in the respective treatment group.
In ßERKO female mice, on the other hand, both doses of EB treatment increased running wheel activity compared with the placebo control treatment [F(2,21) = 14.94; P = 0.0002] as found in ßWT mice. However, absolute numbers of daily revolutions in the EB-treated groups, but not the placebo control group, were significantly lower in ßERKO compared with ßWT [effect of knockout in two EB groups combined: F(1,28) = 4.36; P = 0.046] female mice. As a side point, in placebo control groups there were no genotype differences in running wheel activity between {alpha} ERKO and {alpha} WT female mice (Fig. 2A). This is in contrast to the genotype differences found in pretreatment open field tests (Fig. 1).&-!5n, http://www.100md.com
In males EB treatment significantly increased running wheel activity in both groups of WT mice ({alpha} WT and ßWT; Fig. 2B), although overall {alpha} WT mice were more active than ßWT mice [F(1,34) = 12.02; P = 0.0014]. Effects of gene knockout on the estrogen-inducible activity increase were dependent on which gene was deleted, as revealed by three-way ANOVA [gene x knockout x treatment: F(2,73) = 8.10; P = 0.0007]. Deletion of the ER{alpha} gene affected estrogenic regulation of running wheel activity [knockout x treatment: F(1,37) = 14.19; P < 0.0001]. That is, in {alpha} ERKO mice estrogen failed to increase running wheel activity [treatment: P = NS], whereas in {alpha} WT mice estrogen significantly increased it [treatment: F(2,15) = 24.60; P < 0.0001].
In contrast, deletion of the ERß gene did not affect estrogenic regulation of running wheel activity [treatment: F(2,36) = 16.30; P < 0.0001; knockout and knockout x treatment: P = NS]. In ßERKO mice, both doses of EB treatment significantly increased running wheel activity [treatment: F(2,17) = 14.97; P = 0.0002] as found in ßWT mice [treatment: F(2,19) = 5.10; P = 0.017]. Unlike in females, there was a tendency in the three genotypes of male mice (WT, ßWT, and ßERKO) in which EB increased activity for the lower EB dose (16 ng/d) to be slightly more effective than the higher EB dose (160 ng/d), although there was no statistical difference between these two dose groups.@1y$', http://www.100md.com
Time course of estrogen effects on running wheel activity@1y$', http://www.100md.com
Effects of ER{alpha} and ERß gene knockout on estrogeninducible running wheel activity were further analyzed in each sex and gene by dividing the data into three blocks of 3 d each (Figs. 3 and 4).
fig.ommitteedwh|ed), 百拇医药
Figure 3. Time course of estrogenic regulation of running wheel activity in WT and ERKO (A) and ßWT and ßERKO (B) female mice. Mean daily revolutions of three time blocks of 3 d each are presented for each genotype and treatment group. , Placebo control group; , low dose of EB (16 ng/d) treatment group; , high dose of EB (160 ng/d) treatment group. *, P < 0.05 vs. the respective placebo control group; a, P < 0.05 vs. WT of the respective treatment group.wh|ed), 百拇医药
fig.ommitteedwh|ed), 百拇医药
Figure 4. Time course of estrogenic regulation of running wheel activity in WT and ERKO (A) and ßWT and ßERKO (B) male mice. Mean daily revolutions of three time blocks of 3 d each are presented for each genotype and treatment group. , Placebo control group; , low dose of EB (16 ng/d) treatment group; , high dose of EB (160 ng/d) treatment group. *, P < 0.05 vs. the respective placebo control group; a, P < 0.05 vs. WT of the respective treatment group.wh|ed), 百拇医药
Female ERKO vs. aWTwh|ed), 百拇医药
Regardless of treatment group and genotype, there was a steady increment in running wheel activity along days [block: F(2,68) = 25.45; P < 0.0001; block x treatment: P = NS; block x knockout x treatment: P = NS; Fig. 3A]. Overall genotype differences were detected throughout three blocks [knockout: F(1,34) = 6.65; P = 0.014; block x knockout: P = NS]. Estrogen did not modify running wheel activity in ERKO mice in all three blocks. In {alpha} WT female mice EB treatment effectively increased running wheel activity in all three blocks, although with the lower dose of EB, activity was not different from that in controls during the second block.
Female ßERKO vs. ßWTw1w), 百拇医药
Overall, ßERKO mice were less active than ßWT mice throughout the experiment [knockout: F(1,41) = 4.27; P = 0.045; block x knockout: P = NS; Fig. 3B]. Regardless of genotype, estrogen treatment became more effective toward the end of the testing period [block x treatment: F(4,82) = 2.98; P = 0.024; block x knockout x treatment: P = NS].w1w), 百拇医药
Male ERKO vs. {alpha} WTw1w), 百拇医药
Changes in running wheel activity during all three test blocks varied according to the genotype and treatment group as revealed by the three-way interaction of knockout, treatment, and test block [F(4,74) = 3.66; P = 0.009; Fig. 4A]. In {alpha} ERKO mice all three groups of mice showed a steady increase in running wheel activity along test days (treatment x block: P = NS). In {alpha} WT mice, only EB-treated groups, not the control group, showed an increase in running wheel activity along test days [treatment x block: F(4,30) = 5.03; P = 0.003].
Male ßERKO vs. ßWTm+g62gz, 百拇医药
There were no genotype differences in the time course for estrogenic regulation of running wheel activity (blocks x knockout x treatment: P = NS; Fig. 4B). However, in both genotypes two doses of EB treatment produced their effects in different time course [blocks x treatment: F(4,72) = 3.81; P = 0.0073]. During the first block the higher dose of EB was significantly less effective than the lower dose of EB in both genotypes (at {alpha} = 0.05), whereas during the second and third blocks the two doses were equally effective.m+g62gz, 百拇医药
Effects of estrogen on open field activitym+g62gz, 百拇医药
After the completion of running wheel tests, mice were tested twice for open field activity. Although mice were significantly more active on the first day than on the second day, there were no interactions of test days with genotypes and/or treatment groups. Therefore, mean values of the two tests were used for further analyses.
