Novel Actions of Estrogen Receptor-? on Anxiety-Related Behaviors
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内分泌学杂志 2005年第2期
Departments of Biomedical Sciences (T.D.L., W.C.J.C., R.J.H.) and Chemistry (T.R.), Colorado State University, Fort Collins, Colorado 80523
Address all correspondence and requests for reprints to: Trent D. Lund, Ph.D., Department of Biomedical Science, Colorado State University, Anatomy W103, 1617 Campus Delivery, Fort Collins, Colorado 80523-1670. E-mail: tlund@colostate.edu.
Abstract
Estrogens are reported to have both anxiogenic and anxiolytic properties. This dichotomous neurobiological response to estrogens may be mediated by the existence of two distinct estrogen receptor (ER) systems, ER and ER?. In brain, ER plays a critical role in regulating reproductive neuroendocrine function, whereas ER? may be more important in regulating nonreproductive functions. To determine whether estrogen’s anxiolytic actions could be mediated by ER?, we examined anxiety-related behaviors after treatment with ER subtype-selective agonists. Ovariectomized female rats, divided into four treatment groups, were injected with the selective ER? agonist diarylpropionitrile (DPN), the ER-selective agonist propyl-pyrazole-triol (PPT), 17?-estradiol, or vehicle daily for 4d. After injections, behavior was monitored in the elevated plus maze or open field. Rats treated with DPN showed significantly decreased anxiety-related behaviors in both behavioral paradigms. In the elevated plus maze, DPN significantly increased the number of open arm entries and time spent on the open arms of the maze. Furthermore, DPN significantly reduced, whereas PPT increased, anxiogenic behaviors such as the number of fecal boli and time spent grooming. In the open field, DPN-treated females made more rears, interacted more with a novel object, and spent more time in the middle of the open field than did control or PPT-treated rats. To confirm that DPN's anxiolytic actions are ER mediated, the nonselective ER antagonist tamoxifen was administered alone or in combination with DPN. Tamoxifen blocked the previously identified anxiolytic actions of DPN. Taken together, these findings suggest that the anxiolytic properties of estrogens are ER? mediated.
Introduction
ESTROGENS ARE WELL-ESTABLISHED regulators of mood in humans and animals. Its effects, however, are varied, with both anxiolytic and anxiogenic actions, depending on factors such as age and stage of the reproductive cycle. In women, reduced estrogen levels, particularly at the menopause, are associated with depression, sleep disturbance, irritability, anxiety, panic disorders, and cognitive dysfunction (1, 2, 3). Estrogen replacement therapy in postmenopausal women is consistently reported to improve mood, feelings of general well being, and learning as well as increase general activity levels (3, 4). In contrast, an anxiogenic role for estrogens is suggested by the significantly higher level of depression reported in women compared with men (5, 6, 7), a sex difference that emerges at the time of puberty (5, 6, 7). Furthermore, research by Schmidt et al. (8) identified that women with severe premenstrual dysphoria developed anxiety and other mood symptoms when treated with estradiol in combination with leuprolide, an agonist analog of GnRH.
Similar effects of estradiol on anxiety and mood have been reported in rodents. For example, elevated levels of estradiol achieved during rodent proestrus, or after exogenous hormone injections to ovariectomized (OVX) females, exert anxiolytic actions in the elevated plus maze (9, 10); consistently, when endogenous estrogens are removed, via OVX, behavioral indices of anxiety increase (10, 11, 12, 13, 15). However, these actions of estradiol in rodent models are not always consistent. In the open field test, where activity level has been negatively correlated with anxiety, estradiol treatment of OVX rats has been reported to increase (9, 12), reduce (15), or have no effect on activity (12).
The fact that estrogens have been reported to have both anxiolytic and anxiogenic activity is perhaps explained by the existence of two distinct receptor systems mediating the actions of estrogen: estrogen receptor (ER) and ER?. The roles that both receptors play in regulating mood and anxiety-related behaviors must be considered to fully understand the role of estrogens in behavior and pathology and to explain estrogen’s dichotomous neuropharmacology.
Previous studies have shown that ER is critical for reproductive function as demonstrated by the lack of fecundity in ER knockout mice (16). Female ER knockout mice are anovulatory, have a disruption in the neuroendocrine regulation of LH secretion, and are insensitive to the uterotrophic actions of estradiol (16). Conversely, ER? does not seem to be critical for reproductive function. ER? knockout females have reduced ovulatory function but remain fertile (16). ER? may, however, be critically involved in regulating nonreproductive behaviors and brain development such as synaptic plasticity in the basolateral amygdala and processing of emotional behavior (17).
A previous study has shown that female ER? knockout mice show increased anxiety and reduced cognitive function (17), whereas ER knockouts are not different from wild types. Furthermore, ER? is expressed in the amygdala and paraventricular nucleus of the hypothalamus, nuclei associated with fear and anxiety responses (18, 19). Thus, the abnormal behavior of ER? knockout mice may be the result of the inability of estradiol to act on these brain regions (17).
In the studies presented here, we used recently developed ER-subtype-selective agonists to directly test the hypothesis that either ER or ER? is specifically involved in altering anxiety-related behaviors. Diarylpropionitrile (DPN) is a subtype-selective agonist with a 70-fold greater relative binding affinity and 170-fold greater relative potency in transcription assays for ER? than with ER (20). Additionally, propyl-pyrazole-triol (PPT) (21), is selective for ER, with a 400-fold preference for ER and minimal binding to ER? (22). Before use in behavioral studies, we first characterized these compounds to show that they are ER subtype selective in our hands and can access the brain after peripheral injections.
Materials and Methods
Animals and treatments
Young (60- to 90-d-old) adult male and female Sprague Dawley rats were obtained from Charles Rivers Laboratories (Wilmington, MA) caged in pairs, housed in the Colorado State University vivarium, and maintained on a 12-h dark 12-h light schedule (lights on at 0700 h) with ad libitum access to food and water. One week after arrival, animals were gonadectomized under isofluorane anesthesia as previously described (23, 24).
Hormone treatments
One week after gonadectomy, animals were given a single daily sc injection for 4 d of either dimethylsulfoxide (DMSO; vehicle), 17?-estradiol (E2; 0.25 mg/kg), DPN (1.0 mg/kg), or PPT (1.0 mg/kg) in a total volume of 0.2 ml. The doses of DPN and PPT used in these studies correspond to effective doses, relative to E2, that have been previously established and published (25, 26). PPT at 1 mg/kg had the same effect as an E2 dose of 0.1 mg/kg on uterine weight (25), whereas DPN at 1 mg/kg had the same biological activity as an E2 dose of 0.1 mg/kg (26). One half hour after the final injection, animals underwent behavioral testing. Behavioral testing consisted of three noninvasive paradigms established as indicators of anxiety. Additionally, a subset of OVX females, treated as described above, were given concomitant injections of the nonselective ER antagonist tamoxifen [15.0 mg/kg, a dose known to effectively block ER in the rat brain (27, 28)]. Gonadectomized males were treated with vehicle or DPN and tested in only the elevated plus maze.
DPN synthesis
DPN was synthesized de novo as follows. To a homogeneous solution of sodium hydroxide (250 mmol) and tetrabutylammonium chloride (0.9 mmol) in water (10 ml) at 23 C, 4-(methoxyphenyl)-acetonitrile (60 mmol) and 4-(methoxybenzyl)-chloride (40 mmol) was added dropwise, and the mixture was stirred for 1 h at room temperature. The solution was then diluted with 20 ml of water and extracted three times with 20 ml diethyl ether. The organic phases were combined and washed with a solution of 1 N HCl (20 ml). The resulting solution was then dried over magnesium sulfate, filtered, concentrated in vacuo, and purified by flash column chromatography. Deprotection of the hydroxyl groups was achieved using three to five equivalents of boron tribromide, added dropwise to a solution of 1 equivalent of DPN precursor and 1 equivalent CH2Cl2 (0.1 M) at –78 C (29). The reaction was kept at –78 C for 1 h and then allowed to warm to room temperature while stirring for 16 h. The mixture was then cooled to 0 C and quenched with 15–25 ml water. This mixture was then extracted three times with ethyl acetate, and the combined organic layers were dried over sodium sulfate. After filtration, solvent was then removed in vacuo, and the product was purified using flash chromatography and/or recrystallization from the MeOH/CH2Cl2 mixtures. Purity of the resulting compound was checked with nuclear magnetic resonance. PPT was purchased from Tocris Cookson Inc. (Ellisville, MO); E2 was purchased from Sigma Chemical Co. (St. Louis, MO).
