Mechanisms responsible for progesterone's protection against lordosis-inhibiting effects of restraint

Hormones and Behavior 60 (2011) 219–225

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Hormones and Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y h b e h

Mechanisms responsible for progesterone's protection against lordosis-inhibiting effects of restraint I. Role of progesterone receptors James Hassell, Chandra Suma Johnson Miryala, Cindy Hiegel, Lynda Uphouse ⁎ Department of Biology, Texas Woman's University, Denton, TX 76204, USA

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Article history: Received 30 January 2011 Revised 4 May 2011 Accepted 15 May 2011 Available online 20 May 2011 Keywords: Ovariectomized female rats Hormonal priming Medroxyprogesterone RU486 Stress

a b s t r a c t Progestins and antiprogestins are widely used therapeutic agents in humans. In many cases, these are indicated for the treatment of reproductive activities. However, progesterone has widespread physiological effects including a reduction of the response to stress. We have reported that 5 min of restraint reduced lordosis behavior of ovariectomized rats hormonally primed with estradiol benzoate. When ovariectomized rats received both estradiol benzoate and progesterone priming, restraint had minimal effects on lordosis. Progesterone influences behavior through classical intracellular progesterone receptor-mediated nuclear events as well as extranuclear events. How these multiple events contribute to the response to stress is unclear. The current project was designed to initiate examination of the mechanisms responsible for progesterone's ability to protect against the effects of the restraint. In the first experiment, ovariectomized rats, primed with 10 μg estradiol benzoate, received 500 μg progesterone 4 h, 1 h, or 30 min before restraint. When progesterone was injected 4 h before restraint, progesterone eliminated the effects of restraint. In contrast, progesterone 30 min before restraint offered no protection. Effects of progesterone 1 h before restraint were equivocal allowing the suggestion that less than 4 h of progesterone priming might be sufficient. In the second experiment, the synthetic progestin, medroxyprogesterone, was shown to mimic effects of progesterone in preventing effects of restraint. Finally, the progesterone receptor antagonist, RU486, attenuated progesterone's protection against restraint. These findings offer evidence that ligand-activated progesterone receptor mechanisms contribute to the maintenance of lordosis behavior in the presence of mild stress. © 2011 Elsevier Inc. All rights reserved.

Introduction Female gonadal hormones have wide-spread influences on brain and behavior and are thought to contribute to the higher vulnerability of females to a variety of mood disorders such as premenstrual dysphoric disorder, depression and anxiety (Bannbers et al., in press; Dubrovsky, 2006; Ho et al., 2004; Solomon and Herman, 2009). In rodents, gonadal hormones are critically involved in the timing of sexual behavior (Auger, 2004; Erskine, 1989; Pfaff, 2005; Sodersten, 1981) as well as in the response to stress with estradiol often amplifying and progesterone reducing indices of anxiety and fear (Hiroi and Neumaier, 2006; Picazo and Fernandez-Guasti, 1995; Pluchino et al., 2006; Reddy et al., 2005; Shively and Bethea, 2004). In female rats, cyclic changes in gonadal hormones signal the emergence and termination of sexual behaviors. Induction of lordosis ⁎ Corresponding author. Fax: + 1 940 898 2382. E-mail addresses: [email protected] (J. Hassell), [email protected] (C.S.J. Miryala), [email protected] (C. Hiegel), [email protected] (L. Uphouse). 0018-506X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2011.05.006

behavior requires estradiol priming (Blaustein, 2008; Frye et al., 1998; Sodersten, 1981) while progesterone is especially important for proceptivity (hops and darts) and solicitation behaviors that signal the female's receptive state to the male (Erskine, 1989; Mani et al., 1994; Ogawa et al., 1994). Progesterone also exerts a plethora of nonreproductive effects (Brinton et al., 2008; Frye, 2007; Mani et al., 1997; Pluchino et al., 2009); of special relevance to the current studies is progesterone's anxiolytic action (Bitran et al., 1993; Frye, 2007; Picazo and Fernandez-Guasti, 1995). We have reported that lordosis behavior of ovariectomized female rats, hormonally primed with 10 μg estradiol benzoate, was reduced after 5 min of restraint, but that addition of progesterone to the hormonal priming completely prevented the decline in lordosis behavior (Truitt et al., 2003; White and Uphouse, 2004). Because the female's interaction with a sexually active male can, itself, be stressful (Hennessy et al., 2008), progesterone may also enhance sexual behavior by reducing the impact of stressful stimuli that occur during mating. Progesterone influences behavior through multiple and intersecting mechanisms including ligand-dependent and ligand-independent events, as well as membrane progesterone receptor-mediated actions