In female mice there were significant gene x knockout interactions in total activity [F(1,60) = 4.44; P = 0.039; data not shown], total distance [F(1,60) = 11.91; P = 0.01; Fig. 5A], moving time [F(1,60) = 6.39; P = 0.014; data not shown], and center distance [F(1,60) = 5.99; P = 0.017; Fig. 5B], but not in center time (data not shown). ERKO mice were less active than {alpha} WT, whereas ßERKO mice were not different from ßWT mice. There were also overall treatment effects on total activity [F(2,60) = 4.00; P = 0.023; data not shown], total distance [F(2,60) = 3.79; P = 0.028; Fig. 5A], and moving time [F(2,60) = 3.35; P = 0.042; data not shown]. Mice treated with EB at a dose of 160 ng/d were significantly more active than mice treated with either the low dose of EB (16 ng/d) or placebo regardless of genotype (gene x knockout x treatment: P = NS).e, 百拇医药
fig.ommitteede, 百拇医药
Figure 5. Effects of estrogen treatment on open field activity. Means of 2-d tests are presented for the total moving distance (A for females and C for males) and the center distance (B for females and D for males). , Placebo control group; , low dose of EB (16 ng/d) treatment group; , high dose of EB (160 ng/d) treatment group. a, P < 0.05 vs. WT of the respective treatment group.
In males only the main effect of gene, but not the main effect of knockout or gene x knockout interaction, was statistically significant in all five measurements of open field tests [total activity: F(1,64) = 5.59; P = 0.021; data not shown; total distance: F(1,64) = 5.46; P = 0.023; Fig. 5C; moving time: F(1,64) = 4.14; P = 0.046; data not shown; center distance: F(1,64) = 5.10; P = 0.027; Fig. 5D; center time: F(1,64) = 5.95; P = 0.018; data not shown]. {alpha} WT and {alpha} ERKO mice were more active than ßWT and ßERKO mice. Furthermore, unlike females, there were no significant main effects of treatment or interactions with gene and/or knockout in any measurement.a|, http://www.100md.com
Discussiona|, http://www.100md.com
Roles of ER and ERß in estrogenic regulation of running wheel activitya|, http://www.100md.com
It was found in this study that estrogen greatly potentiated running wheel activity in mice, as previously reported in rats (2, 3, 4, 5). The present study demonstrates for the first time a differential dependence on the ER{alpha} vs. the ERß gene in the estrogenic regulation of running wheel activity. In both sexes of mice ER{alpha} gene disruption completely abolished the estrogen-inducible increase in running wheel activity, whereas ERß gene disruption did not affect it. These results suggest that estrogen may facilitate running wheel activity primarily by acting through ER{alpha} , presumably in the mPOA (7, 8, 9). As it is known that responsiveness to EB for the potentiation of running wheel activity is not affected by neonatal steroid manipulation (4), it is likely that ER{alpha} activation at the time of testing in adulthood is critical for estrogenic regulation of this behavior. The present study, however, does not completely rule out the possibility that the lack of EB effects in {alpha} ERKO mice may also be due to the lack of ER{alpha} stimulation during perinatal development regardless of sex.
The present findings also suggest that activation of ERß by itself is not sufficient to increase running wheel activity in mice. This idea is consistent with the results of our studies in which an ERß-specific compound (Ogawa et al., unpublished data) failed to increase running wheel activity in Swiss-Webster mice. However, this does not rule out the possibility that ERß participates in the regulation of running wheel activity. In fact, in female ßERKO mice, estrogen was slightly less effective than in ßWT female mice in terms of absolute number of daily revolutions after EB treatment. Therefore, it is possible that simultaneous activation of ER{alpha} and ERß might be necessary for complete estrogenic regulation of running wheel activity at least in female mice. It should be noted that ER{alpha} and ERß are both localized in the mPOA (10, 11, 12, 13, 14), although potential synergistic actions of the two receptors could well be localized in different parts of the brain.
It should be noted that the doses of estrogen found to be quite effective to increase running wheel activity in wild-type and ßERKO mice in the present study were much lower than the doses normally used to induce female sexual behavior. By the last day of the running wheel activity test, mice in the lower and higher dose estrogen-treated groups received a total of 0.27 or 2.7 µg, respectively. Serum estradiol levels determined in some of the mice used in the present study on 26 d after pellet implant were indeed less than the detectable level of the assay (<5 pg/ml) even in the higher estrogen dose group (160 ng/d). Nevertheless, we found that stimulatory effects of estrogen on running wheel activity levels in WT, ßWT, and ßERKO mice of both sexes were more obvious during the second and third blocks compared with the first block. Except in {alpha} WT female mice, in which the interaction between block and treatment was not statistically significant, the placebo groups of these genotypes did not show such an increment in running wheel activity during the day. In the present study we used EB to ensure stimulatory action of systemically administered estrogen on running wheel activity, particularly in {alpha} ERKO mice, in which most of the estrogen-inducible behaviors were severely disrupted. As EB has a longer half-life in the body than 17ß-estradiol, accumulation of the steroid over days might cause a gradual increase in running wheel activity in estrogen-treated groups. In {alpha} ERKO mice, on the other hand, all three treatment groups, including the placebo groups, showed a gradual increase in running wheel activity in both sexes. These findings suggest that running experience could itself stimulate running wheel activity in {alpha} ERKO mice even though estrogen failed to modify it.
