INTRODUCTION

Specific receptor subtypes of the serotonergic and noradrenergic systems have been found to be involved in the antidepressant-like action of several antidepressant drugs. For example, it is accepted that the 5-HT1A, 5-HT2, and 5-HT3 receptors all participate in the antidepressant-like action induced by selective serotonin re-uptake inhibitors (SSRIs) such as fluoxetine (FLX), paroxetine or sertraline (Blier, 2001; Blier and Ward, 2003; Duman et al, 1997; Li et al, 1996, 1997a, 1997b; Rupprecht et al, 2001; Stahl, 1998a). In particular, the 5-HT1A receptor has been proposed as a target of antidepressants that exhibit a clear therapeutic effect (for reviews see Blier and Ward, 2003; Cryan and Leonard, 2000). In this sense, in vitro and in vivo studies have shown that several SSRIs promote a desensitization of the presynaptic 5-HT1A receptor subtype (Blier, 2001; Le Poul et al, 1995; Li et al, 1996, 1997a), and that facilitates a general enhancement of serotonin levels. These changes in serotonin transmission appear to promote either sensitization (Castro et al, 2003; Shen et al, 2002) or desensitization of the postsynaptic serotonergic 5-HT1A receptors (Li et al, 1997a; Serres et al, 2000; Shen et al, 2002), depending on the brain area.

More evidence that sustains the participation of 5-HT1A receptors in the effect of serotonergic antidepressants results from the use of antagonists to the 5-HT1A receptor subtype, such as pindolol (Artigas et al, 1994, 1996) and WAY 100635 (Davidson and Stamford, 1995; Dawson and Nguyen, 1998; Gundlah et al, 1997; Hjorth et al, 1997; Millan et al, 1998), which, by blocking presynaptic serotonergic receptors, can facilitate the antidepressant action of serotonergic compounds in both clinical and preclinical studies (for a review, see Cryan and Leonard, 2000).

Furthermore, several behavioral reports indicate that full or partial 5-HT1A agonists, such as buspirone, gepirone, indorenate, and 8-OH-DPAT, produce antidepressant-like effects in several animal models of depression, such as the FST and learned helplessness test (Lucki and Wieland, 1990; Lucki et al, 1994; Martin et al, 1990; Martínez-Mota et al, 2002; Wieland and Lucki, 1990).

The FST is an animal model used for the screening of antidepressant treatments, which was initially proposed as an animal model of depression (Lucki, 1997; Porsolt et al, 1977, 1978; Porsolt and Lenegre, 1992). Recently, it was proposed that, with the scoring of active behaviors in this model (for example, swimming and climbing) in addition to immobility behavior (indicative of the behavioral despair), it is possible to infer the neurotransmitter systems that mediate the antidepressant-like action of drugs (Detke et al, 1995; Lucki, 1997). Accordingly, noradrenergic re-uptake inhibitors like desipramine or maprotiline decrease the immobility behavior and increase at the same time climbing (Detke et al, 1995; Espejo and Miñano, 1999). In contrast, drugs that inhibit the re-uptake of serotonin, like FLX, sertraline, or paroxetine (Detke et al, 1995), agonists to the 5-HT1A receptor subtype, such as indorenate or 8-OH-DPAT (Martínez-Mota et al, 2002) and agonists to the 5-HT2C receptor subtype, such as WAY 161503, RO 60-0175, and RO 60-0332 (Cryan and Lucki, 2000), all decrease immobility and increase swimming behavior. In addition, the depletion of serotonin with para-chlorophenylalanine in rats cancels the actions of FLX, but not those of DMI (Page et al, 1999). On these bases, it appears that the FST is useful to identify the neurotransmitter system that mediates the antidepressant-like effect of a given drug.