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(Conneely et al., 2003; Dressing et al., 2011; Mani et al., 1997; Pluchino et al., 2009). The best known such mechanism is progesterone's activation of intracellular progesterone receptors (PR) which then function as transcription factors influencing gene expression (Mani and Portillo, 2010; Mani et al., 1997; Tetel, 2009). Two different PRs (PR-A and PR-B) vary in their relative tissue distribution, response to antagonists, and contribution to ligand-dependent and ligandindependent effects of progesterone (Leonhardt et al., 2003; Mani et al., 2006). Classical PR-mediated effects of progesterone in the ventromedial nucleus of the hypothalamus play a prominent role in progesterone's facilitation of sexual behavior in estradiol-primed, ovariectomized female rodents (Blaustein, 2008; Mani et al., 1994, 1997; Ogawa et al., 1994). Investigators have concluded that PR-A plays the more dominant role in facilitation of female sexual behavior, but both isoforms appear to participate (Mani et al., 2006). In addition, progesterone actions include a variety of extranuclear signaling mechanisms that may or may not require participation of the intracellular PR (Gonzalez-Flores et al., 2010; Intlekofer and Petersen, 2010; Mani and Portillo, 2010; Pluchino et al., 2006; Tetel, 2009; Zheng, 2009). Several of these nonclassical progesterone mechanisms contribute to progesterone's facilitation of sexual behavior (Auger, 2004; Frye et al., 1998; Frye and Vongher, 1999; Gonzalez-Flores et al., 2010; Tetel et al., 2009) but which of these contribute to the hormone's protection against 5 min restraint is not known. Given the extensive use of progestins (as well as progestin receptor modulators) in the human population (Gellersen et al., 2009), it is important to understand how these compounds influence the response to stress. In the current studies, we have begun to address the mechanism through which progesterone's protection against lordosis-inhibiting effects of restraint is exerted. Because behavioral consequences of classical ligand-dependent activation of the intracellular PR are thought to occur slowly, we began by determining a time-course for progesterone's protection against the effect of 5 min restraint. We then investigated effects of the nonmetabolizable progestin, medroxyprogesterone (Lee et al., 1999), and the PR antagonist, RU486 (Meyer et al., 1990). If classical intracellular PR-mediated responses to progesterone were required for protection against the mild stress, it was hypothesized that (1) progesterone's effects would require time to develop; (2) medroxyprogesterone would mimic the effects of progesterone; and (3) RU486 would attenuate effects of progesterone. These findings were presented, in part, at the Society for Neuroscience meeting in 2010. Materials and methods Materials Estradiol benzoate, progesterone, medroxyprogesterone acetate (17α-hydroxy-6α-methyl-4-pregnene-3,20-dione), RU486 (11β-(4Dimethylamino)phenyl-17β-hydroxy-17-(1-propynyl)estra-4,9dien-3-one), dimethyl sulfoxide (DMSO), and sesame seed oil were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Propylene glycol was obtained from Eastman Kodak Company (Rochester, NY). Isoflurane (AErrane®) was purchased from Henry Schein (Melville, NY). Decapicone® restrainers were from Braintree Scientific, Inc. (Braintree, MA). Other supplies came from Fisher Scientific (Houston, TX). General methods Animals and housing Adult Fischer (F-344) female rats, purchased from Charles River Laboratories (Wilmington, MA), were housed 2 or 3 per cage in polycarbonate shoebox cages (45.72 × 24.13 × 2.59 cm) in a colony room maintained at 25 °C and 55% humidity, with lights on from 12

midnight to 12 noon. Cage mates were tested at the same time. Food and water were available ad lib. All procedures were conducted according to PHS policy and were approved by the IACUC at Texas Woman's University. Surgical procedures and hormone treatments When approximately 80 to 90 days of age (2 to 3 weeks after arrival in the colony), females were anesthetized with AErrane® and ovariectomized as previously described (White and Uphouse, 2004). Approximately two weeks later (when rats were 94 to 111 days of age), rats were used in the experiments. For hormonal priming, estradiol benzoate (EB) and progesterone were dissolved in sesame seed oil and injected subcutaneously (s.c.) in a volume of 0.1 ml/rat. Restraint procedures Restraint procedures were as previously described (Uphouse et al., 2007). The female was placed head first into a Decapicone® so that her nose was flush with the small opening at the tip of the cone. The base of the cone was gathered around the female's tail and secured tightly with tape. The process of wrapping the female required between 30 and 60 s. The wrapped female was set aside for 5 min of restraint. Testing for sexual receptivity Pretesting for sexual receptivity, as previously described (Uphouse et al., 1992), was initiated within 1 to 3 h after colony lights off and prior to the restraint experience. Experimenter visibility was aided by red lighting. In the pretest, females were placed into the home cages of sexually experienced Sprague–Dawley male rats. Sprague–Dawley males are more sexually active and engage in more mounting behavior per 5 min interval than Fischer males (personal observations). For this reason, Sprague–Dawley males have been preferred for our testing of sexual behavior. After placement in the male's cage, the rat's behavior was monitored for 10 min or until the male had accomplished 10 mounts; rats with a pretest lordosis to mount (L/M) ratio of 0.7 or higher were used to examine the effects of restraint. Lordosis quality and proceptivity (hopping and darting), as previously described (White and Uphouse, 2004), were also recorded. Proceptivity was measured as present or absent for the pre-restraint and post-restraint intervals. After restraint, sexual behavior was monitored for 15 consecutive min. Statistical procedures Data were evaluated in two separate ways. First, pretest effects of treatment for all animals were evaluated with univariate ANOVA (when multiple groups were included) or Student's t-test (when only two groups were included in the experiment). This procedure was performed to determine if treatments had influenced sexual behavior prior to the restraint experience. Only rats with a pretest L/M ratio ≥ 0.7 were then included in the restraint procedures and the pretest for this subset of rats was used as the data for the pre-restraint interval. For evaluation of effects of restraint, L/M ratios, lordosis quality scores, and number of mounts by the male were grouped into the pre-restraint interval and five-min intervals after restraint; data were then evaluated by repeated measures ANOVA with time after restraint as the repeated factor. Post-hoc comparisons were made with Dunnett's test for within group time comparisons or with Tukey's test for comparison between groups, within time interval. Proceptive behavior was compared with Chi-square procedures; comparison of proceptivity before and after restraint was accomplished with the Wilcoxon Signed Ranks test. Only rats with a pretest L/M ≥ 0.7 were included in the analyses for the effects of restraint. Statistical analyses were conducted with SPSS 17 for Macintosh or SPSS 15 for PC. Independent pair-wise comparisons were performed manually (Zar, 1999). An alpha level of 0.05 was required for rejection of the null hypothesis.