Elevated plus maze
Maze performance was evaluated as previously described (30, 31). The following measures were quantitated: (1) latency to enter the first arm; (2) the number of open and closed arm entries; (3) total time spent in open arms, closed arms and the center; (4) the total time spent grooming; (5) the number of fecal boli.
Open field test
The open field test was conducted as previously described (32). Measures scored in the test included 1) activity (total square crossings), 2) rearing, 3) grooming, 4) active sniffing, 5) gnawing, 6) head dips, and 7) number of fecal boli.
Light/dark box
Activity in the light/dark box was determined as previously described (33, 34). The following measures were scored: 1) initial latency to enter the dark compartment, 2) number of compartment entries, and 3) total time spent in each compartment. All behavioral testing was done between 0900 and 1200 h.
Uterine weight
Immediately after death, uteri were removed from the animal, dissected free of fat and connective tissue, and weighed.
Plasma corticosterone analysis
At killing, trunk blood was collected into ice-chilled tubes containing 0.5 M EDTA (200 μl) and 4 μg/ml aprotinin (100 μl). Blood was centrifuged and plasma removed and stored at –20 C until assayed for corticosterone via RIA as previously described (35, 36) using rabbit anticorticosterone serum (ICN Biomedicals, Inc., Costa Mesa, CA) at a final dilution of 1: 2,000 according to manufacturer’s protocol. Standard curves were constructed from dilutions (5–500 ng/ml) of corticosterone (4-pregnen-11?, 21-diol-3, 20-dione; Steraloids, Wilton, NH).
Progesterone receptor (PR) immunocytochemistry
PR expression in the medial preoptic nucleus (MPN) was visualized in a subset of females after hormone treatments using a polyclonal antibody raised in rabbit and directed against the DNA-binding domain (amino acids 533–547) of the human PR-A and PR-B (37, 38). Fresh-frozen cryostat sections (16 μm) through the MPN were 1) immersion fixed in 0.1 M phosphate buffer containing 5% acrolein for 2 h at room temperature (RT) and rinsed three times for 10 min each with Tris-buffered saline (TBS; 0.05 M Tris, 0.9% NaCl, pH 7.6), 2) treated with 0.1% NaBH4 made in TBS for 15 min, 3) rinsed three times for 10 min each with TBS, 4) treated with 0.3% H2O2 made in TBS for 15 min, 5) rinsed three times for 10 min each with TBS, 6) incubated with TBS containing 20% normal goat serum and 0.3% Triton X-100 (Sigma) for 1 h at RT, 7) incubated with 1:1000 purified rabbit anti-PR polyclonal (Dako Corp., Glostrup, Denmark) diluted with TBS containing 2% normal goat serum and 0.3% Triton X-100 (Sigma) for two times overnight at 4 C, 8) rinsed three times for 10 min each with TBS, 9) incubated with biotinylated goat antirabbit (Vector Laboratories, Burlingame, CA) 1:600 diluted in TBS containing 2% normal goat serum and 0.3% Triton X-100 for 60 min at RT, 10) rinsed three times for 10 min each with TBS, 11) incubated with ABC Elite kit (Vector) 1:800 diluted with TBS for 60 min at RT, 12) rinsed three times for 10 min each with TBS, 13) reacted with 0.05% diaminobenzidine, 0.25% nickel ammonium sulfate, 0.01% H2O2 made in TBS for 10 min, 14) rinsed three times for 10 min each with TBS, and 15) dehydrated with increasing grades of alcohol and xylene and coverslipped using entellan (Merck, Darmstadt, Germany).
Binding assay
Cytosolic and nuclear ER concentration for hypothalamic preoptic area (HPOA), septum, and amygdala were determined by in vitro binding assay (35, 36). Protein was determined by the method of Lowry et al. (39), and DNA was determined by the method of Burton (40).
Synthesis of hormone receptor proteins
Full-length human ER expression vector (pSG5-ER; R. H. Price, University of California, San Francisco) and rat ER? expression vector (pcDNA-ER?; T. A. Brown, Pfizer, Groton, CT) were synthesized in vitro using the TnT-coupled reticulocyte lysate system (Promega, Madison, WI) with T7-RNA polymerase, during a 90-min reaction at 30 C. Translation reaction mixtures were stored at –80 C until used.
Saturation isotherms
To calculate and confirm the binding affinity of PPT and DPN for ER subtypes, 100-μl aliquots of reticulocyte lysate were incubated at optimal time and temperature, 90 min at room temperature (ER?) and 18 h at 4 C (ER), with increasing (0.01–50 nM) concentrations of [3H]E2. These times were determined empirically and represent optimal binding of receptor with E2. Nonspecific binding was assessed using a 200-fold excess of the ER agonist, diethylstilbestrol, in parallel tubes. After incubation, bound and unbound [3H]E2 were separated by passing the incubation reaction through a 1-ml lipophilic Sephadex LH-20 (Sigma) column according to previously published protocols (23, 41).
Statistics and analysis
Where appropriate, data were analyzed by ANOVA statistics followed by Newman-Keuls post hoc tests. Significance was set at P < 0.05. Curve fitting, scientific graphing, and analysis were completed using GraphPad Software (GraphPad Prism 3.0, San Diego, CA).
Results
Screen of subtype-selective agonist binding
Competition binding studies using in vitro translated ERs were conducted to calculate and confirm the ER binding properties and subtype preference of PPT and DPN. Our findings corroborate the previously published affinities and selectivity for both PPT and DPN (20, 21, 22, 25, 26). Based on the ability of PPT to compete with [3H]E2 for ER binding, its selectivity for ER subtypes was confirmed, with PPT having a much higher affinity for ER than for ER?. In contrast, DPN bound with higher affinity to ER? than to ER. E2 had a similar affinity for both ER and ER?. A summary of these results is presented in Table 1.
TABLE 1. Binding affinities of selected compounds for ER and ER?
Peripherally administered DPN can bind ER in the brain
PPT appears to cross the blood-brain barrier based upon its ability to up-regulate hypothalamic PR mRNA expression after peripheral administration (25). To determine whether peripherally administered DPN can also access the brain, we examined the concentration of occupied and unoccupied ER at several time points after DPN treatment using a standard in vitro binding assay (34, 35). This approach uses differential centrifugation to separate unoccupied receptor, which is found in the cytosolic fraction after high-speed centrifugation, from occupied receptor, which must be salt extracted from the cell nuclear fraction.
A single injection of DPN to OVX rats produced a significant increase in occupied, nuclear ER in the HPOA (one-way ANOVA F4,23 = 13.51; P < 0.0001) (Fig. 1A). This was accompanied by a stoichiometric decrease in unoccupied cytosolic ER (F4,23 = 8.01; P < 0.01) (Fig. 1B) in this same area compared with controls. Furthermore, both occupied and unoccupied receptors showed changes in level within 30 min (nuclear, P < 0.01; cytosolic, P < 0.01) after injection. This response was maintained (albeit decreased) at 6 h (occupied receptor, P < 0.01; unoccupied receptor, P < 0.01) and 12 h (occupied receptor, P < 0.01; unoccupied receptor, P < 0.01) after the injection. A similar pattern was identified in amygdala [occupied, F4,23 = 3.97, P < 0.01 (Fig. 1C); unoccupied, F4,23 = 6.52, P < 0.01 (Fig. 1D)] and septum (occupied, F4,23 = 3.67, P < 0.01; unoccupied, F4,23 = 24.00, P < 0.01) ER (septum data not shown graphically). Based on these results, we calculate that the half-life of DPN binding in brain is 7.87 (± 1.03) h.
FIG. 1. DPN causes time-dependent stoichiometric changes in occupied and unoccupied estrogen receptors. Binding of [3H]E2 from the nuclear (occupied receptor) fraction (A and C) and the cytosolic fraction (unoccupied receptor) was determined in the HPOA (A and B), amygdala (C and D), and septum (data not shown) at 30 min and 6, 12, or 24 h after a single injection of DPN (1 mg/kg). *, Significant difference compared with controls (white bars; untreated).
DPN does not up-regulate PR immunoreactivity (PR-ir) in brain
Using immunocytochemistry, we found that peripheral administration of E2 (0.25 mg/kg) and PPT (1 mg/kg) up-regulated PR-ir in the MPN of OVX females (Fig. 2, A–C). However, DPN treatment failed to increase PR-ir at the dose used in all of our studies (1 mg/kg body weight). PR-ir was indistinguishable between DPN-treated and DMSO-treated (Fig. 2A) females. Thus, because PR up-regulation seems to be an ER-mediated event, the failure of DPN to up-regulate PR suggests strongly that DPN has no ER activity at the dose used here.