Using this model (FST), the estrogens 17β-estradiol (E2) and ethynil-estradiol (EE2) have been found to produce antidepressant-like actions (Estrada-Camarena et al, 2003). An important difference between these two estrogens is that the natural estrogen E2 is more potent at the serotonergic than at the noradrenergic system in decreasing re-uptake sites (Ghraf et al, 1983; Michel et al, 1987), while the synthetic steroidal compound EE2 inhibits with a comparable efficacy the serotonergic and catecholaminergic sites (Ghraf et al, 1983; Michel et al, 1987). Both estrogens have been used in clinical practice as contraceptive and/or hormonal restitution therapies (Soares et al, 2001). In the FST, Estrada-Camarena et al (2003) reported that the behavioral profile induced by both estrogens was similar to that induced by the antidepressants FLX and desipramine. Thus, both E2 and EE2 decreased immobility behavior but, while E2 promoted a concomitant increase in swimming, EE2 increased both swimming and climbing behaviors.

Hence, considering the possible participation of the serotonergic system in the antidepressant-like action induced by these estrogens in the FST, the aim of the present work was to analyze the participation of the 5-HT1A receptor subtype in the antidepressant-like actions induced by E2 and EE2. In addition, it was evaluated whether the antidepressant action of estrogens on 5-HT1A receptors occurs immediately or requires several hours to be established. This receptor subtype was chosen, since in vitro and in vivo studies have shown that estrogens like E2 and estradiol benzoate interact with pre- and postsynaptic 5-HT1A receptors. For example, estradiol can modulate the activity of this receptor depending on the dose used. In this sense, Jackson and Uphouse (1998) showed that the systemic administration of low doses of E2 may synergize with low doses of 8-OH-DPAT to inhibit lordosis behavior (Jackson and Uphouse, 1996, 1998). However, when high doses of E2 were administered, this compound blocked the actions of 8-OH-DPAT on lordosis behavior (Jackson and Uphouse, 1998; Uphouse, 2000). In relation to 5-HT1A presynaptic receptors, it has been shown that estradiol benzoate reduces the ability of 8-OH-DPAT to decrease the firing of dorsal raphe neurons (DRN) in rats (Lakoski, 1988) and promotes the desensitization of these receptors in the DRN of female monkeys (Lu and Bethea, 2002). As to the postsynaptic receptors, the effect of estrogens varies depending on the area studied and the treatment used. Thus, E2 promotes a downregulation of mRNA expression and decreases the binding to this receptor subtype in some limbic areas such as the medial amygdala, the piriform cortex, and the perirhinal cortex (Österlund and Hurd, 1998), but not in others, such as the hippocampus and prefrontal cortex (Landry and Di Paolo, 2003). Interestingly, some of these actions have been reported to occur in minutes (Mize and Alper, 2000, 2001), while others appear after several hours (Raap et al, 2000; Uphouse, 2000). Finally, it is important to mention that, at present, there are no reports on the interaction between EE2 and the 5-HT1A receptor subtype.

MATERIALS AND METHODS

Animals

Ovariectomized female Wistar rats (250–300 g, 3-months old), bred in the animal facilities of the ‘Centro de Investigación y Estudios Avanzados’ (Mexico City, Mexico), were housed in groups of six in polycarbonate cages and maintained on a 12 h light–dark cycle (lights on at 2200 h) in a temperature-controlled (22°C) room. Rats had free access to food and water, and were handled for 3–5 days prior to behavioral testing. All experimental procedures were performed in accordance with the Mexican official norm for animal care and handling (NOM-062-ZOO-1999) and approved by the Institutional Ethics Committee of the CINVESTAV-IPN.

Surgical Procedures

Ovariectomy was performed under ethylic ether anesthesia. Briefly, a single midline incision was made in the ventral area, oviducts were exposed, and ovaries removed. The complete extraction of the ovaries was corroborated by visual inspection. After 3 weeks, animals were randomly assigned to an experimental group and behavioral studies performed.

Forced Swimming Test

Swimming sessions were conducted by placing rats in individual glass cylinders (46 cm height × 20 cm diameter) containing water at 23–25°C, 30 cm deep, so that rats could not support themselves by touching the bottom with their paws or tail. Two swimming sessions were conducted: an initial 15-min pre-test, followed 24 h later by a 5-min test. Following each swimming session, the rats were removed from the cylinders, dried with paper towels and placed into heated cages for 30 min, and then returned to their home cages. Test sessions were run between 1200 and 1500 h and videotaped for later scoring. A single observer, who was blind to the treatment conditions, did all the behavioral scoring.