J. Hassell et al. / Hormones and Behavior 60 (2011) 219–225

Specific experiments Experiment 1: Time course for progesterone's attenuation of the effects of restraint Ninety rats were used in the experiment. All rats were hormonally primed with 10 μg EB. Two days later, rats were injected with 500 μg progesterone 4 h (EP 4 h rats), 1 h (EP 1 h rats) or 30 min (EP 30 min rats) before a 5 min restraint experience. To control for effects of injections, every rat received three injections: (1) EB, (2) sesame seed oil (SSO) or progesterone, and (3) SSO or progesterone. EP 4 h rats received progesterone 4 h before restraint and an injection of SSO either 1 h or 30 min before restraint and were combined for statistical analysis. EP 1 h rats and EP 30 min rats received SSO at 4 h before restraint and progesterone either 1 h or 30 min before restraint. Two additional groups (EO rats) received the SSO vehicle at 4 h and at either 1 h or 30 min before restraint and were combined for analysis. The pretest for sexual receptivity was conducted 1 h before restraint except for 30 min injections where the pretest took place immediately after the injection. After the 5 min restraint, rats were again tested for sexual receptivity for 15 consecutive min. Pretest data for all animals were first evaluated for effects of hormonal treatments on lordosis behavior and proceptivity. For assessment of effects of restraint, only data from rats with a pretest ≥ 0.7 were included. L/M ratios, lordosis quality and mounts were analyzed by repeated measures ANOVA with type of hormonal priming as the independent factor and time after restraint as the repeated factor. Of the original 90 rats, 26 rats were not included in the evaluation of the effects of restraint. Fifteen of these rats had pretest L/M ratios lower than 0.7 and were eliminated from the ANOVA. An additional 11 rats failed to receive sufficient mounts after restraint. Of these latter 11 rats, 7 were in the EO group, perhaps indicative of their lesser attractiveness to the males (Kavaliers et al., 1994).

Experiment 2: Substitution of medroxyprogesterone for progesterone A total of 22 rats were used. Rats were ovariectomized and hormonally primed with 10 μg EB as for Experiment 1. However, two days later, instead of progesterone priming, rats were injected s.c. with either the propylene glycol vehicle (E-Vehicle rats, n = 10) or 615 μg medroxyprogesterone in propylene glycol (E-Medroxy rats, n = 12). Injections were given s.c. in a volume of 2.0 ml/kg. Four to six hours later, rats were pretested for sexual behavior, restrained and retested as in the first experiment. Pretest data for all animals were compared with Student's t-test. Effects of restraint were evaluated by repeated measures ANOVA with treatment as the independent factor and time after restraint as the repeated factor. Two of the E-Vehicle and one of the E-Medroxy rats

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failed to have L/M ratios of a least 0.7 in the pretest and were not restrained. One additional E-Medroxy rat had too few mounts after restraint and was also excluded from the ANOVA. Experiment 3: Effect of RU486 on the response to progesterone A total of 35 ovariectomized rats were hormonally primed with 10 μg EB followed 48 h later with 500 μg progesterone. One hour before progesterone, rats were injected with vehicle (13.5% DMSO/ propylene glycol) or 2.5 or 5.0 mg/kg RU486 in the DMSO/propylene glycol vehicle. Four to six hours after progesterone, rats were pretested for sexual behavior, restrained for 5 min, and immediately tested for 15 consecutive min as in prior experiments. One rat injected with 2.5 mg/kg RU486 and 4 rats given 5.0 mg/kg RU486 had L/M ratios b 0.7 during the pretest and were not restrained. Five additional rats had too few mounts after restraint and their data were excluded from the ANOVA. Results Experiment 1: Time course for progesterone's attenuation of the effects of restraint Behavior during the pretest Table 1 shows the behavioral characteristics of rats during the pretest. Regardless of hormonal priming, most rats exhibited some evidence of sexual receptivity during the pretest. There were no significant group differences in L/M ratios (F3,86 = 2.20, p N 0.05), in lordosis quality (F3,84 = 1.72, p N 0.05) or in number of rats showing a L/M ratio of at least 0.7 during the pretest (Chi-square = 1.68, df = 3, p N 0.05). However, there was a significant effect of hormonal treatment on the number of rats showing proceptivity in the pretest (Chi square = 11.27, df = 3, p ≤ 0.001) with EP 4 h rats showing more proceptivity than the other three groups. Since pretesting occurred immediately after the progesterone injection of EP 1 h and EP 30 min rats, proceptivity of these rats was not expected to differ from EO rats. Effects of restraint Characteristics of the 64 rats included in evaluation of the effects of restraint are shown in Table 1. With the exception of EP 4 h rats, most rats had not shown proceptivity during the pretest leading to a significant pre-restraint treatment effect for the rats that were subjected to restraint (Pearson's Chi-square = 9.86, df = 3, p ≤ 0.02). In EP 4 h rats, proceptivity was reduced after restraint (Wilcoxon Signed Ranks test, Z = 2.64, p ≤ 0.008) so that group differences disappeared (Chi-square = 4.96, df = 3, p N 0.05). There were significant group differences in the proportion of rats that showed a decline in lordosis behavior after restraint (Chi-