FIG. 2. Representative photomicrographs of PR-ir in the medial preoptic area of OVX females after four daily injections of DMSO (A) (vehicle control), E2 (B), PPT (C), or DPN (D). Arrows indicate representative PR-ir cells. 3v, Third ventricle. Original magnification, x400.
DPN is not uterotrophic
Uterus weights differed significantly among OVX females treated sc once per day with DMSO (control) E2, PPT, and DPN (F3,28 = 12.81; P < 0.01) for 4 d. Treatments of E2 and PPT caused a significant increase in weight compared with controls [E2, 0.631 ± 0.014 g (mean ± SEM); PPT, 0.628 ± 0.063 g; control, 0.287 ± 0.014 g], whereas DPN (0.222 ± 0.010 g) treatment did not differ from controls.
DPN treatment decreases fear and anxiety behavior
To test our hypothesis that ER subtypes play differing but crucial roles in mediating fear and anxiety behaviors, we tested animals in three behavioral paradigms: the elevated plus maze, the open field test, and the light/dark box (a different set of animals was used for each paradigm).
Elevated plus maze.
In the elevated plus maze, rodents avoid the open, elevated, and brightly lit arms of the maze and prefer to remain in the more darkly lit, closed arms. A reduction in anxiety is indicated by an animal’s tendency to spend more time interacting with their environment through exploration (rearing and head dips) of the maze that leads to their entering and spending more time in the open arms of the maze.
When tested in the elevated plus maze, OVX females treated with DPN displayed significantly fewer anxiety-related behaviors. This was evidenced by increased numbers of entries onto the open arms (F3,31 = 9.58; P < 0.01) (Fig. 3A) and increased time spent on the open arms (F3,31 = 19.75; P < 0.01) (Fig. 3B) compared with control and PPT-treated animals. DPN-treated females also displayed more rearing behavior (F3,31 = 6.23; P < 0.01) (Fig. 3C) and more head dips (F3,31 = 6.73; P < 0.01) (Fig. 3D) compared with control and PPT-treated females. Furthermore, DPN significantly reduced, whereas PPT increased, anxiogenic behaviors such as the number of fecal boli (F3,31 = 15.34; P < 0.01) (Fig. 3E) and time spent grooming (F3,31 = 7.52; P < 0.01) (Fig. 3F) compared with control females. Consistent with this, plasma samples collected one half hour after removal from the maze showed that corticosterone levels were significantly reduced in DPN-treated animals and significantly increased in E2- and PPT-treated animals (F3,30 = 12.63; P < 0.01) (Fig. 4) relative to control females.
FIG. 3. DPN treatment of female rats reduced anxiety-related behavior in the elevated plus maze. The following behaviors were quantitated: open arm entries (A), time spent on the open arms (B), rears (C), head dips (D), fecal boli (E), and time spent grooming (F) after four daily injections of DPN, PPT, E2, or vehicle. n = 9 animals per treatment group; for PPT, n = 8. *, Significant difference (P < 0.01) compared with control treatment.
FIG. 4. DPN decreases plasma levels of corticosterone. Plasma corticosterone levels were determined from plasma samples taken one half hour after removal from the maze. Nonstressed controls were killed directly from their home cages. n = 5 animals per treatment group. *, Significant difference (P < 0.01) compared with control treatment 30 min after stress.
All animals tested in the maze had significantly higher plasma corticosterone levels compared with nonstressed controls (F1,30 = 555; P < 0.01) (Fig. 4) killed directly from their home cages. Nonstress corticosterone levels did not differ among treatments.
Open field test.
DPN treatment reduced anxiety-related behavior in the open field paradigm in a fashion similar to that observed in the elevated plus maze. In the open field, measure of time in the center of the arena vs. time in the perimeter gives a measure of anxiety-related behaviors. In a brightly lit open area, rats tend to stay near the walls of the open field and avoid the center. Less anxious animals spend more time engaging in exploratory behaviors as evidenced by increased time in the center, increased rearing, and increased encounters with novel objects.
DPN- and E2-treated females exhibited more rearing (F3,20 = 9.71; P < 0.01) (Fig. 5A) and more interaction with a novel object (F3,20 = 5.03; P < 0.01) (Fig. 5B) and spent more time in the middle of the maze of the open field (F3,20 = 7.30; P < 0.01) (Fig. 5C) than did control or PPT-treated females. However, in the assessment of general locomotor activity, PPT-treated females engaged in significantly more activity (total square crossings, F3,20 = 7.83; P < 0.01) (Fig. 5D) compared with all other treatment groups.
FIG. 5. DPN treatment of female rats reduced anxiety-related behavior in the novel open field. Animals were tested in a novel open field after four daily injections of DPN, PPT, E2, or vehicle. The number of rears (A), time spent exploring a novel item (B), time in the middle squares (C), and overall locomotor activity (total square crossings (D) were quantitated. n = 6 animals per treatment group. *, Significant difference (P < 0.01) compared with control treatment.
Light/dark box.
Consistent with our results above, OVX female rats treated with DPN showed significantly less anxious behavior in the light/dark box. The light/dark exploration task represents a naturalistic conflict between the tendencies of rodents to explore a novel environment vs. the tendency of rodents to avoid the brightly lit open area. DPN-treated females spent more time in (F1,8 = 27.57; P < 0.01) and made more transitions to (F1,8 = 10.00; P < 0.01) to the lit compartment than did control females (data not presented graphically).
DPN's anxiolytic actions are ER mediated
To establish that DPN's anxiolytic actions are ER mediated, we administered the nonselective ER antagonist tamoxifen alone or in combination with DPN or E2 to OVX females. In this study, we replicated our finding that DPN enhanced anxiolytic behaviors and also found that tamoxifen blocked the anxiolytic actions of both E2 and DPN. DPN and E2-treated females made more entries onto (F5,24 = 4.14; P < 0.01) (Fig. 6A) and spent more time on (F5,24 = 5.86; P < 0.01) (Fig. 6B) the open arms than control or tamoxifen-alone treated groups. Concomitant treatment of E2 or DPN with tamoxifen prevented these effects. Furthermore, the DPN-treated females displayed more head dips (F5,24 = 5.17; P < 0.01) (Fig. 6C) and fewer fecal boli (F5,24 = 2.50; P < 0.01) (Fig. 6D). Again, these effects were blocked by concomitant treatment with tamoxifen.
FIG. 6. The ER antagonist tamoxifen eliminated the anxiolytic action of DPN and E2 in the elevated plus maze. Animals were injected for four consecutive days with DPN, E2, or vehicle with or without concomitant injections of tamoxifen. Open arm entries (A), time spent on open arms (B), head dips (C), and fecal boli (D) were quantitated. n = 4–6 animals per treatment group Striped bars are used solely to indicate group location and represent no occurrences of behavior. *, Significant difference (P < 0.01) compared with control treatment.
DPN treatment decreases fear and anxiety behavior in male rats
To determine whether ER? regulates anxiety-related behaviors in gonadectomized male rats, similar to that observed in OVX females, we examined the ability of DPN to alter the behavior of 75-d-old adult male rat’s performance in the elevated plus maze. Gonadectomized males treated with DPN made significantly more entries onto the open arms (F1,14 = 5.03; P < 0.05) (Fig. 7A) and spent more time on the open arms (F1,14 = 6.22; P < 0.05) (Fig. 7B) than did vehicle-treated males. DPN-treated males also displayed more rearing behavior (F1,14 = 30.37; P < 0.01) (Fig. 7C), more head dips (F1,14 = 5.09; P < 0.05) (Fig. 7D), and less grooming behavior (F1,14 = 11.07; P < 0.01) and exhibited a trend toward a decrease in the number of fecal boli (F1,14 = 1.94; P = 0.10) compared with vehicle-treated males.
FIG. 7. DPN reduced anxiety-related behavior of male rats tested in the elevated plus maze. Gonadectomized male rats were treated with DPN or vehicle daily for four consecutive days and then tested for behavior on the elevated plus maze. The following behaviors were quantitated: open arm entries (A), time spent on the open arms (B), rears (C), and head dips (D). n = 8 animals per treatment group. *, Significant difference (P < 0.05) compared with control treatment.
Discussion
The results from the present study support the hypothesis that estrogen’s dichotomous action in regulating mood and anxiety-related behaviors are mediated by the opposing and distinct roles of ER and ER?. Our data show that the ER?-subtype-selective agonist DPN can cross the blood-brain barrier and occupy neural ERs with a half-life of approximately 8 h. In doing so, it is able to inhibit anxiogenic and enhance anxiolytic behaviors on three different tasks. These actions of DPN are blocked by concomitant treatment with tamoxifen, indicating that this is truly an ER-mediated event.