Behavioral Scoring

A time-sampling technique was employed to score three different behaviors (Detke et al, 1995). During the test session, the scorer rated at the end of each 5-s period the following behaviors of the rat: (1) immobility—floating in the water without struggling, and doing only those movements necessary to keep the head above the water; (2) swimming—showing active swimming motions, more than those necessary to merely keep the head above water, that is, moving around in the cylinder or diving; and (3) climbing—presenting active movements with the forepaws in and out of the water, usually directed against the walls.

Drugs

The estrogenic compounds E2 and EE2 (Sigma-Aldrich, St Louis, MO, USA) were dissolved in corn oil and s.c. injected (0.2 ml/rat). (±)8-hydroxy-2-(di-n)-propil-aminotetraline hydrobromide (Sigma-Aldrich, St Louis, MO, USA) was dissolved in saline solution, whereas WAY 100635 maleate (Sigma-Aldrich, St Louis, MO, USA) was dissolved in distilled water and s.c. (2 ml/kg) administered. All solutions were freshly prepared before each experimental series.

Experimental Design

Experiment 1. Participation of the 5-HT1A receptor in the antidepressant-like action of 8-OH-DPAT

In the first experiment, the effect of an agonist and a selective antagonist to the 5-HT1A receptor subtype, 8-OH-DPAT, and WAY 100635, respectively, were evaluated in ovariectomized female rats subjected to the FST. It is important to mention that, in previous studies, it has been reported that it is necessary to use a subacute schedule of high doses of antidepressants in order to observe the antidepressant-like actions in the FST (Detke et al, 1997). Therefore, independent groups (n=7–13) were formed and received three injections between the FST pretest and test sessions of one of the following doses of 8-OH-DPAT: 0.0625, 0.125, 0.25, and 0.5 mg/kg, or WAY 100635 (0.5 and 1.0 mg/kg) at 23, 5, and 1 h before the test session.

In a second experiment, the effect of WAY 100635 on the action of 8-OH-DPAT was analyzed (n=7–12). In this case, two doses of WAY 100635 (0.5 and 1.0 mg/kg) were combined with an effective dose of 8-OH-DPAT (0.5 mg/kg). Both compounds were administered simultaneously at 23, 5, and 1 h before the FST.

Control groups were treated with distilled water and saline solution following the same schedule of administration as the experimental groups.

Experiment 2. Effect of WAY 100635 on the antidepressant-like actions of E2 and EE23

Since estrogens have effects on serotonergic receptors after both short (in terms of minutes) and long (in terms of hours or few days) latencies, and in order to evaluate the possible involvement of the 5-HT1A receptor in the antidepressant-like effect of E2 and EE2 in the FST, the experiment was divided into two parts. In the first one, designed to study the short-term effects of estrogens on 5-HT1A receptors, WAY 100635 was administered simultaneously with an effective dose of E2 (10 μg/rat, n=8–10) or EE2 (5 μg/rat, n=6–10) and the test was performed 48 h later. Doses and latencies were chosen according to previous studies (Estrada-Camarena et al, 2003). In this experiment, the animals were assigned to independent groups that received one of the following treatments: saline+corn-oil (control), E2 (10 μg/rat)+saline, E2 (10 μg/rat)+WAY 100635 (0.5 and 1.0 mg/kg), EE2 (5 μg/rat)+saline, EE2 (5 μg/rat)+WAY 100635 (0.5 and 1.0 mg/kg). In the second part, aimed to study the long-term actions of estrogens on this serotonergic receptor subtype, E2 or EE2 (n=6–10) was administered 48 h before the FST and WAY 100635 was injected 30 min before the test session. The experimental groups included in this experiment were saline+corn-oil (control), E2 (10 μg/rat)+saline, E2 (10 μg/rat)+WAY 100635 (1 mg/kg), EE2 (5 μg/rat)+saline, EE2 (5 μg/kg)+WAY 100635 (1 mg/kg) (see Figure 1).