Sex comparisons across the estrogenic regulation of running wheel activity4, 百拇医药
In the present study, there were no obvious sex differences in estrogen effects on running wheel activity. Both male and female ERKO failed to respond to EB treatment, whereas WT, ßWT, and ßERKO mice of both sexes showed responses to EB. These findings are consistent with a previous study in rats showing no sex differences in the response to estrogen after gonadectomy (4). The study by Gentry and Wade (4) also revealed that neonatal androgenization of female rats did not change the magnitude of the response to estrogen later in adulthood for the potentiation of running wheel activity, although they needed a longer latency to respond. Taken together, these findings suggest that facilitation of running wheel activity by estrogen is not a sexually dimorphic trait.4, 百拇医药
A number of studies in golden hamsters and rats have shown that there is a clear sex difference in estrogenic regulation of free running circadian activity rhythms (21, 22, 23, 24). In these studies it is implied that responsiveness to estrogen (e.g. shortened free running period, ) in adulthood is regulated by the neonatal steroid condition. Both males and neonatally androgenized females failed to show shortened {tau} in response to estrogen in adulthood during free running circadian rhythm tests. In the present study we measured only daily activity under a normal 12-h light, 12-h dark entrained condition. Therefore, it remains to be determined in future studies whether circadian systems measured by the use of running wheel activity are also affected by ER{alpha} and/or ERß gene disruption, and if so, if there is any sex difference. Potential roles of ER{alpha} and ERß genes in the regulation of the clock gene expression (25, 26) in the suprachiasmatic nucleus as well as other brain regions receiving input from it (e.g. paraventricular nucleus) need to be further investigated.
Although there was no sex difference in EB effects on running wheel activity, there was sex/knockout interaction in placebo groups of WT and ERKO mice. Unlike in open field activity, as discussed below, there was no genotype difference in running wheel activity in the placebo groups of female mice. On the other hand, the placebo control group of male ERKO mice tended to be more active than {alpha} WT mice in running wheel tests, particularly during the last 3 d of 9-d tests. These results suggest that running wheel activity in gonadectomized mice might be modulated by ligand-independent effects of ER{alpha} gene disruption in male mice.xf?{k, 百拇医药
Roles of two types of ERs in the regulation of open field activityxf?{k, 百拇医药
In contrast to a great potentiation of running wheel activity (up to 2- to 2.5-fold) by estrogen in both sexes, open field activity was only slightly affected by estrogen in females. In males there was no effect of estrogen in any genotype of mice. Differential effects of estrogen on running wheel activity and open field activity were also described with brain sitespecific implants of estrogen in female rats (9) and systemic estrogen administration in female mice (16).
We found that open field activity, both total and in the center area, before estrogen implants was significantly reduced in {alpha} ERKO females, whereas it tended to increase in ßERKO females compared with that in the respective WT control mice. A similar trend was noticed in the placebo control groups during the postimplant open field tests. On the other hand, no such genotype differences were observed in running wheel activity in the placebo control groups of female mice regardless of gene. We found previously that {alpha} ERKO female mice tended to be less active, particularly in the light compartment, during the dark/light transition tests regardless of gonadal state (20). Taken together, these findings suggest that the lack of ER{alpha} gene expression might by itself ligand-independently modify baseline activity in a novel environment (open field and dark/light transition tests), but not home cage activity (running wheel), in female mice. Furthermore, genotype differences between {alpha} ERKO and {alpha} WT female mice in both open field and dark/light transition (20) tests were consistent regardless of gonadal states, i.e. gonadally intact, gonadectomized, and gonadectomized plus estrogen treatment. These findings suggest that the reduction of activity in {alpha} ERKO female mice in these novel environment tests may be due to a combination of the lack of responsiveness to estrogen in adulthood as well as developmental effects of ER{alpha} gene disruption. During preimplant open field tests in the present study, {alpha} ERKO female mice resembled male {alpha} WT and {alpha} ERKO mice more than female {alpha} WT mice in terms of total activity, total distance, and moving time. We speculate that this type of behavioral masculinization in {alpha} ERKO female mice might be due to elevated stimulation of AR during neonatal development. Starting on postnatal d 9–21, the number of ligand-bound AR-immunoreactive cells in {alpha} ERKO female mouse brains was significantly higher than that in {alpha} WT females in the mPOA among other brain regions and was almost equivalent to that in male mouse brains (27).
ERß gene disruption, on the other hand, tended to increase open field activity in female mice. This is in marked contrast to a great reduction of open field activity and elevation of anxiety reported in ERß knockout female mice (28, 29), which were developed separately from those used in the present study. There are three important factors that might influence the differences in open field activity between these two studies. In the present study mice were tested under red light, whereas in the study by Krezel et al. (29), they were tested under brightly illuminated white light conditions. It is assumed that the latter is more sensitive than the former to reveal traits such as anxiety or emotionality. Secondly, we tested the mice after gonadectomy, but Krezel et al. tested gonadally intact mice. However, this may not be a major factor in explaining the behavioral differences, as the effects of ERß gene disruption might also be independent of the gonadal state at the time of testing, as we found in the case of ER{alpha} (see above). Thirdly, a more important difference between the two studies may be the age of the mice. Mice in the present study were tested at 10–12 wk, whereas those in Krezel’s study were tested when they were more than 7 months old. In fact, we found that in older ßERKO females (>30 wk of age), open field activity and radial maze activity during a spatial learning paradigm, both of which were performed under bright light illumination, were greatly reduced (30). It is possible, therefore, that the effects of ERß gene disruption may best appear in older female mice.