These studies used recently developed ER-subtype-selective agonists rather than knockout models to directly test the hypothesis that either ER or ER? is specifically involved in altering anxiety-related behaviors. Although a valuable resource, the use of knockout mouse models is of limited utility to address the present question. The absence of one receptor subtype may result in unrecognized differences in brain ontogeny or the potential development of compensatory mechanisms, or altered hormonal profiles (42, 43) may complicate the interpretation of data resulting from their use. Nonetheless, our data are largely consistent with that of Krezel et al. (17), who showed that female ER? knockout mice have an increase in anxiogenic behaviors in the elevated plus maze. In contrast, Krezel et al. (17) did not find that male ER? knockout mice had increased anxiogenic behaviors. However, our results clearly show that male rats can respond to DPN with an increase in anxiolytic behaviors, suggesting that ER? is not sex specific in its anxiolytic actions. The differences between our results and those of Krezel et al. (17) may be because of the presence vs. absence (our study) of testes, species differences, or the inherent differences between studying knockout models vs. wild-type animals.
DPN crosses the blood-brain barrier
Although PPT has been shown to have neural effects (e.g. the induction of PR mRNA) after systemic administration in vivo (25), it has not been previously shown whether peripherally administered DPN could effectively cross the blood-brain barrier. Our data show that within 30 min after peripheral administered DPN, increases in salt-extractable nuclear ER binding are detectable in brain, thus indicating that DPN accesses the brain. Increases in nuclear ER are accompanied by a stoichiometric decrease in unoccupied receptor in the cytosolic fraction of HPOA, amygdala, and septum, further indicating that these changes are not because of alterations in the total numbers of receptor, but rather because of activation of the existing receptor population. In all brain regions sampled, both occupied and unoccupied receptors showed changes within 30 min of the injection, a response that was maintained up to 12 h after the injection. These brain areas were chosen for sampling because ER? mRNA and protein are reportedly expressed at high levels within these brain areas (44, 45), and nuclei within these areas have been associated with fear and anxiety-related behaviors (18, 19).
DPN does not activate ER in brain
Notwithstanding DPN's effectiveness in crossing the blood-brain barrier and binding ERs, DPN, at the dose given in these studies (1 mg/kg), did not up-regulate PR-ir in the MPN. In contrast, we show that PPT can effectively increase PR-ir, which confirms previous findings identifying PPT’s ability to induce PR mRNA (25). Furthermore, because both PPT and E2, but not DPN, up-regulate PR-ir, this strongly suggests that PR-ir in the MPN is ER mediated. Such findings also indicate that DPN does not activate ER at the dose used here. In light of DPN's ability to bind ER in brain, our data suggest that DPN's action on behaviors is ER? mediated. Additionally, DPN, unlike E2 and PPT, did not alter uterine weights, suggesting that the dose of DPN given did not activate ER peripherally.
ER? mediates anxiety-related behavior
Peripheral administration of the selective ER? agonist DPN decreased anxiety-related behaviors in three different behavioral paradigms based on the natural conflict between their desires to explore a novel environment vs. avoidance of the brightly lit arena. In the light/dark box paradigm, DPN-treated females displayed less anxious behavior than control animals, spending more time on and making more transitions into the lit compartment. In the open field paradigm, similar observations were made such that both E2 and DPN decreased anxiety behavior. DPN- and E2-treated females spent more time in the middle, open squares and engaged with a novel object and exhibited more rearing behaviors than control females and females treated with the ER-selective ligand PPT. It is likely that this increased explorative behavior by E2- and DPN-treated females was responsible for the observed decrease in general locomotor activity. Because PPT-treated females engaged in less novel object exploration and rearing behavior than control or E2- or DPN-treated females, it follows that more of their time would be spent in general activity assessed as number of square crossings. It is, therefore, unlikely that the observed anxiolytic effects of DPN can be attributed solely to a general increase in locomotor activity.
In the elevated plus maze, when an additional anxiety-producing component was introduced by elevating the maze 3 ft above the ground, DPN- and E2-treated females had decreased levels of anxiety. Females treated with DPN had an increased number of rears and head dips and made more entries onto the open arms and subsequently spent more time on the open arms of the maze than PPT-treated and control animals. Additionally, the behavior of E2-treated females, in spending significantly more time on the open arms of the maze than control or PPT-treated females, also suggests a decrease in anxiety in E2-treated females relative to control and PPT-treated females. Furthermore, the anxiolytic actions of DPN are not limited to females. Males treated with DPN displayed far less anxiety-related behavior compared with controls. DPN-treated males relative to controls made more arm entries, spent more time on the open arms, and had increased rearing and head dips and decreased grooming and fecal boli. In contrast to DPN and in comparison with control and E2, OVX females treated with PPT displayed some increased anxiogenic behaviors. Although these behaviors, number of fecal boli, and time spent grooming are well established indicators of increased anxiety (30, 31, 46), PPT’s anxiogenic effect may not have manifested itself behaviorally in other measures because of floor effects in the test paradigm.
DPN's anxiolytic actions are ER mediated
The possibility exists that DPN's actions as an anxiolytic may not be entirely ER? mediated. For instance, it is possible that DPN may act in a manner similar to the benzodiazepines to reinforce transmission at GABA(A) receptors, or as serotonin (5-HT)(1A) receptor agonists and 5-HT reuptake inhibitors similar to the principal drugs currently employed in the management of anxiety disorders. In support of this, 5-HT-containing neurons in the raphe nucleus have been shown to contain ER? (47, 48, 49), and GABA-containing interneurons also appear to be ER? positive (50, 51). Nonetheless, the ability of tamoxifen to block DPN's anxiolytic actions strongly suggests that an ER is mediating its effects.
Although there are compounds that are superior to tamoxifen (i.e. ICI 182,780) in their ability to act as pure antagonists of ER, these compounds are incapable of crossing the blood-brain barrier. Unfortunately, to our knowledge there exists no available ER?-selective antagonist at the present time. Therefore, because it was critical that we block ER in brain, we were limited in these studies to the use of tamoxifen. In all treatment paradigms, tamoxifen had no effect of its own.
It is also highly likely that DPN's actions are because of binding of ER? and not because of nonselective interactions with ER. Although a recent publication suggests that a lower dose of DPN could also work to regulate mood (52), the higher dose used in these studies appears to be acting solely via ER?. The ER-selective agonist did not mimic the actions of DPN and often had effects opposite that of DPN. Furthermore, DPN's actions were different qualitatively and quantitatively from the mixed agonist E2. Moreover, DPN was ineffective in inducing PR expression in the MPN. Taken together, the above findings support the hypothesis that ER? is a critical factor in the regulation of anxiety-related behaviors and that in some cases ER and ER? may work in opposition to provide precise control of such behaviors.
DPN decreases plasma levels of corticosterone
After physical or psychological stressors, females typically display a more robust hormonal response than males (53, 54, 55). It appears that in males, androgens act to inhibit (53, 54, 55), whereas in females, estrogens function to enhance (35, 36, 53) the activity of the hypothalamo-pituitary adrenal axis. Endocrine manipulation studies in females show that OVX can reduce the stress-induced secretion of corticosterone, a reduction that is reversed via E2 administration (35, 36, 53). In the present study, 30 min after testing in the elevated plus maze, corticosterone levels were increased in females treated with E2 and PPT treatments but decreased by DPN-treated animals relative to OVX controls. This finding suggests that the aforementioned increase in circulating levels of corticosterone is a function of ER activation and that ER?’s action is consistently opposite that of ER and inhibitory to stress-induced hormone secretion. Because DPN decreases circulating levels of corticosterone, the possibility does exist that DPN's anxiolytic effects are caused at least in part by regulation of adrenal secretions, or the result could be epiphenomenal to the reduced anxiety in these animals. Research has identified that the anxiolytic action of other compounds (ipsapirone and buspirone) are prevented by adrenalectomy (14), making this a viable explanation of DPN's mechanism of action. Studies examining such an interaction are currently underway.
In summary, activation of ER? by the selective agonist DPN decreases anxiety-related behavior. Our results demonstrate that DPN crosses the blood-brain barrier to have its effect on behaviors. Furthermore, DPN decreases anxiety-related behavior in both sexes and across a variety of behavioral paradigms. Because the anxiolytic effects of DPN are extinguished by the ER antagonist tamoxifen, DPN's effects are likely ER mediated. Moreover, based upon DPN's selectivity to ER?, it is ER? that mediates the anxiolytic effects of estrogens. Thus, selectively targeting neural ER? activation in women may be an important factor to consider in the design of future therapeutics for anxiety and depression.