Table 1 Time-dependent effects of progesterone and the response to restraint.

Pretest behavior (all animals)a Initial pretest n Pretest mean (S.E.) L/M ratio Pretest mean (S.E.) lordosis quality Number (%) proceptive in pretest Number (%) with pretest L/M ≥ 0.7

EP 4 h

EP 1 h

EP 30 min

EO

Total

22 0.93 (0.04) 2.84 (0.10) 12 (54.5%) 20 (90.9%)

15 0.84 (0.07) 2.72 (0.08) 1 (6.7%) 13 (86.7%)

20 0.78 (0.04) 2.54 (0.11) 2 (10.0%) 16 (80.0%)

33 0.79 (0.04) 2.67 (0.08) 11 (33.3%) 26 (78.8%)

90

2 11 1 (9.1%) 1 (9.1%)

2 14 1 (7.1%) 0

7 19 6 (31.6%) 0

11 64

Behavior of restrained rats (rats with pretest L/M ratio ≥ 0.7)b Number missing mounts after restraint 0 Final n for restraint analysis 20 Number (%) proceptive before restraint 12 (60.0%) Number (%) proceptive after restrainta 2 (10.0%) a b

Behavior of all animals before restraint. Only rats with pretest L/M ≥ 0.7 were included in the restraint procedures; pretest behavior of this subset of rats is included in describing the data before restraint.

75

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square = 13.58, df = 3, p ≤ 0.004) with EP 4 h rats showing the least inhibition. This was reflected in a significant decline in the L/M ratios after restraint (ANOVA for time, F3,180 = 19.63, p ≤ 0.001) (Fig. 1). L/M ratios of EO and EP 30 min rats were significantly different from their pretest at every test interval after restraint while EP 1 h rats showed only a transient decline in the L/M ratio 5 min after restraint (Dunnett's test to the pretest value, q180,4 ≥ 2.35, p ≤ 0.05). L/M ratios of EP 4 h rats were never significantly different from their prerestraint L/M ratios. Within time intervals, EO and EP 30 min rats differed significantly from EP 4 h rats at every test interval after restraint (Tukey's q180,4 ≥ 3.63, p ≤ 0.05). L/M ratios of EP 1 h rats were consistently lower than those of EP 4 h rats, but L/M ratios for these two groups were never significantly different (Tukey's, all p N 0.05). Although there were significant effects of hormonal treatment on lordosis quality (F3,55 = 4.51, p ≤ 0.007; data not shown), this primarily reflected the higher lordosis quality of EP 4 h rats. Neither time (indicative of the effect of restraint) nor the time by treatment interaction was significant (all p N 0.05). Mean ± S.E. lordosis quality for EP 4 h, EP 1 h, EP 30 min and EO rats, respectively, were 2.88 ± 0.60, 2.64 ± 0.08, 2.56 ± 0.09, and 2.63 ± 0.14. Although across all groups there was a decline in the number of mounts received across time (F3,180 = 18.77, p ≤ 0.001), there were no group differences in the number of mounts received by the females. The mean ± S.E. number of mounts per interval for EP 4 h, EP 1 h, EP 30 min and EO rats, respectively, were 8.01 ± 0.42, 8.64 ± 0.57, 7.75 ± 0.50, and 6.80 ± 0.43. Experiment 2: Substitution of medroxyprogesterone for progesterone Behavior during the pretest Consistent with prior reports that medroxyprogesterone can substitute for progesterone in the facilitation of lordosis behavior (Pazol et al., 2006), pretest L/M ratios were slightly, but significantly, higher in rats treated with medroxyprogesterone than they were in vehicle-treated, EB primed rats (t = 4.25, df = 20, p ≤ 0.001) (Table 2). Similarly, lordosis quality was slightly higher for medroxyprogesterone-treated rats (t = 2.05, df = 20, p ≤ 0.05). There was not a significant difference in proceptivity during the pretest.

Table 2 Effects of medroxyprogesterone and the response to restraint.