Acknowledgmentsl:4}j[, 百拇医药
The authors are thankful to Dr. C. J. Krebs for his molecular biological expertise, and Dr. C. Pavlides and Ms. L. Frank for critical and editorial reading of the manuscript.l:4}j[, 百拇医药
Received May 15, 2002.l:4}j[, 百拇医药
Accepted for publication September 23, 2002.l:4}j[, 百拇医药
Referencesl:4}j[, 百拇医药
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Sacrestano D, McKenna E, Ogawa S, Gustafsson J-Å, Smithies O, Korach KS, Pfaff DW 2000 Spatial learning in gene knockout mice of estrogen receptors {alpha} ({alpha} ERKO) and ß (ßERKO). Soc Neurosci Abstr 26:1746(Sonoko Ogawa Johnny Chan Jan-Åke Gustafsson Kenneth S. Korach and Donald W. Pfaff)
Estrogens are known to increase running wheel activity of rodents primarily by acting on the medial preoptic area (mPOA). The mechanisms of this estrogenic regulation of running wheel activity are not completely understood. In particular, little is known about the separate roles of two types of estrogen receptors, ER{alpha} and ERß, both of which are expressed in mPOA neurons. In the present study the effects of continuous estrogen treatment on running wheel activity were examined in male and female mice specifically lacking either the ER (ERKO) or the ERß (ßERKO) gene. Mice were gonadectomized and 1 wk later implanted with either a low dose (16 ng/d) or a high dose (160 ng/d) of estradiol benzoate (EB) or with a placebo control pellet. Home cage running wheel activity was recorded for 9 d starting 10 d after EB implants. The same mice were also tested for open field activity before and after EB implants. In both female and male {alpha} ERKO mice, running wheel activity was not different from that in corresponding wild-type (WT) mice in placebo control groups. In both females and males it was increased by EB only in WT, not ERKO, mice. In ßERKO mice, on the other hand, both doses of EB equally increased running wheel activity in both sexes just as they did in ßWT mice. Absolute numbers of daily revolutions of EB-treated groups, however, were significantly lower in ßERKO females compared with ßWT females. Before EB treatment, gonadectomized ERKO female were significantly less active than WT mice in open field tests, whereas ßERKO females tended to be more active than ßWT mice. In male mice there were no effect of ER or ERß gene knockout on open field activity. Unlike its effect on running wheel activity, EB treatment induced only a small increase in open field activity in female, but not male, mice. These findings indicate that 1) in both sexes estrogenic regulation of running wheel activity is primarily mediated through the ER{alpha} , not the ERß; and 2) hormone/genotype effects are specific to the type of locomotor activity (i.e. home cage running wheel activity and open field activity) measured.
Introductionwdd, 百拇医药
IT IS WELL established that estrogen regulates running wheel activity in female and male rats. Gonadally intact female rats show the highest activity during proestrus, when plasma levels of estrogen are elevated (1). In gonadectomized female and male rats, it has been shown that estrogen treatment increases running wheel activity (2, 3, 4, 5), although estrogen failed to do so in ferrets of both sexes (6). A number of lesion (7), electrophysiological (8), and site-specific steroid implant studies (9) revealed that the medial preoptic area (mPOA) is the primary brain site responsible for this behavioral effect of estrogen, although the anterior hypothalamic area just posterior to mPOA may also be involved. Two types of nuclear estrogen receptors, ER{alpha} and ERß, are both localized in the mPOA as shown by in situ hybridization as well as immunocytochemical studies (10, 11, 12, 13, 14). It is not known, however, which ER is responsible for estrogenic regulation of running wheel activity. In the present study we examined the effects of estrogen treatment on running wheel activity in male and female mice specifically lacking either ER{alpha} ({alpha} ERKO) or ERß (ßERKO) genes.
Previous studies have shown that estrogen also regulates open field activity in female rats (15). It is generally assumed that estrogen may control open field activity and running wheel activity through different mechanisms. Open field activity measured in an unfamiliar testing apparatus represent an animal’s response to a new environment, whereas running wheel activity is the animal’s home cage activity. Brain site-specific estrogen implants effective in increasing running wheel activity in gonadectomized female rats failed to increase open field activity (9). These findings in rats suggest that estrogenic regulation of running wheel activity may be primarily localized in the mPOA, whereas that of open field activity may involve more than one specific brain site. Furthermore, a recent study in C57BL/6J mice showed that systemic estrogen treatments stimulating running wheel activity did not necessarily increase or could even decrease certain measurements of open field activity (16). It is assumed that activity measured in open field tests may be confounded by a number of factors, such as fear and emotionality, depending on testing conditions, e.g. lighting intensity and size of the apparatus (17). Indeed, it is reported that open field activity levels were highly correlated with behavioral responses in other fear-related behavioral test paradigms, such as fear conditioning and dark/light transition tests, rather than with home cage running wheel activity levels (16). Taken together, these findings suggest that two types of activity may be differentially regulated by estrogen via two types of ERs.
Although the effects of estrogen are not yet known, our previous studies suggested that ER{alpha} gene disruption might modify baseline open field activity in a gender-dependent fashion. In gonadally intact male mice, ER{alpha} , but not ERß, gene disruption significantly increased open field activity (18, 19). In females, on the other hand, ER{alpha} gene disruption tended to decrease both open field activity in gonadally intact mice and chamber transitions in the dark/light transition tests in both gonadally intact and gonadectomized mice (20). In the present study we first pretested gonadectomized mice of both sexes for open field activity to assess sex- and/or gene-dependent effects of gene knockout. We then assigned mice to one of three treatment groups based on activity levels and examined whether estrogen might affect running wheel and open field activities separately by using the same mice in both tests.8d*, 百拇医药
Materials and Methods8d*, 百拇医药
Mice
A total of 172 mice deficient in the ER{alpha} ({alpha} ERKO) or ERß (ßERKO) gene and their respective wild-type (WT) littermates of both sexes were used. They were obtained from the {alpha} ERKO and ßERKO breeding colonies maintained at Rockefeller University by mating heterozygous male and female mice. Original breeding pairs (mixed background of C57BL/6J and 129) were obtained from the NIEHS. Animal rooms were maintained on a 12-h light, 12-h dark cycle at a constant temperature (22 C). Experimental mice were group-housed with the same sex and mixed genotype cage mates from weaning (21 d old) until used for the experiment. Food and water were available ad libitum throughout the studies.%@3, 百拇医药
Procedure%@3, 百拇医药
At the age of 9–11 wk, mice were gonadectomized (d 1; see Table 1). On the sixth day all mice were tested once for open field behavior (see below) and assigned to 1 of 3 treatment groups based on their activity levels (see below) and body weights. The numbers of animals in each treatment group of each genotype are indicated in parentheses in Tables 2 and 3. Two days later the mice were implanted with either a ß-estradiol 3-benzoate (EB) capsule with a dose of 16 or 160 ng/d or a placebo capsule (Innovative Research of America, Toledo, OH). At the same time, they were weighed, single-housed in plastic cages (27 x 16 x 13 cm), and given a phytoestrogen-free diet (AIN76A, Ralston Purina Co., St. Louis, MO). On the 15th day (1 wk after the implant) mice were transferred to running wheel activity cages (Mini Mitter, Bend, OR) and monitored daily for the number of wheel revolutions (d 16–25; data starting on d 17 were included for the analysis). The day after the last recording, they were transferred again to regular single-house cages and tested for open field behavior twice 1 wk later (d 33 and 34).