Acknowledgments
We thank D. Munson, J. Evans, M. McNulty, and D. Moore for their expert technical assistance.
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Address all correspondence and requests for reprints to: Trent D. Lund, Ph.D., Department of Biomedical Science, Colorado State University, Anatomy W103, 1617 Campus Delivery, Fort Collins, Colorado 80523-1670. E-mail: tlund@colostate.edu.
Abstract
Estrogens are reported to have both anxiogenic and anxiolytic properties. This dichotomous neurobiological response to estrogens may be mediated by the existence of two distinct estrogen receptor (ER) systems, ER and ER?. In brain, ER plays a critical role in regulating reproductive neuroendocrine function, whereas ER? may be more important in regulating nonreproductive functions. To determine whether estrogen’s anxiolytic actions could be mediated by ER?, we examined anxiety-related behaviors after treatment with ER subtype-selective agonists. Ovariectomized female rats, divided into four treatment groups, were injected with the selective ER? agonist diarylpropionitrile (DPN), the ER-selective agonist propyl-pyrazole-triol (PPT), 17?-estradiol, or vehicle daily for 4d. After injections, behavior was monitored in the elevated plus maze or open field. Rats treated with DPN showed significantly decreased anxiety-related behaviors in both behavioral paradigms. In the elevated plus maze, DPN significantly increased the number of open arm entries and time spent on the open arms of the maze. Furthermore, DPN significantly reduced, whereas PPT increased, anxiogenic behaviors such as the number of fecal boli and time spent grooming. In the open field, DPN-treated females made more rears, interacted more with a novel object, and spent more time in the middle of the open field than did control or PPT-treated rats. To confirm that DPN's anxiolytic actions are ER mediated, the nonselective ER antagonist tamoxifen was administered alone or in combination with DPN. Tamoxifen blocked the previously identified anxiolytic actions of DPN. Taken together, these findings suggest that the anxiolytic properties of estrogens are ER? mediated.
Introduction
ESTROGENS ARE WELL-ESTABLISHED regulators of mood in humans and animals. Its effects, however, are varied, with both anxiolytic and anxiogenic actions, depending on factors such as age and stage of the reproductive cycle. In women, reduced estrogen levels, particularly at the menopause, are associated with depression, sleep disturbance, irritability, anxiety, panic disorders, and cognitive dysfunction (1, 2, 3). Estrogen replacement therapy in postmenopausal women is consistently reported to improve mood, feelings of general well being, and learning as well as increase general activity levels (3, 4). In contrast, an anxiogenic role for estrogens is suggested by the significantly higher level of depression reported in women compared with men (5, 6, 7), a sex difference that emerges at the time of puberty (5, 6, 7). Furthermore, research by Schmidt et al. (8) identified that women with severe premenstrual dysphoria developed anxiety and other mood symptoms when treated with estradiol in combination with leuprolide, an agonist analog of GnRH.
Similar effects of estradiol on anxiety and mood have been reported in rodents. For example, elevated levels of estradiol achieved during rodent proestrus, or after exogenous hormone injections to ovariectomized (OVX) females, exert anxiolytic actions in the elevated plus maze (9, 10); consistently, when endogenous estrogens are removed, via OVX, behavioral indices of anxiety increase (10, 11, 12, 13, 15). However, these actions of estradiol in rodent models are not always consistent. In the open field test, where activity level has been negatively correlated with anxiety, estradiol treatment of OVX rats has been reported to increase (9, 12), reduce (15), or have no effect on activity (12).
The fact that estrogens have been reported to have both anxiolytic and anxiogenic activity is perhaps explained by the existence of two distinct receptor systems mediating the actions of estrogen: estrogen receptor (ER) and ER?. The roles that both receptors play in regulating mood and anxiety-related behaviors must be considered to fully understand the role of estrogens in behavior and pathology and to explain estrogen’s dichotomous neuropharmacology.
Previous studies have shown that ER is critical for reproductive function as demonstrated by the lack of fecundity in ER knockout mice (16). Female ER knockout mice are anovulatory, have a disruption in the neuroendocrine regulation of LH secretion, and are insensitive to the uterotrophic actions of estradiol (16). Conversely, ER? does not seem to be critical for reproductive function. ER? knockout females have reduced ovulatory function but remain fertile (16). ER? may, however, be critically involved in regulating nonreproductive behaviors and brain development such as synaptic plasticity in the basolateral amygdala and processing of emotional behavior (17).
A previous study has shown that female ER? knockout mice show increased anxiety and reduced cognitive function (17), whereas ER knockouts are not different from wild types. Furthermore, ER? is expressed in the amygdala and paraventricular nucleus of the hypothalamus, nuclei associated with fear and anxiety responses (18, 19). Thus, the abnormal behavior of ER? knockout mice may be the result of the inability of estradiol to act on these brain regions (17).
In the studies presented here, we used recently developed ER-subtype-selective agonists to directly test the hypothesis that either ER or ER? is specifically involved in altering anxiety-related behaviors. Diarylpropionitrile (DPN) is a subtype-selective agonist with a 70-fold greater relative binding affinity and 170-fold greater relative potency in transcription assays for ER? than with ER (20). Additionally, propyl-pyrazole-triol (PPT) (21), is selective for ER, with a 400-fold preference for ER and minimal binding to ER? (22). Before use in behavioral studies, we first characterized these compounds to show that they are ER subtype selective in our hands and can access the brain after peripheral injections.
Materials and Methods
Animals and treatments
Young (60- to 90-d-old) adult male and female Sprague Dawley rats were obtained from Charles Rivers Laboratories (Wilmington, MA) caged in pairs, housed in the Colorado State University vivarium, and maintained on a 12-h dark 12-h light schedule (lights on at 0700 h) with ad libitum access to food and water. One week after arrival, animals were gonadectomized under isofluorane anesthesia as previously described (23, 24).
Hormone treatments
One week after gonadectomy, animals were given a single daily sc injection for 4 d of either dimethylsulfoxide (DMSO; vehicle), 17?-estradiol (E2; 0.25 mg/kg), DPN (1.0 mg/kg), or PPT (1.0 mg/kg) in a total volume of 0.2 ml. The doses of DPN and PPT used in these studies correspond to effective doses, relative to E2, that have been previously established and published (25, 26). PPT at 1 mg/kg had the same effect as an E2 dose of 0.1 mg/kg on uterine weight (25), whereas DPN at 1 mg/kg had the same biological activity as an E2 dose of 0.1 mg/kg (26). One half hour after the final injection, animals underwent behavioral testing. Behavioral testing consisted of three noninvasive paradigms established as indicators of anxiety. Additionally, a subset of OVX females, treated as described above, were given concomitant injections of the nonselective ER antagonist tamoxifen [15.0 mg/kg, a dose known to effectively block ER in the rat brain (27, 28)]. Gonadectomized males were treated with vehicle or DPN and tested in only the elevated plus maze.
DPN synthesis
DPN was synthesized de novo as follows. To a homogeneous solution of sodium hydroxide (250 mmol) and tetrabutylammonium chloride (0.9 mmol) in water (10 ml) at 23 C, 4-(methoxyphenyl)-acetonitrile (60 mmol) and 4-(methoxybenzyl)-chloride (40 mmol) was added dropwise, and the mixture was stirred for 1 h at room temperature. The solution was then diluted with 20 ml of water and extracted three times with 20 ml diethyl ether. The organic phases were combined and washed with a solution of 1 N HCl (20 ml). The resulting solution was then dried over magnesium sulfate, filtered, concentrated in vacuo, and purified by flash column chromatography. Deprotection of the hydroxyl groups was achieved using three to five equivalents of boron tribromide, added dropwise to a solution of 1 equivalent of DPN precursor and 1 equivalent CH2Cl2 (0.1 M) at –78 C (29). The reaction was kept at –78 C for 1 h and then allowed to warm to room temperature while stirring for 16 h. The mixture was then cooled to 0 C and quenched with 15–25 ml water. This mixture was then extracted three times with ethyl acetate, and the combined organic layers were dried over sodium sulfate. After filtration, solvent was then removed in vacuo, and the product was purified using flash chromatography and/or recrystallization from the MeOH/CH2Cl2 mixtures. Purity of the resulting compound was checked with nuclear magnetic resonance. PPT was purchased from Tocris Cookson Inc. (Ellisville, MO); E2 was purchased from Sigma Chemical Co. (St. Louis, MO).
Elevated plus maze
Maze performance was evaluated as previously described (30, 31). The following measures were quantitated: (1) latency to enter the first arm; (2) the number of open and closed arm entries; (3) total time spent in open arms, closed arms and the center; (4) the total time spent grooming; (5) the number of fecal boli.