Pretest behavior (all animals)a Initial n Pretest mean (S.E.) L/M ratio Pretest mean (S.E.) lordosis quality Number (%) proceptive in pretest Number (%) with pretest L/M ≥ 0.7 Number missing mounts after restraint

E-Vehicle

E-Medroxy

Total

10 0.81 (0.05) 2.57 (0.15) 3 (30%) 8 (80%) 1

12 0.99 (0.008) 2.89 (0.07) 8 (66.7%) 12 (100%) 1

22

Behavior of restrained rats (rats with pretest L/M ratio ≥ 0.7)b Number missing mounts after restraint 1 1 Final n for restraint analysis 7 11 Number (%) proceptive before restraint 1 (14.3%) 10 (90.9%) Number (%) proceptive after restraint 2 (28.6%) 5 (45.4%)

Behavior of all animals before restraint. Only rats with pretest L/M ≥ 0.7 were included in the restraint procedures; pretest behavior of this subset of rats is included in describing the data before restraint. b

Effects of restraint In agreement with the first experiment, restraint significantly reduced sexual behavior of E-Vehicle rats (Fig. 2). Substitution of medroxyprogesterone for progesterone resembled the effect of progesterone in attenuating this restraint-induced decline in lordosis behavior. For L/M ratios, there was a significant effect of type of priming (F1,16 = 16.18, p ≤ 0.001), time after restraint (F3,48 = 11.19, p ≤ 0.001), and their interaction (F3,48 = 7.81, p ≤ 0.001). Similar to findings in the first experiment, rats treated with EB and vehicle had lower L/M ratios after restraint than their pre-restraint behavior (Tukey's, all q48,4 ≥ 3.79, p ≤ 0.05). L/M ratios of rats primed with EB and medroxyprogesterone were never significantly different from their pre-restraint values (p N 0.05) and were significantly different from L/M ratios of E-Vehicle rats during each of the post-restraint test intervals (Tukey's all q48,2 ≥ 2.86, p ≤ 0.05). Two E-Vehicle rats had L/M ratios of zero during the test and were excluded from the analysis of lordosis quality. For the remaining rats, lordosis quality was significantly higher in E-Medroxy than in EVehicle rats (F1,14 = 9.96, p ≤ 0.007; data not shown) but there was

1.0

* **

*

0.6

* ** EP 4 hr

0.4

EP 1 hr EP 30 min

0.2

* **

* **

* ** * **

EO

LORDOSIS / MOUNT RATIO

LORDOSIS / MOUNT RATIO

1.0

0.8

2 18

a

E-MEDROXY

RESTRAINT

20 2

E-VEHICLE

RESTRAINT

0.8

**

* **

0.6

0.4

* **

0.2

0.0 PRE

5

10

15

TIME (min)

0.0 PRE

5

10

15

TIME (min) Fig. 1. Time-dependent effects of progesterone on the lordosis response to restraint. Ovariectomized rats were hormonally primed with 10 μg EB 52 to 54 h before restraint. Progesterone (500 μg, EP) or oil (EO) was injected 4 h, 1 h, or 30 min before the restraint experience. Rats were pretested for sexual behavior, restrained for 5 min, and immediately tested for 15 min. Data are the mean ± S.E. L/M ratios for the pretest (PRE) and 3 consecutive 5 min intervals after restraint. N's for rats injected with progesterone 4 h (EP 4 h), 1 h (EP 1 h), or 30 min (EP 30 min) before restraint were 20, 11 and 14, respectively. A total of 19 rats were included in the EO group. *Indicates a significant difference from the pretest, within treatment condition. **Indicates a significant difference from EP 4 h rats, within time interval.

Fig. 2. Effects of medroxyprogesterone on the response to restraint. Ovariectomized rats were hormonally primed with 10 μg EB. Two days later, instead of progesterone priming, rats were injected s.c. with either the propylene glycol vehicle (E-Vehicle) (n = 7) or 615 μg medroxyprogesterone (E-Medroxy) (n = 11) in propylene glycol. Four to six hours later, rats were pretested for sexual behavior (PRE), restrained for 5 min, and retested for 15 consecutive min. Data are the mean ± S.E. L/M ratios for each of the test intervals. *Indicates significant differences from the pretest L/M ratio. **Indicates significant differences between rats primed only with EB and those primed with EB and medroxyprogesterone.

J. Hassell et al. / Hormones and Behavior 60 (2011) 219–225

Experiment 3: Effect of RU486 on the response to progesterone Behavior during the pretest Data for the pretest experience prior to restraint are shown in Table 3. There was a small, but significant, effect of RU486 on both the pretest L/M ratios (F2,32 = 4.45 p ≤ 0.02) and on lordosis quality (F2,32 = 5.54, p ≤ 0.009) (Table 3). There was not a significant difference in the proportion of rats showing proceptivity in the pretest (Chi square, df = 2, p N 0.05). Effects of restraint As seen in Table 3, ten rats were not included in the evaluation of effects of restraint. The sample size for rats given 5.0 mg/kg RU486 was the most affected with 4 rats showing L/M ratios below 0.7 in the pretest; and 2 more rats had low numbers of mounts following restraint. For the remaining rats, treatment with RU486 before restraint reduced progesterone's protective effect against the mild stressor. Rats treated with RU486 showed a decline in sexual behavior that resembled that of rats primed only with EB (see EO rats in Fig. 1 and E-Vehicle rats in Fig. 2). As a consequence, there was a significant effect of treatment (F2,22 = 4.94, p ≤ 0.02) and time relative to restraint (F 3,66 = 8.00, p ≤ 0.001), but not their interaction (F6,66 = 1.48, p N 0.05). Inhibitory effects of restraint in RU486-treated were primarily evident during the 5 min interval immediately following restraint. This was the only time point where the L/M ratios were lower than the respective pre-restraint values (for 2.5 and Table 3 Effects of RU486 and the response to restraint. EP-Vehicle Pretest behavior (all animals)a Initial n Pretest mean (S.E.) L/M ratio