fig.ommitteed], http://www.100md.com
Table 1. Experimental procedure], http://www.100md.com
fig.ommitteed], http://www.100md.com
Table 2. Preimplant body weight], http://www.100md.com
fig.ommitteed], http://www.100md.com
Table 3. Preimplant open field activity (total distance)], http://www.100md.com
Open field behavior tests], http://www.100md.com
Mice were tested for 5 min in an open field apparatus (40.5 x 40.5 cm, 30-cm high wall), which was illuminated with red light from the top at the center of the apparatus. At the beginning of the test a mouse was placed gently in a corner square with its head facing the corner. Activity was monitored automatically with infrared beams, and data were analyzed and stored using a Digiscan Analyzer and Digiscan software (Digiscan model RXYZCM, Accuscan Instruments, Columbus, OH). The total horizontal and vertical moving distances (total activity), total horizontal moving distance (total distance), cumulative duration of horizontal moving (moving time), moving distance in the center area (center distance), and time spent in the center area (center time) were recorded for each mouse. The first three measurements primarily indicated general exploratory activity levels in a novel environment, whereas the two measurements in the center area were interpreted to also be related to the animals’ anxiety and fear levels. The center area was defined as the area more than 1 in. away from the wall. After completion of the open field test, mice were weighed and assigned to one of three treatment groups balanced in terms of total distance and body weight.
Running wheel activity[]am, http://www.100md.com
Mice were housed individually in plastic cages (32 x 17 x 14 cm) equipped with a running wheel (25-cm diameter; Mini Mitter). Each wheel revolution was registered by a magnetic switch, which was connected to a counter. The number of revolutions was recorded daily for 9 d as described above.[]am, http://www.100md.com
Statistics[]am, http://www.100md.com
Data from pretreatment behavioral tests were analyzed in each sex with two-way ANOVAs for the main effects of gene (ER{alpha} vs. ERß) and gene knockout (KO vs. WT) and their interactions. Data from posttreatment behavioral tests were analyzed in each sex by three-way ANOVAs for the main effects of gene, knockout, treatment group, and their interactions. Repeated measurement data were analyzed in each sex and gene with three-way ANOVA for the main effects of gene knockout (KO vs. WT) treatment group, test block (as a repeated measure), and their interactions. If applicable, post hoc one- or two-way ANOVAs were performed. Turkey’s test was used for post hoc pairwise comparisons.
Resultsu, 百拇医药
Preestrogen treatment levels of open-field activity and body weightsu, 百拇医药
Gene disruption of ER{alpha} and ERß had opposite effects on open field activity in gonadectomized female, but not male, mice (Fig. 1). In four open field activity measurements, but not in the measurement of center time, there were significant interactions of gene and knockout [total activity: F(1,84) = 9.05; P = 0.004; Fig. 1A; total distance: F(1,84) = 6.31; P = 0.014; Fig. 1B; moving time: F(1,84) = 6.61; P = 0.012; Fig. 1C center distance: F(1,84) = 8.11; P = 0.006; Fig. 1D]. ERKO females were less active than {alpha} WT females, whereas ßERKO female mice tended to be more active than ßWT females.u, 百拇医药
fig.ommitteedu, 百拇医药
Figure 1. Effects of ER{alpha} and ERß gene disruption on open field activity in gonadectomized mice of both sexes. A, Total activity; B, total moving distance; C, cumulative moving time in seconds; D, moving distance in the center area; E, cumulative time spent in the center area; F, body weight. *, P < 0.05 vs. respective WT; {dagger} , P < 0.10 vs. respective WT.
In males, on the other hand, there were no significant effects of gene knockout in either ER{alpha} or ERß genes, except that ßERKO mice tended to show shorter moving times than ßWT. Instead, there were significant main effects of genetic background [total activity: F(1,81) = 5.21; P = 0.025; Fig. 1A; total distance: F(1,81) = 2.97; P = 0.089; Fig. 1B; center distance: F(1,81) = 5.05; P = 0.027 (Fig. 1D); center time: F(1,81) = 11.43; P = 0.001; Fig. 1E]. ßWT and ßERKO were less active than {alpha} WT and {alpha} ERKO mice.{x, 百拇医药
Body weights of female mice were not different among the four genotype groups. In males, there were significant main effects of genetic background [F(1,81) = 14.60; P = 0.0003; Fig. 1F]. Overall, ßWT and ßERKO were heavier than {alpha} WT and {alpha} ERKO mice.{x, 百拇医药
Based on the results of open field tests, mice were assigned to one of three treatment groups, balanced in terms of total moving distance and body weight (Tables 2 and 3). Tables 2 and 3 also indicate the numbers of animals in each treatment group of each genotype.
Effects of estrogen on overall (running wheel, d 1–9) running wheel activity|vnv5, http://www.100md.com
EB treatment significantly increased running wheel activity in both {alpha} WT [F(2,17) = 5.54; P = 0.014] and ßWT [F(2,20) = 6.14; P = 0.009] female mice, although in the former group only the higher dose of EB (160 ng/d) was significantly different from the placebo control (Fig. 2A). Deletion of ER{alpha} completely abolished the facilitatory action of estrogen on running wheel activity. For {alpha} ERKO female mice, there were no differences between the EB-treated groups and the placebo control group.|vnv5, http://www.100md.com
fig.ommitteed|vnv5, http://www.100md.com
Figure 2. Effects of ER and ERß gene disruption on estrogenic regulation of running wheel activity. Mean daily revolutions during the 9-d test period were shown for females (A) and males (B). , Placebo control group; , low dose of EB (16 ng/d) treatment group; , high dose of EB (160 ng/d) treatment group. *, P < 0.05 vs. the respective placebo control group; a, P < 0.05 vs. WT in the respective treatment group.