Open field test
The open field test was conducted as previously described (32). Measures scored in the test included 1) activity (total square crossings), 2) rearing, 3) grooming, 4) active sniffing, 5) gnawing, 6) head dips, and 7) number of fecal boli.
Light/dark box
Activity in the light/dark box was determined as previously described (33, 34). The following measures were scored: 1) initial latency to enter the dark compartment, 2) number of compartment entries, and 3) total time spent in each compartment. All behavioral testing was done between 0900 and 1200 h.
Uterine weight
Immediately after death, uteri were removed from the animal, dissected free of fat and connective tissue, and weighed.
Plasma corticosterone analysis
At killing, trunk blood was collected into ice-chilled tubes containing 0.5 M EDTA (200 μl) and 4 μg/ml aprotinin (100 μl). Blood was centrifuged and plasma removed and stored at –20 C until assayed for corticosterone via RIA as previously described (35, 36) using rabbit anticorticosterone serum (ICN Biomedicals, Inc., Costa Mesa, CA) at a final dilution of 1: 2,000 according to manufacturer’s protocol. Standard curves were constructed from dilutions (5–500 ng/ml) of corticosterone (4-pregnen-11?, 21-diol-3, 20-dione; Steraloids, Wilton, NH).
Progesterone receptor (PR) immunocytochemistry
PR expression in the medial preoptic nucleus (MPN) was visualized in a subset of females after hormone treatments using a polyclonal antibody raised in rabbit and directed against the DNA-binding domain (amino acids 533–547) of the human PR-A and PR-B (37, 38). Fresh-frozen cryostat sections (16 μm) through the MPN were 1) immersion fixed in 0.1 M phosphate buffer containing 5% acrolein for 2 h at room temperature (RT) and rinsed three times for 10 min each with Tris-buffered saline (TBS; 0.05 M Tris, 0.9% NaCl, pH 7.6), 2) treated with 0.1% NaBH4 made in TBS for 15 min, 3) rinsed three times for 10 min each with TBS, 4) treated with 0.3% H2O2 made in TBS for 15 min, 5) rinsed three times for 10 min each with TBS, 6) incubated with TBS containing 20% normal goat serum and 0.3% Triton X-100 (Sigma) for 1 h at RT, 7) incubated with 1:1000 purified rabbit anti-PR polyclonal (Dako Corp., Glostrup, Denmark) diluted with TBS containing 2% normal goat serum and 0.3% Triton X-100 (Sigma) for two times overnight at 4 C, 8) rinsed three times for 10 min each with TBS, 9) incubated with biotinylated goat antirabbit (Vector Laboratories, Burlingame, CA) 1:600 diluted in TBS containing 2% normal goat serum and 0.3% Triton X-100 for 60 min at RT, 10) rinsed three times for 10 min each with TBS, 11) incubated with ABC Elite kit (Vector) 1:800 diluted with TBS for 60 min at RT, 12) rinsed three times for 10 min each with TBS, 13) reacted with 0.05% diaminobenzidine, 0.25% nickel ammonium sulfate, 0.01% H2O2 made in TBS for 10 min, 14) rinsed three times for 10 min each with TBS, and 15) dehydrated with increasing grades of alcohol and xylene and coverslipped using entellan (Merck, Darmstadt, Germany).
Binding assay
Cytosolic and nuclear ER concentration for hypothalamic preoptic area (HPOA), septum, and amygdala were determined by in vitro binding assay (35, 36). Protein was determined by the method of Lowry et al. (39), and DNA was determined by the method of Burton (40).
Synthesis of hormone receptor proteins
Full-length human ER expression vector (pSG5-ER; R. H. Price, University of California, San Francisco) and rat ER? expression vector (pcDNA-ER?; T. A. Brown, Pfizer, Groton, CT) were synthesized in vitro using the TnT-coupled reticulocyte lysate system (Promega, Madison, WI) with T7-RNA polymerase, during a 90-min reaction at 30 C. Translation reaction mixtures were stored at –80 C until used.
Saturation isotherms
To calculate and confirm the binding affinity of PPT and DPN for ER subtypes, 100-μl aliquots of reticulocyte lysate were incubated at optimal time and temperature, 90 min at room temperature (ER?) and 18 h at 4 C (ER), with increasing (0.01–50 nM) concentrations of [3H]E2. These times were determined empirically and represent optimal binding of receptor with E2. Nonspecific binding was assessed using a 200-fold excess of the ER agonist, diethylstilbestrol, in parallel tubes. After incubation, bound and unbound [3H]E2 were separated by passing the incubation reaction through a 1-ml lipophilic Sephadex LH-20 (Sigma) column according to previously published protocols (23, 41).
Statistics and analysis
Where appropriate, data were analyzed by ANOVA statistics followed by Newman-Keuls post hoc tests. Significance was set at P < 0.05. Curve fitting, scientific graphing, and analysis were completed using GraphPad Software (GraphPad Prism 3.0, San Diego, CA).
Results
Screen of subtype-selective agonist binding
Competition binding studies using in vitro translated ERs were conducted to calculate and confirm the ER binding properties and subtype preference of PPT and DPN. Our findings corroborate the previously published affinities and selectivity for both PPT and DPN (20, 21, 22, 25, 26). Based on the ability of PPT to compete with [3H]E2 for ER binding, its selectivity for ER subtypes was confirmed, with PPT having a much higher affinity for ER than for ER?. In contrast, DPN bound with higher affinity to ER? than to ER. E2 had a similar affinity for both ER and ER?. A summary of these results is presented in Table 1.
TABLE 1. Binding affinities of selected compounds for ER and ER?
Peripherally administered DPN can bind ER in the brain
PPT appears to cross the blood-brain barrier based upon its ability to up-regulate hypothalamic PR mRNA expression after peripheral administration (25). To determine whether peripherally administered DPN can also access the brain, we examined the concentration of occupied and unoccupied ER at several time points after DPN treatment using a standard in vitro binding assay (34, 35). This approach uses differential centrifugation to separate unoccupied receptor, which is found in the cytosolic fraction after high-speed centrifugation, from occupied receptor, which must be salt extracted from the cell nuclear fraction.
A single injection of DPN to OVX rats produced a significant increase in occupied, nuclear ER in the HPOA (one-way ANOVA F4,23 = 13.51; P < 0.0001) (Fig. 1A). This was accompanied by a stoichiometric decrease in unoccupied cytosolic ER (F4,23 = 8.01; P < 0.01) (Fig. 1B) in this same area compared with controls. Furthermore, both occupied and unoccupied receptors showed changes in level within 30 min (nuclear, P < 0.01; cytosolic, P < 0.01) after injection. This response was maintained (albeit decreased) at 6 h (occupied receptor, P < 0.01; unoccupied receptor, P < 0.01) and 12 h (occupied receptor, P < 0.01; unoccupied receptor, P < 0.01) after the injection. A similar pattern was identified in amygdala [occupied, F4,23 = 3.97, P < 0.01 (Fig. 1C); unoccupied, F4,23 = 6.52, P < 0.01 (Fig. 1D)] and septum (occupied, F4,23 = 3.67, P < 0.01; unoccupied, F4,23 = 24.00, P < 0.01) ER (septum data not shown graphically). Based on these results, we calculate that the half-life of DPN binding in brain is 7.87 (± 1.03) h.
FIG. 1. DPN causes time-dependent stoichiometric changes in occupied and unoccupied estrogen receptors. Binding of [3H]E2 from the nuclear (occupied receptor) fraction (A and C) and the cytosolic fraction (unoccupied receptor) was determined in the HPOA (A and B), amygdala (C and D), and septum (data not shown) at 30 min and 6, 12, or 24 h after a single injection of DPN (1 mg/kg). *, Significant difference compared with controls (white bars; untreated).
DPN does not up-regulate PR immunoreactivity (PR-ir) in brain
Using immunocytochemistry, we found that peripheral administration of E2 (0.25 mg/kg) and PPT (1 mg/kg) up-regulated PR-ir in the MPN of OVX females (Fig. 2, A–C). However, DPN treatment failed to increase PR-ir at the dose used in all of our studies (1 mg/kg body weight). PR-ir was indistinguishable between DPN-treated and DMSO-treated (Fig. 2A) females. Thus, because PR up-regulation seems to be an ER-mediated event, the failure of DPN to up-regulate PR suggests strongly that DPN has no ER activity at the dose used here.
FIG. 2. Representative photomicrographs of PR-ir in the medial preoptic area of OVX females after four daily injections of DMSO (A) (vehicle control), E2 (B), PPT (C), or DPN (D). Arrows indicate representative PR-ir cells. 3v, Third ventricle. Original magnification, x400.