10 0.99 (0.007) Pretest mean (S.E.) lordosis quality 2.99 (0.029) Number (%) proceptive in pretest 5 (50%) Number (%) with pretest L/M ≥ 0.7 10 (100%) Number missing mounts after 2 restraint

EP-RU486 EP-RU486 Total (2.5 mg/kg) (5.0 mg/kg) 12 0.92 (0.029) 2.81 (0.055) 8 (66.7%) 11 (91.7%) 1

Behavior of restrained rats (rats with pretest L/M ratio ≥ 0.7)b Number missing mounts 2 1 after restraint Final n for restraint 8 10 Number (%) proceptive 4 (50%) 7 (70%) before restraint Number (%) proceptive 7 (87.5%) 1 (10%) after restraint a

13 0.83 (0.050) 2.69 (0.082) 7 (53.8%) 9 (69.2%) 2

2 7 4 (57.1%)

35

30 5

5 25

1 (14.2%)

Behavior of all animals before restraint. Only rats with pretest L/M ≥ 0.7 were included in the restraint procedures; pretest behavior of this subset of rats is included in describing the data before restraint. b

1.0

LORDOSIS / MOUNT RATIO

not a significant effect of time after restraint (F3,42 = 0.62, p N 0.05) or of the interaction between type of priming and time after restraint (F3,42 = 0.18, p N 0.05). The average ± S.E. lordosis quality per test interval for E-Medroxy and E-Vehicle rats, respectively, was 2.85 ± 0.04 and 2.64 ± 0.06. For rats included in the restraint experience, significantly more EMedroxy rats than E-Vehicle rats showed proceptivity prior to restraint (Chi square = 10.56, df = 1, p ≤ 0.001; Table 2), but there were no significant differences in the two groups after restraint (Chi square, p N 0.05). Across all groups, mounts declined during testing (F3,54 = 4.74, p ≤ 0.005), but type of priming did not affect the number of mounts received; and there was no interaction between type of priming and time after restraint (both p N 0.05). The average mounts per interval for E-Medroxy and E-Vehicle rats, respectively, were 8.18 ± 0.61 and 7.53 ± 0.75.

223

RESTRAINT

0.8

* * **

0.6

EP VEHICLE

0.4

2.5 mg/kg RU486 0.2 5 mg/kg RU486 0.0 PRE

5

10

15

TIME (min) Fig. 3. RU486 reduces progesterone's protection against restraint stress. Ovariectomized rats were hormonally primed with 10 μg EB followed 48 h later with 500 μg progesterone. One hour before progesterone, rats were injected with vehicle (13.5% DMSO/propylene glycol), 2.5 or 5.0 mg/kg RU486. N's for EP-Vehicle, 2.5 and 5.0 mg/kg RU486, respectively, were 8, 10, and 7. Four to six hours after progesterone, rats were pretested for sexual behavior (PRE), restrained for 5 min, and immediately tested for 15 consecutive min. Data are the mean ± S.E. L/M ratios for each of the test intervals. *Indicates significant differences from the pretest L/M ratio. **Indicates significant differences from rats primed with EB and progesterone.