In ßERKO female mice, on the other hand, both doses of EB treatment increased running wheel activity compared with the placebo control treatment [F(2,21) = 14.94; P = 0.0002] as found in ßWT mice. However, absolute numbers of daily revolutions in the EB-treated groups, but not the placebo control group, were significantly lower in ßERKO compared with ßWT [effect of knockout in two EB groups combined: F(1,28) = 4.36; P = 0.046] female mice. As a side point, in placebo control groups there were no genotype differences in running wheel activity between {alpha} ERKO and {alpha} WT female mice (Fig. 2A). This is in contrast to the genotype differences found in pretreatment open field tests (Fig. 1).&-!5n, http://www.100md.com
In males EB treatment significantly increased running wheel activity in both groups of WT mice ({alpha} WT and ßWT; Fig. 2B), although overall {alpha} WT mice were more active than ßWT mice [F(1,34) = 12.02; P = 0.0014]. Effects of gene knockout on the estrogen-inducible activity increase were dependent on which gene was deleted, as revealed by three-way ANOVA [gene x knockout x treatment: F(2,73) = 8.10; P = 0.0007]. Deletion of the ER{alpha} gene affected estrogenic regulation of running wheel activity [knockout x treatment: F(1,37) = 14.19; P < 0.0001]. That is, in {alpha} ERKO mice estrogen failed to increase running wheel activity [treatment: P = NS], whereas in {alpha} WT mice estrogen significantly increased it [treatment: F(2,15) = 24.60; P < 0.0001].
In contrast, deletion of the ERß gene did not affect estrogenic regulation of running wheel activity [treatment: F(2,36) = 16.30; P < 0.0001; knockout and knockout x treatment: P = NS]. In ßERKO mice, both doses of EB treatment significantly increased running wheel activity [treatment: F(2,17) = 14.97; P = 0.0002] as found in ßWT mice [treatment: F(2,19) = 5.10; P = 0.017]. Unlike in females, there was a tendency in the three genotypes of male mice (WT, ßWT, and ßERKO) in which EB increased activity for the lower EB dose (16 ng/d) to be slightly more effective than the higher EB dose (160 ng/d), although there was no statistical difference between these two dose groups.@1y$', http://www.100md.com
Time course of estrogen effects on running wheel activity@1y$', http://www.100md.com
Effects of ER{alpha} and ERß gene knockout on estrogeninducible running wheel activity were further analyzed in each sex and gene by dividing the data into three blocks of 3 d each (Figs. 3 and 4).
fig.ommitteedwh|ed), 百拇医药
Figure 3. Time course of estrogenic regulation of running wheel activity in WT and ERKO (A) and ßWT and ßERKO (B) female mice. Mean daily revolutions of three time blocks of 3 d each are presented for each genotype and treatment group. , Placebo control group; , low dose of EB (16 ng/d) treatment group; , high dose of EB (160 ng/d) treatment group. *, P < 0.05 vs. the respective placebo control group; a, P < 0.05 vs. WT of the respective treatment group.wh|ed), 百拇医药
fig.ommitteedwh|ed), 百拇医药
Figure 4. Time course of estrogenic regulation of running wheel activity in WT and ERKO (A) and ßWT and ßERKO (B) male mice. Mean daily revolutions of three time blocks of 3 d each are presented for each genotype and treatment group. , Placebo control group; , low dose of EB (16 ng/d) treatment group; , high dose of EB (160 ng/d) treatment group. *, P < 0.05 vs. the respective placebo control group; a, P < 0.05 vs. WT of the respective treatment group.wh|ed), 百拇医药
Female ERKO vs. aWTwh|ed), 百拇医药
Regardless of treatment group and genotype, there was a steady increment in running wheel activity along days [block: F(2,68) = 25.45; P < 0.0001; block x treatment: P = NS; block x knockout x treatment: P = NS; Fig. 3A]. Overall genotype differences were detected throughout three blocks [knockout: F(1,34) = 6.65; P = 0.014; block x knockout: P = NS]. Estrogen did not modify running wheel activity in ERKO mice in all three blocks. In {alpha} WT female mice EB treatment effectively increased running wheel activity in all three blocks, although with the lower dose of EB, activity was not different from that in controls during the second block.
Female ßERKO vs. ßWTw1w), 百拇医药
Overall, ßERKO mice were less active than ßWT mice throughout the experiment [knockout: F(1,41) = 4.27; P = 0.045; block x knockout: P = NS; Fig. 3B]. Regardless of genotype, estrogen treatment became more effective toward the end of the testing period [block x treatment: F(4,82) = 2.98; P = 0.024; block x knockout x treatment: P = NS].w1w), 百拇医药
Male ERKO vs. {alpha} WTw1w), 百拇医药
Changes in running wheel activity during all three test blocks varied according to the genotype and treatment group as revealed by the three-way interaction of knockout, treatment, and test block [F(4,74) = 3.66; P = 0.009; Fig. 4A]. In {alpha} ERKO mice all three groups of mice showed a steady increase in running wheel activity along test days (treatment x block: P = NS). In {alpha} WT mice, only EB-treated groups, not the control group, showed an increase in running wheel activity along test days [treatment x block: F(4,30) = 5.03; P = 0.003].