DPN is not uterotrophic
Uterus weights differed significantly among OVX females treated sc once per day with DMSO (control) E2, PPT, and DPN (F3,28 = 12.81; P < 0.01) for 4 d. Treatments of E2 and PPT caused a significant increase in weight compared with controls [E2, 0.631 ± 0.014 g (mean ± SEM); PPT, 0.628 ± 0.063 g; control, 0.287 ± 0.014 g], whereas DPN (0.222 ± 0.010 g) treatment did not differ from controls.
DPN treatment decreases fear and anxiety behavior
To test our hypothesis that ER subtypes play differing but crucial roles in mediating fear and anxiety behaviors, we tested animals in three behavioral paradigms: the elevated plus maze, the open field test, and the light/dark box (a different set of animals was used for each paradigm).
Elevated plus maze.
In the elevated plus maze, rodents avoid the open, elevated, and brightly lit arms of the maze and prefer to remain in the more darkly lit, closed arms. A reduction in anxiety is indicated by an animal’s tendency to spend more time interacting with their environment through exploration (rearing and head dips) of the maze that leads to their entering and spending more time in the open arms of the maze.
When tested in the elevated plus maze, OVX females treated with DPN displayed significantly fewer anxiety-related behaviors. This was evidenced by increased numbers of entries onto the open arms (F3,31 = 9.58; P < 0.01) (Fig. 3A) and increased time spent on the open arms (F3,31 = 19.75; P < 0.01) (Fig. 3B) compared with control and PPT-treated animals. DPN-treated females also displayed more rearing behavior (F3,31 = 6.23; P < 0.01) (Fig. 3C) and more head dips (F3,31 = 6.73; P < 0.01) (Fig. 3D) compared with control and PPT-treated females. Furthermore, DPN significantly reduced, whereas PPT increased, anxiogenic behaviors such as the number of fecal boli (F3,31 = 15.34; P < 0.01) (Fig. 3E) and time spent grooming (F3,31 = 7.52; P < 0.01) (Fig. 3F) compared with control females. Consistent with this, plasma samples collected one half hour after removal from the maze showed that corticosterone levels were significantly reduced in DPN-treated animals and significantly increased in E2- and PPT-treated animals (F3,30 = 12.63; P < 0.01) (Fig. 4) relative to control females.
FIG. 3. DPN treatment of female rats reduced anxiety-related behavior in the elevated plus maze. The following behaviors were quantitated: open arm entries (A), time spent on the open arms (B), rears (C), head dips (D), fecal boli (E), and time spent grooming (F) after four daily injections of DPN, PPT, E2, or vehicle. n = 9 animals per treatment group; for PPT, n = 8. *, Significant difference (P < 0.01) compared with control treatment.
FIG. 4. DPN decreases plasma levels of corticosterone. Plasma corticosterone levels were determined from plasma samples taken one half hour after removal from the maze. Nonstressed controls were killed directly from their home cages. n = 5 animals per treatment group. *, Significant difference (P < 0.01) compared with control treatment 30 min after stress.
All animals tested in the maze had significantly higher plasma corticosterone levels compared with nonstressed controls (F1,30 = 555; P < 0.01) (Fig. 4) killed directly from their home cages. Nonstress corticosterone levels did not differ among treatments.
Open field test.
DPN treatment reduced anxiety-related behavior in the open field paradigm in a fashion similar to that observed in the elevated plus maze. In the open field, measure of time in the center of the arena vs. time in the perimeter gives a measure of anxiety-related behaviors. In a brightly lit open area, rats tend to stay near the walls of the open field and avoid the center. Less anxious animals spend more time engaging in exploratory behaviors as evidenced by increased time in the center, increased rearing, and increased encounters with novel objects.
DPN- and E2-treated females exhibited more rearing (F3,20 = 9.71; P < 0.01) (Fig. 5A) and more interaction with a novel object (F3,20 = 5.03; P < 0.01) (Fig. 5B) and spent more time in the middle of the maze of the open field (F3,20 = 7.30; P < 0.01) (Fig. 5C) than did control or PPT-treated females. However, in the assessment of general locomotor activity, PPT-treated females engaged in significantly more activity (total square crossings, F3,20 = 7.83; P < 0.01) (Fig. 5D) compared with all other treatment groups.
FIG. 5. DPN treatment of female rats reduced anxiety-related behavior in the novel open field. Animals were tested in a novel open field after four daily injections of DPN, PPT, E2, or vehicle. The number of rears (A), time spent exploring a novel item (B), time in the middle squares (C), and overall locomotor activity (total square crossings (D) were quantitated. n = 6 animals per treatment group. *, Significant difference (P < 0.01) compared with control treatment.
Light/dark box.
Consistent with our results above, OVX female rats treated with DPN showed significantly less anxious behavior in the light/dark box. The light/dark exploration task represents a naturalistic conflict between the tendencies of rodents to explore a novel environment vs. the tendency of rodents to avoid the brightly lit open area. DPN-treated females spent more time in (F1,8 = 27.57; P < 0.01) and made more transitions to (F1,8 = 10.00; P < 0.01) to the lit compartment than did control females (data not presented graphically).
DPN's anxiolytic actions are ER mediated
To establish that DPN's anxiolytic actions are ER mediated, we administered the nonselective ER antagonist tamoxifen alone or in combination with DPN or E2 to OVX females. In this study, we replicated our finding that DPN enhanced anxiolytic behaviors and also found that tamoxifen blocked the anxiolytic actions of both E2 and DPN. DPN and E2-treated females made more entries onto (F5,24 = 4.14; P < 0.01) (Fig. 6A) and spent more time on (F5,24 = 5.86; P < 0.01) (Fig. 6B) the open arms than control or tamoxifen-alone treated groups. Concomitant treatment of E2 or DPN with tamoxifen prevented these effects. Furthermore, the DPN-treated females displayed more head dips (F5,24 = 5.17; P < 0.01) (Fig. 6C) and fewer fecal boli (F5,24 = 2.50; P < 0.01) (Fig. 6D). Again, these effects were blocked by concomitant treatment with tamoxifen.
FIG. 6. The ER antagonist tamoxifen eliminated the anxiolytic action of DPN and E2 in the elevated plus maze. Animals were injected for four consecutive days with DPN, E2, or vehicle with or without concomitant injections of tamoxifen. Open arm entries (A), time spent on open arms (B), head dips (C), and fecal boli (D) were quantitated. n = 4–6 animals per treatment group Striped bars are used solely to indicate group location and represent no occurrences of behavior. *, Significant difference (P < 0.01) compared with control treatment.
DPN treatment decreases fear and anxiety behavior in male rats
To determine whether ER? regulates anxiety-related behaviors in gonadectomized male rats, similar to that observed in OVX females, we examined the ability of DPN to alter the behavior of 75-d-old adult male rat’s performance in the elevated plus maze. Gonadectomized males treated with DPN made significantly more entries onto the open arms (F1,14 = 5.03; P < 0.05) (Fig. 7A) and spent more time on the open arms (F1,14 = 6.22; P < 0.05) (Fig. 7B) than did vehicle-treated males. DPN-treated males also displayed more rearing behavior (F1,14 = 30.37; P < 0.01) (Fig. 7C), more head dips (F1,14 = 5.09; P < 0.05) (Fig. 7D), and less grooming behavior (F1,14 = 11.07; P < 0.01) and exhibited a trend toward a decrease in the number of fecal boli (F1,14 = 1.94; P = 0.10) compared with vehicle-treated males.
FIG. 7. DPN reduced anxiety-related behavior of male rats tested in the elevated plus maze. Gonadectomized male rats were treated with DPN or vehicle daily for four consecutive days and then tested for behavior on the elevated plus maze. The following behaviors were quantitated: open arm entries (A), time spent on the open arms (B), rears (C), and head dips (D). n = 8 animals per treatment group. *, Significant difference (P < 0.05) compared with control treatment.
Discussion
The results from the present study support the hypothesis that estrogen’s dichotomous action in regulating mood and anxiety-related behaviors are mediated by the opposing and distinct roles of ER and ER?. Our data show that the ER?-subtype-selective agonist DPN can cross the blood-brain barrier and occupy neural ERs with a half-life of approximately 8 h. In doing so, it is able to inhibit anxiogenic and enhance anxiolytic behaviors on three different tasks. These actions of DPN are blocked by concomitant treatment with tamoxifen, indicating that this is truly an ER-mediated event.