5.0 mg/kg RU486, respectively, Dunnett's q66,4 = 4.56 and 2.38, p ≤ 0.05) (Fig. 3). One rat treated with 2.5 mg/kg RU486 had a L/M of zero after restraint and was excluded from the quality analysis. For the remaining rats, there was a significant effect of time after restraint (F3,63 = 4.74, p ≤ 0.005), but no other factors were significant. The mean ± S.E. lordosis quality for EP-Vehicle, 2.5 mg/kg RU486 and 5.0 mg/kg RU486, respectively, was 2.82 ± 0.109, 2.63 ± 0.103, and 2.55 ± 0.012. Similarly, there was a time-dependent decline in the number of mounts per test interval (F3,81 = 29.51, p ≤ 0.001), but no other effects were significant. The mean ± S.E. number of mounts per test interval for EP-Vehicle, 2.5 mg/kg RU486 and 5.0 mg/kg RU486, respectively, was 6.22 ± 0.58, 6.56 ± 0.56, and 6.19 ± 0.62. Prior to restraint, there were no significant differences in proceptivity (p ≤ 0.05), but group differences emerged following restraint (Chi square = 13.57, df = 2, p b 0.001). This group difference resulted primarily from a surprising increase in proceptivity of EPVehicle-treated rats after restraint. Proceptivity of rats treated with RU486 was reduced after restraint (Wilcoxon Signed Ranks Test, Z = 2.49, p ≤ 0.02). Discussion Progesterone is known for its anxiolytic effects and the responsible mechanisms are often attributed to the action of progesterone metabolites (Frye et al., 2008; Picazo and Fernandez-Guasti, 1995; Reddy et al., 2005). However, given the wide-spread signaling cascades of progesterone receptors (Brinton et al., 2008; Leonhardt et al., 2003; Mani, 2006), the role of progesterone receptors per se in the reduced response to stressful experiences remains unclear. The current studies were initiated to test the hypothesis that progesterone's ability to protect against the lordosis-inhibiting effect of 5 min restraint required progesterone's interaction with the intracellular progesterone receptor. Although progesterone is not required for expression of female rat sexual behavior, progesterone facilitates estradiol's induction of lordosis behavior and is thought to be required for proceptive behavior (Blaustein, 2008; Erskine, 1989). In agreement with earlier studies (White and Uphouse, 2004), when ovariectomized rats were hormonally primed only with EB, 5 min restraint produced a rapid and robust inhibition of lordosis behavior. Progesterone, given 4 h, but not 30 min, before restraint,

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completely eliminated the effect of restraint on lordosis behavior. This observation appears to rule out relatively rapid effects of progesterone. However, when progesterone was injected 1 h earlier, progesterone did offer partial protection against the lordosis-inhibiting effect of restraint. Thus, these findings allow the suggestion that some of the restraint-protective effects of progesterone may require no more than an hour of progesterone exposure. It is important to note, however, that in a majority of studies where rapid effects of progesterone have been reported, the steroid was administered either intracranially or intravenously (Frye and Vongher, 1999; Picazo and Fernandez-Guasti, 1995). However, rapid effects of progesterone have been previously observed when testing occurred as early as 30 min after ip injection (Gomez et al., 2002; Starkey and Bridges, 2010). Nevertheless, the absence of a protective effect of progesterone after 30 min cannot exclusively rule out progesterone metabolites as a contributing factor; but PR-mediated events appear to be required as indicated by the effects of medroxyprogesterone and RU486. Medroxyprogesterone is a synthetic progestin that binds to PR, glucocorticoid receptors (GR) and androgen receptors (AR) (Koubovec et al., 2005; Pridjian et al., 1987; Selman et al., 1996). Medroxyprogesterone's use in contraceptives stems from its ability to bind to the PR and mimic many (but not all) ligand-mediated PR responses (Koubovec et al., 2005). Similar to the effects of progesterone, medroxyprogesterone can acutely facilitate but later inhibit female rat sexual behavior (Pazol et al., 2006). In the current study, medroxyprogesterone was as effective as progesterone in attenuating the lordosis-inhibiting effects of restraint. Since medroxyprogesterone inhibits enzymes involved in progesterone metabolism (Jarrell, 1984; Lee et al., 1999), progesterone metabolites do not appear to be required for progesterone's attenuation of the lordosis-inhibitory effects of restraint. This outcome was surprising given the welldefined role of progesterone metabolites, such as allopregnanolone, in reducing the response to stressful stimuli (Barbaccia, 2004; Dubrovsky, 2006; Eser et al., 2008; Frye, 2007). Instead, the current data are consistent with a role for ligand-dependent PR-mediated events involved in the protection. However, the current data do not rule out a participation of progesterone metabolites. RU486 has antiprogestin activity by inhibiting PR-mediated transcriptional events (Leonhardt et al., 2003) and progesterone's facilitation of female rodent lordosis behavior (Gonzalez-Mariscal et al., 1989). Mechanisms are thought to include inhibition of PR interaction with cofactors essential for the mediation of genomic responses (Leonhardt et al., 2003). In the current experiment, RU486, administered 1 h before progesterone, attenuated progesterone's protection against the lordosis-inhibiting effects of the 5 min restraint. RU486, as well as other antiprogestins, has a greater impact on transcriptional activity of PR-A than PR-B with PR-B being relatively resistant to antagonists (Leonhardt et al., 2003; Mani et al., 2006). Thus, the effectiveness of RU486 in the current study may be evidence of a PR-A involvement in protecting against the effects of restraint. However, in contrast to earlier studies on effects of RU486 (Auger, 2004), in the current experiment, RU486 had relatively minor effects on lordosis behavior in the absence of restraint. This is not surprising since 10 μg estradiol benzoate, alone, is adequate for elicitation of lordosis responding in ovariectomized Fischer female rats. However, at the highest dose (5 mg/kg) of RU486, a substantial number of rats failed to exhibit high levels of lordosis responding during the initial pretest. The requirement that rats show a L/M ratio of at least 0.7 to be included in the restraint experiment may have resulted in an unplanned selection bias which could explain why effects of restraint were slightly greater in the 2.5 mg/kg group than in the 5 mg/kg group. Alternatively, under some conditions, RU486 has been reported to have partial agonist activity (Leonhardt et al., 2003; Meyer et al., 1990; Taylor et al., 1998) and this could have contributed to the lesser effects of 5 mg/kg RU486.