Male ßERKO vs. ßWTm+g62gz, 百拇医药
There were no genotype differences in the time course for estrogenic regulation of running wheel activity (blocks x knockout x treatment: P = NS; Fig. 4B). However, in both genotypes two doses of EB treatment produced their effects in different time course [blocks x treatment: F(4,72) = 3.81; P = 0.0073]. During the first block the higher dose of EB was significantly less effective than the lower dose of EB in both genotypes (at {alpha} = 0.05), whereas during the second and third blocks the two doses were equally effective.m+g62gz, 百拇医药
Effects of estrogen on open field activitym+g62gz, 百拇医药
After the completion of running wheel tests, mice were tested twice for open field activity. Although mice were significantly more active on the first day than on the second day, there were no interactions of test days with genotypes and/or treatment groups. Therefore, mean values of the two tests were used for further analyses.
In female mice there were significant gene x knockout interactions in total activity [F(1,60) = 4.44; P = 0.039; data not shown], total distance [F(1,60) = 11.91; P = 0.01; Fig. 5A], moving time [F(1,60) = 6.39; P = 0.014; data not shown], and center distance [F(1,60) = 5.99; P = 0.017; Fig. 5B], but not in center time (data not shown). ERKO mice were less active than {alpha} WT, whereas ßERKO mice were not different from ßWT mice. There were also overall treatment effects on total activity [F(2,60) = 4.00; P = 0.023; data not shown], total distance [F(2,60) = 3.79; P = 0.028; Fig. 5A], and moving time [F(2,60) = 3.35; P = 0.042; data not shown]. Mice treated with EB at a dose of 160 ng/d were significantly more active than mice treated with either the low dose of EB (16 ng/d) or placebo regardless of genotype (gene x knockout x treatment: P = NS).e, 百拇医药
fig.ommitteede, 百拇医药
Figure 5. Effects of estrogen treatment on open field activity. Means of 2-d tests are presented for the total moving distance (A for females and C for males) and the center distance (B for females and D for males). , Placebo control group; , low dose of EB (16 ng/d) treatment group; , high dose of EB (160 ng/d) treatment group. a, P < 0.05 vs. WT of the respective treatment group.
In males only the main effect of gene, but not the main effect of knockout or gene x knockout interaction, was statistically significant in all five measurements of open field tests [total activity: F(1,64) = 5.59; P = 0.021; data not shown; total distance: F(1,64) = 5.46; P = 0.023; Fig. 5C; moving time: F(1,64) = 4.14; P = 0.046; data not shown; center distance: F(1,64) = 5.10; P = 0.027; Fig. 5D; center time: F(1,64) = 5.95; P = 0.018; data not shown]. {alpha} WT and {alpha} ERKO mice were more active than ßWT and ßERKO mice. Furthermore, unlike females, there were no significant main effects of treatment or interactions with gene and/or knockout in any measurement.a|, http://www.100md.com
Discussiona|, http://www.100md.com
Roles of ER and ERß in estrogenic regulation of running wheel activitya|, http://www.100md.com
It was found in this study that estrogen greatly potentiated running wheel activity in mice, as previously reported in rats (2, 3, 4, 5). The present study demonstrates for the first time a differential dependence on the ER{alpha} vs. the ERß gene in the estrogenic regulation of running wheel activity. In both sexes of mice ER{alpha} gene disruption completely abolished the estrogen-inducible increase in running wheel activity, whereas ERß gene disruption did not affect it. These results suggest that estrogen may facilitate running wheel activity primarily by acting through ER{alpha} , presumably in the mPOA (7, 8, 9). As it is known that responsiveness to EB for the potentiation of running wheel activity is not affected by neonatal steroid manipulation (4), it is likely that ER{alpha} activation at the time of testing in adulthood is critical for estrogenic regulation of this behavior. The present study, however, does not completely rule out the possibility that the lack of EB effects in {alpha} ERKO mice may also be due to the lack of ER{alpha} stimulation during perinatal development regardless of sex.
The present findings also suggest that activation of ERß by itself is not sufficient to increase running wheel activity in mice. This idea is consistent with the results of our studies in which an ERß-specific compound (Ogawa et al., unpublished data) failed to increase running wheel activity in Swiss-Webster mice. However, this does not rule out the possibility that ERß participates in the regulation of running wheel activity. In fact, in female ßERKO mice, estrogen was slightly less effective than in ßWT female mice in terms of absolute number of daily revolutions after EB treatment. Therefore, it is possible that simultaneous activation of ER{alpha} and ERß might be necessary for complete estrogenic regulation of running wheel activity at least in female mice. It should be noted that ER{alpha} and ERß are both localized in the mPOA (10, 11, 12, 13, 14), although potential synergistic actions of the two receptors could well be localized in different parts of the brain.
It should be noted that the doses of estrogen found to be quite effective to increase running wheel activity in wild-type and ßERKO mice in the present study were much lower than the doses normally used to induce female sexual behavior. By the last day of the running wheel activity test, mice in the lower and higher dose estrogen-treated groups received a total of 0.27 or 2.7 µg, respectively. Serum estradiol levels determined in some of the mice used in the present study on 26 d after pellet implant were indeed less than the detectable level of the assay (<5 pg/ml) even in the higher estrogen dose group (160 ng/d). Nevertheless, we found that stimulatory effects of estrogen on running wheel activity levels in WT, ßWT, and ßERKO mice of both sexes were more obvious during the second and third blocks compared with the first block. Except in {alpha} WT female mice, in which the interaction between block and treatment was not statistically significant, the placebo groups of these genotypes did not show such an increment in running wheel activity during the day. In the present study we used EB to ensure stimulatory action of systemically administered estrogen on running wheel activity, particularly in {alpha} ERKO mice, in which most of the estrogen-inducible behaviors were severely disrupted. As EB has a longer half-life in the body than 17ß-estradiol, accumulation of the steroid over days might cause a gradual increase in running wheel activity in estrogen-treated groups. In {alpha} ERKO mice, on the other hand, all three treatment groups, including the placebo groups, showed a gradual increase in running wheel activity in both sexes. These findings suggest that running experience could itself stimulate running wheel activity in {alpha} ERKO mice even though estrogen failed to modify it.