Figure 1
figure 1

Schedules of antagonist administrations in experiment 2. In order to evaluate the short-term actions of estrogens, steroids and the 5-HT1A receptor antagonist WAY 100635 were administered simultaneously (left panel). For the evaluation of long-term actions of estrogens, steroids were administered 47.5 h before the antagonist (right panel).

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Experiment 3. Effect of the combination of the 8-OH-DPAT with E2 or EE2

This experiment was designed to establish if the effect of a subthreshold dose of the 5-HT1A agonist 8-OH-DPAT could synergize with that of a noneffective dose of E2 or EE2 to produce an antidepressant-like action in the FST. As aforementioned, the actions produced by estrogens on the serotonergic system may involve minutes to days. On this basis, as in the previous experiment, the putative synergism of estrogens with 8-OH-DPAT was analyzed at two different times. In the first part, E2 (n=7–11) or EE2 (n=8–11) was applied 48 h before the FST and 8-OH-DPAT (0.0625 mg/kg) at 23, 5, and 1 h before the FST. In this case, the experimental groups were: saline+corn-oil (control), 8-OH-DPAT (0.0625 mg/kg)+corn-oil, E2 (2.5 μg/rat)+saline, E2 (2.5 μg/rat)+8-OH-DPAT (0.0625 mg/kg), EE2 (1.25 μg/rat)+saline, and EE2 (1.25 μg/rat)+8-OH-DPAT (0.0625 mg/kg). In the second part, only one injection of 8-OH-DPAT and one of estrogen were applied simultaneously 48 h before the FST. For this experiment, the groups used were: saline+corn oil (control), corn oil+8-OH-DPAT (0.0625 mg/kg), E2 (2.5 μg/rat)+8-OH-DPAT (0.0625 mg/kg), EE2 (1.25 μg/rat)+8-OH-DPAT (0.0625 mg/kg).

Doses and latencies for 8-OH-DPAT, WAY 100635, and estrogens were selected on the basis of previous reports (Estrada-Camarena et al, 2003; Martínez-Mota et al, 2002) and the present study.

Statistics

Statistical analyses were performed using the SigmaStat 2.03. computational program. A one-way ANOVA on ranks, Kruskall–Wallis test followed by the Dunn’s test, was used, and only the values with p<0.05 were accepted as significant.

RESULTS

Figure 2 shows the effect of several doses of 8-OH-DPAT (panel a), WAY 100635 (panel b), and the combination of both drugs (panel c) in the FST. As it can be seen, 8-OH-DPAT significantly decreased the immobility behavior (H=16.90, df=4, p=0.002) and increased the swimming behavior (H=19.92, df=4, p<0.001). This effect reached statistical significance at doses of 0.25 and 0.5 mg/kg. No changes were observed in the climbing behavior after 8-OH-DPAT (H=3.91, df=4, NS). On the other hand, the antagonist to the 5-HT1A receptor subtype, WAY 100635, significantly decreased swimming behavior at the dose of 1.0 mg/kg in the FST (one-way ANOVA values for immobility, H=4.69, df=2, NS; for swimming, H=15.11, df=2, p<0.001; and for climbing, H=0.059, df=2, NS). Finally, in panel c, the effect of WAY 100635 on the antidepressant-like action induced by an effective dose of 8-OH-DPAT can be observed. The decrease of immobility behavior and the concomitant increase of swimming behavior induced by 8-OH-DPAT were completely blocked by WAY 100635 at the two doses used. Significant changes in the climbing behavior were not observed (one-way ANOVA values for immobility, H=14.98, df=3, p=0.002; swimming, H=24.3, df=3, p<0.001; and climbing, H=5.73, df=3, NS).