In contrast to progesterone's protection against effects of restraint on L/M ratios, comparable protection was not evident for proceptivity. A restraint-induced decline in proceptivity was present in Experiment 1 in spite of progesterone administration 4 h earlier, in Experiment 2 following medroxyprogesterone treatment, and in Experiment 3 in RU486-treated rats. However, a decrease in proceptivity was not apparent in EP-Vehicle-treated rats in Experiment 3. The reason for this difference is not clear. Collectively, these series of experiments implicate ligand-dependent activation of PR-mediated genomic responses in progesterone's attenuation of the lordosis-inhibiting effects of 5 min restraint. However, both medroxyprogesterone (Pridjian et al., 1987; Selman et al., 1996) and RU486 (Lee et al., 2009; Zhang et al., 2007) can interact with glucocorticoid receptors (GR). Since progesterone can also bind to GR (Zhang et al., 2007), we cannot rule out the possibility that GR, rather than PR, are responsible for progesterone's suppression of the lordosis-inhibiting effect of restraint. Moreover, depending on cellular conditions, both medroxyprogesterone and RU486 can exhibit partial agonist action at GRs (Leonhardt et al., 2003; Zhang et al., 2007) so that additional studies with more selective PR antagonists such as CDB-4124, which is devoid of GR action, (Attardi et al., 2004; Attardi et al., 2002) would be important in differentiating these two receptor-mediated events. Acknowledgments This research was supported by NIH HD28419, by NIH GM55380, and by a TWU institutional research support grant to LU. Special appreciation is given to Dr. Jutatip Guptarak for her training of JH and for her critical reading of a prior version of this manuscript. The authors thank Ms. Sarah Adams for technical assistance and for critical reading of the manuscript. We thank Ms. Karolina Blaha-Black and Mr. Dan Wall for animal care. References Attardi, B.J., Burgenson, J., Hild, S.A., Reel, J.R., Blye, R.P., 2002. CDB-4124 and its putative monodemethylated metabolite, CDB-4453, are potent antiprogestins with reduced antiglucocorticoid activity: in vitro comparison to mifepristone and CDB-2914. Mol. Cell. Endocrinol. 188, 111–123. Attardi, B.J., Burgenson, J., Hild, S.A., Reel, J.R., 2004. In vitro antiprogestational/ antiglucocorticoid activity and progestin and glucocorticoid receptor binding of the putative metabolites and synthetic derivatives of CDB-2914, CDB-4124, and mifepristone. J. Steroid Biochem. Mol. Biol. 88, 277–288. Auger, A.P., 2004. Steroid receptor control of reproductive behavior. Horm. Behav. 45, 168–172. Bannbers, E., Kask, K., Wikstrom, J., Risbrough, V., Sundstrom Poromaa, I., in press. Patients with premenstrual dysphoric disorder have increased startle modulation during anticipation in the late luteal phase period in comparison to control subjects. Psychoneuroendocrinology. DOI: S0306-4530(11)00082-5 [pii]10.1016/ j.psyneuen.2011.02.011. Barbaccia, M.L., 2004. Neurosteroidogenesis: relevance to neurosteroid actions in brain and modulation by psychotropic drugs. Crit. Rev. Neurobiol. 16, 67–74. Bitran, D., Purdy, R.H., Kellogg, C.K., 1993. Anxiolytic effect of progesterone is associated with increases in cortical allopregnanolone and GABAA receptor function. Pharmacol. Biochem. Behav. 45, 423–428. Blaustein, J.D., 2008. Neuroendocrine regulation of feminine sexual behavior: lessons from rodent models and thoughts about humans. Annu. Rev. Psychol. 59, 93–118. Brinton, R.D., Thompson, R.F., Foy, M.R., Baudry, M., Wang, J., Finch, C.E., Morgan, T.E., Pike, C.J., Mack, W.J., Stanczyk, F.Z., Nilsen, J., 2008. Progesterone receptors: form and function in brain. Front. Neuroendocrinol. 29, 313–339. Conneely, O.M., Mulac-Jericevic, B., Lydon, J.P., 2003. Progesterone-dependent regulation of female reproductive activity by two distinct progesterone receptor isoforms. Steroids 68, 771–778. Dressing, G.E., Goldberg, J.E., Charles, N.J., Schwertfeger, K.L., Lange, C.A., 2011. Membrane progesterone receptor expression in mammalian tissues: a review of regulation and physiological implications. Steroids 76, 11–17. Dubrovsky, B., 2006. Neurosteroids, neuroactive steroids, and symptoms of affective disorders. Pharmacol. Biochem. Behav. 84, 644–655. Erskine, M.S., 1989. Solicitation behavior in the estrous female rat: a review. Horm. Behav. 23, 473–502. Eser, D., Baghai, T.C., Schule, C., Nothdurfter, C., Rupprecht, R., 2008. Neuroactive steroids as endogenous modulators of anxiety. Curr. Pharm. Des. 14, 3525–3533. Frye, C.A., 2007. Progestins influence motivation, reward, conditioning, stress, and/or response to drugs of abuse. Pharmacol. Biochem. Behav. 86, 209–219.

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