Sex comparisons across the estrogenic regulation of running wheel activity4, 百拇医药
In the present study, there were no obvious sex differences in estrogen effects on running wheel activity. Both male and female ERKO failed to respond to EB treatment, whereas WT, ßWT, and ßERKO mice of both sexes showed responses to EB. These findings are consistent with a previous study in rats showing no sex differences in the response to estrogen after gonadectomy (4). The study by Gentry and Wade (4) also revealed that neonatal androgenization of female rats did not change the magnitude of the response to estrogen later in adulthood for the potentiation of running wheel activity, although they needed a longer latency to respond. Taken together, these findings suggest that facilitation of running wheel activity by estrogen is not a sexually dimorphic trait.4, 百拇医药
A number of studies in golden hamsters and rats have shown that there is a clear sex difference in estrogenic regulation of free running circadian activity rhythms (21, 22, 23, 24). In these studies it is implied that responsiveness to estrogen (e.g. shortened free running period, ) in adulthood is regulated by the neonatal steroid condition. Both males and neonatally androgenized females failed to show shortened {tau} in response to estrogen in adulthood during free running circadian rhythm tests. In the present study we measured only daily activity under a normal 12-h light, 12-h dark entrained condition. Therefore, it remains to be determined in future studies whether circadian systems measured by the use of running wheel activity are also affected by ER{alpha} and/or ERß gene disruption, and if so, if there is any sex difference. Potential roles of ER{alpha} and ERß genes in the regulation of the clock gene expression (25, 26) in the suprachiasmatic nucleus as well as other brain regions receiving input from it (e.g. paraventricular nucleus) need to be further investigated.
Although there was no sex difference in EB effects on running wheel activity, there was sex/knockout interaction in placebo groups of WT and ERKO mice. Unlike in open field activity, as discussed below, there was no genotype difference in running wheel activity in the placebo groups of female mice. On the other hand, the placebo control group of male ERKO mice tended to be more active than {alpha} WT mice in running wheel tests, particularly during the last 3 d of 9-d tests. These results suggest that running wheel activity in gonadectomized mice might be modulated by ligand-independent effects of ER{alpha} gene disruption in male mice.xf?{k, 百拇医药
Roles of two types of ERs in the regulation of open field activityxf?{k, 百拇医药
In contrast to a great potentiation of running wheel activity (up to 2- to 2.5-fold) by estrogen in both sexes, open field activity was only slightly affected by estrogen in females. In males there was no effect of estrogen in any genotype of mice. Differential effects of estrogen on running wheel activity and open field activity were also described with brain sitespecific implants of estrogen in female rats (9) and systemic estrogen administration in female mice (16).
We found that open field activity, both total and in the center area, before estrogen implants was significantly reduced in {alpha} ERKO females, whereas it tended to increase in ßERKO females compared with that in the respective WT control mice. A similar trend was noticed in the placebo control groups during the postimplant open field tests. On the other hand, no such genotype differences were observed in running wheel activity in the placebo control groups of female mice regardless of gene. We found previously that {alpha} ERKO female mice tended to be less active, particularly in the light compartment, during the dark/light transition tests regardless of gonadal state (20). Taken together, these findings suggest that the lack of ER{alpha} gene expression might by itself ligand-independently modify baseline activity in a novel environment (open field and dark/light transition tests), but not home cage activity (running wheel), in female mice. Furthermore, genotype differences between {alpha} ERKO and {alpha} WT female mice in both open field and dark/light transition (20) tests were consistent regardless of gonadal states, i.e. gonadally intact, gonadectomized, and gonadectomized plus estrogen treatment. These findings suggest that the reduction of activity in {alpha} ERKO female mice in these novel environment tests may be due to a combination of the lack of responsiveness to estrogen in adulthood as well as developmental effects of ER{alpha} gene disruption. During preimplant open field tests in the present study, {alpha} ERKO female mice resembled male {alpha} WT and {alpha} ERKO mice more than female {alpha} WT mice in terms of total activity, total distance, and moving time. We speculate that this type of behavioral masculinization in {alpha} ERKO female mice might be due to elevated stimulation of AR during neonatal development. Starting on postnatal d 9–21, the number of ligand-bound AR-immunoreactive cells in {alpha} ERKO female mouse brains was significantly higher than that in {alpha} WT females in the mPOA among other brain regions and was almost equivalent to that in male mouse brains (27).
ERß gene disruption, on the other hand, tended to increase open field activity in female mice. This is in marked contrast to a great reduction of open field activity and elevation of anxiety reported in ERß knockout female mice (28, 29), which were developed separately from those used in the present study. There are three important factors that might influence the differences in open field activity between these two studies. In the present study mice were tested under red light, whereas in the study by Krezel et al. (29), they were tested under brightly illuminated white light conditions. It is assumed that the latter is more sensitive than the former to reveal traits such as anxiety or emotionality. Secondly, we tested the mice after gonadectomy, but Krezel et al. tested gonadally intact mice. However, this may not be a major factor in explaining the behavioral differences, as the effects of ERß gene disruption might also be independent of the gonadal state at the time of testing, as we found in the case of ER{alpha} (see above). Thirdly, a more important difference between the two studies may be the age of the mice. Mice in the present study were tested at 10–12 wk, whereas those in Krezel’s study were tested when they were more than 7 months old. In fact, we found that in older ßERKO females (>30 wk of age), open field activity and radial maze activity during a spatial learning paradigm, both of which were performed under bright light illumination, were greatly reduced (30). It is possible, therefore, that the effects of ERß gene disruption may best appear in older female mice.
Acknowledgmentsl:4}j[, 百拇医药
The authors are thankful to Dr. C. J. Krebs for his molecular biological expertise, and Dr. C. Pavlides and Ms. L. Frank for critical and editorial reading of the manuscript.l:4}j[, 百拇医药
Received May 15, 2002.l:4}j[, 百拇医药
Accepted for publication September 23, 2002.l:4}j[, 百拇医药
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