These studies used recently developed ER-subtype-selective agonists rather than knockout models to directly test the hypothesis that either ER or ER? is specifically involved in altering anxiety-related behaviors. Although a valuable resource, the use of knockout mouse models is of limited utility to address the present question. The absence of one receptor subtype may result in unrecognized differences in brain ontogeny or the potential development of compensatory mechanisms, or altered hormonal profiles (42, 43) may complicate the interpretation of data resulting from their use. Nonetheless, our data are largely consistent with that of Krezel et al. (17), who showed that female ER? knockout mice have an increase in anxiogenic behaviors in the elevated plus maze. In contrast, Krezel et al. (17) did not find that male ER? knockout mice had increased anxiogenic behaviors. However, our results clearly show that male rats can respond to DPN with an increase in anxiolytic behaviors, suggesting that ER? is not sex specific in its anxiolytic actions. The differences between our results and those of Krezel et al. (17) may be because of the presence vs. absence (our study) of testes, species differences, or the inherent differences between studying knockout models vs. wild-type animals.
DPN crosses the blood-brain barrier
Although PPT has been shown to have neural effects (e.g. the induction of PR mRNA) after systemic administration in vivo (25), it has not been previously shown whether peripherally administered DPN could effectively cross the blood-brain barrier. Our data show that within 30 min after peripheral administered DPN, increases in salt-extractable nuclear ER binding are detectable in brain, thus indicating that DPN accesses the brain. Increases in nuclear ER are accompanied by a stoichiometric decrease in unoccupied receptor in the cytosolic fraction of HPOA, amygdala, and septum, further indicating that these changes are not because of alterations in the total numbers of receptor, but rather because of activation of the existing receptor population. In all brain regions sampled, both occupied and unoccupied receptors showed changes within 30 min of the injection, a response that was maintained up to 12 h after the injection. These brain areas were chosen for sampling because ER? mRNA and protein are reportedly expressed at high levels within these brain areas (44, 45), and nuclei within these areas have been associated with fear and anxiety-related behaviors (18, 19).
DPN does not activate ER in brain
Notwithstanding DPN's effectiveness in crossing the blood-brain barrier and binding ERs, DPN, at the dose given in these studies (1 mg/kg), did not up-regulate PR-ir in the MPN. In contrast, we show that PPT can effectively increase PR-ir, which confirms previous findings identifying PPT’s ability to induce PR mRNA (25). Furthermore, because both PPT and E2, but not DPN, up-regulate PR-ir, this strongly suggests that PR-ir in the MPN is ER mediated. Such findings also indicate that DPN does not activate ER at the dose used here. In light of DPN's ability to bind ER in brain, our data suggest that DPN's action on behaviors is ER? mediated. Additionally, DPN, unlike E2 and PPT, did not alter uterine weights, suggesting that the dose of DPN given did not activate ER peripherally.
ER? mediates anxiety-related behavior
Peripheral administration of the selective ER? agonist DPN decreased anxiety-related behaviors in three different behavioral paradigms based on the natural conflict between their desires to explore a novel environment vs. avoidance of the brightly lit arena. In the light/dark box paradigm, DPN-treated females displayed less anxious behavior than control animals, spending more time on and making more transitions into the lit compartment. In the open field paradigm, similar observations were made such that both E2 and DPN decreased anxiety behavior. DPN- and E2-treated females spent more time in the middle, open squares and engaged with a novel object and exhibited more rearing behaviors than control females and females treated with the ER-selective ligand PPT. It is likely that this increased explorative behavior by E2- and DPN-treated females was responsible for the observed decrease in general locomotor activity. Because PPT-treated females engaged in less novel object exploration and rearing behavior than control or E2- or DPN-treated females, it follows that more of their time would be spent in general activity assessed as number of square crossings. It is, therefore, unlikely that the observed anxiolytic effects of DPN can be attributed solely to a general increase in locomotor activity.
In the elevated plus maze, when an additional anxiety-producing component was introduced by elevating the maze 3 ft above the ground, DPN- and E2-treated females had decreased levels of anxiety. Females treated with DPN had an increased number of rears and head dips and made more entries onto the open arms and subsequently spent more time on the open arms of the maze than PPT-treated and control animals. Additionally, the behavior of E2-treated females, in spending significantly more time on the open arms of the maze than control or PPT-treated females, also suggests a decrease in anxiety in E2-treated females relative to control and PPT-treated females. Furthermore, the anxiolytic actions of DPN are not limited to females. Males treated with DPN displayed far less anxiety-related behavior compared with controls. DPN-treated males relative to controls made more arm entries, spent more time on the open arms, and had increased rearing and head dips and decreased grooming and fecal boli. In contrast to DPN and in comparison with control and E2, OVX females treated with PPT displayed some increased anxiogenic behaviors. Although these behaviors, number of fecal boli, and time spent grooming are well established indicators of increased anxiety (30, 31, 46), PPT’s anxiogenic effect may not have manifested itself behaviorally in other measures because of floor effects in the test paradigm.
DPN's anxiolytic actions are ER mediated
The possibility exists that DPN's actions as an anxiolytic may not be entirely ER? mediated. For instance, it is possible that DPN may act in a manner similar to the benzodiazepines to reinforce transmission at GABA(A) receptors, or as serotonin (5-HT)(1A) receptor agonists and 5-HT reuptake inhibitors similar to the principal drugs currently employed in the management of anxiety disorders. In support of this, 5-HT-containing neurons in the raphe nucleus have been shown to contain ER? (47, 48, 49), and GABA-containing interneurons also appear to be ER? positive (50, 51). Nonetheless, the ability of tamoxifen to block DPN's anxiolytic actions strongly suggests that an ER is mediating its effects.
Although there are compounds that are superior to tamoxifen (i.e. ICI 182,780) in their ability to act as pure antagonists of ER, these compounds are incapable of crossing the blood-brain barrier. Unfortunately, to our knowledge there exists no available ER?-selective antagonist at the present time. Therefore, because it was critical that we block ER in brain, we were limited in these studies to the use of tamoxifen. In all treatment paradigms, tamoxifen had no effect of its own.
It is also highly likely that DPN's actions are because of binding of ER? and not because of nonselective interactions with ER. Although a recent publication suggests that a lower dose of DPN could also work to regulate mood (52), the higher dose used in these studies appears to be acting solely via ER?. The ER-selective agonist did not mimic the actions of DPN and often had effects opposite that of DPN. Furthermore, DPN's actions were different qualitatively and quantitatively from the mixed agonist E2. Moreover, DPN was ineffective in inducing PR expression in the MPN. Taken together, the above findings support the hypothesis that ER? is a critical factor in the regulation of anxiety-related behaviors and that in some cases ER and ER? may work in opposition to provide precise control of such behaviors.
DPN decreases plasma levels of corticosterone
After physical or psychological stressors, females typically display a more robust hormonal response than males (53, 54, 55). It appears that in males, androgens act to inhibit (53, 54, 55), whereas in females, estrogens function to enhance (35, 36, 53) the activity of the hypothalamo-pituitary adrenal axis. Endocrine manipulation studies in females show that OVX can reduce the stress-induced secretion of corticosterone, a reduction that is reversed via E2 administration (35, 36, 53). In the present study, 30 min after testing in the elevated plus maze, corticosterone levels were increased in females treated with E2 and PPT treatments but decreased by DPN-treated animals relative to OVX controls. This finding suggests that the aforementioned increase in circulating levels of corticosterone is a function of ER activation and that ER?’s action is consistently opposite that of ER and inhibitory to stress-induced hormone secretion. Because DPN decreases circulating levels of corticosterone, the possibility does exist that DPN's anxiolytic effects are caused at least in part by regulation of adrenal secretions, or the result could be epiphenomenal to the reduced anxiety in these animals. Research has identified that the anxiolytic action of other compounds (ipsapirone and buspirone) are prevented by adrenalectomy (14), making this a viable explanation of DPN's mechanism of action. Studies examining such an interaction are currently underway.
In summary, activation of ER? by the selective agonist DPN decreases anxiety-related behavior. Our results demonstrate that DPN crosses the blood-brain barrier to have its effect on behaviors. Furthermore, DPN decreases anxiety-related behavior in both sexes and across a variety of behavioral paradigms. Because the anxiolytic effects of DPN are extinguished by the ER antagonist tamoxifen, DPN's effects are likely ER mediated. Moreover, based upon DPN's selectivity to ER?, it is ER? that mediates the anxiolytic effects of estrogens. Thus, selectively targeting neural ER? activation in women may be an important factor to consider in the design of future therapeutics for anxiety and depression.
Acknowledgments
We thank D. Munson, J. Evans, M. McNulty, and D. Moore for their expert technical assistance.
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