Figure 2
figure 2

Behavioral effects produced by several doses of 8-OH-DPAT (8-OH, −23, −5, and −1 h; panel a), WAY 100635 (WAY, −23, −5, and −1 h, panel b) and 8-OH plus WAY 100635 (−23, −5, and −1 h, panel c) in the FST. Data correspond to mean of counts (+1SEM) of immobility, swimming, and climbing behaviors when sampled every 5 s during a 5-min test period (7–13 animals per group). Dunn's test, *p<0.05.

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Figure 3 shows the effect of the simultaneous application, 48 h before the FST, of the selective antagonist to the 5-HT1A receptor, WAY 100635, and E2 (panel a) or EE2 (panel b). As it can be seen, the decrease in immobility behavior and the increase in swimming behavior induced by an effective dose of E2 (10 μg/rat, panel a) were blocked by both doses of WAY 100635 (0.5 and 1.0 mg/kg). There were no changes in the climbing behavior (one-way ANOVA for the immobility behavior, H=17.3, df=3, p<0.001, for swimming behavior, H=17.59, df=3, p<0.001, and for climbing, H=0.85, df=3, NS). In relation to the effect of WAY 100635 on the actions of EE2 (panel b), the results showed that WAY 100635 was unable to cancel the effect of this steroid on immobility, swimming, and climbing behaviors (one-way ANOVA for immobility, H=14.44, df=3, p=0.002; for swimming, H=11.26, df=3, p=0.01; for climbing, H=4.87, df=3, NS).

Figure 3
figure 3

Behavioral effect of the simultaneous application of WAY 100635 (WAY; −48 h) and an effective dose of E2 (−48 h, panel a) or EE2 (−48 h; panel b) in the FST. Data represent the mean of counts (+1SEM) of immobility, swimming, and climbing behaviors when sampled every 5 s during a 5-min test period (6–10 animals per group). Dunn's test, *p<0.05.

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The effects of the application of WAY 100635 several hours (47.5 h) after injecting E2 or EE2 are shown in Table 1. As it can be seen, WAY 100635 did not cancel the antidepressant-like action of the natural estrogen, E2. Thus, the decrease in the immobility behavior accompanied by the increase in swimming behavior remained in the group that received WAY 100635 several hours after E2. In contrast, under this administration schedule, the antagonist to the 5-HT1A receptor subtype blocked the effects of EE2. In this case, when WAY 100635 was applied several hours after EE2, the steroid did not decrease the immobility behavior in comparison to the control group. In addition, a decrease in swimming behavior was observed. One-way ANOVA on ranks is shown in Table 1.

Table 1 Effect of the WAY 100635 Applied 30 min before the FST on the Antidepressant-Like Action of E2 or EE2
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Figure 4 illustrates the effect of the application of estrogens, E2 (panel a) and EE2 (panel b), 48 h before the FST, followed by the administration of three injections of a suboptimal dose of 8-OH-DPAT. Low doses of either E2 or 8-OH-DPAT had no effects in the FST (panel a); however, when combined, a decrease in immobility behavior (H=15.74, df=3, p=0.001) with a concomitant increase in swimming behavior (H=15.54, df=3, p=0.001) and no changes in climbing behavior (H=1.99, df=3, NS) were observed. A similar effect was found when a suboptimal dose of the synthetic estrogen EE2 was combined with a noneffective treatment of 8-OH-DPAT (panel b), that is, a decrease in immobility behavior (H=13.23, df=3, p=0.004) accompanied by an increase of swimming behavior (H=12.55, df=3, p=0.006) was observed. None of the treatments induced changes in climbing behavior (H=2.02, df=3, NS).

Figure 4
figure 4

Behavioral effect of the combination of a subthreshold dose of E2 (−48 h; panel a) or EE2 (−48 h, panel b) with noneffective doses of 8-OH-DPAT (8-OH; −23, −5, and −1 h) in the FST. Data represent the mean of counts (+1SEM) of immobility, swimming, and climbing behaviors when sampled every 5 s during a 5-min test period (7–11 animals per group). Dunn's test, *p<0.05.

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The results obtained after the simultaneous injection of a suboptimal dose of 8-OH-DPAT with a noneffective dose of E2 or EE2 48 h before the FST can be seen in Table 2. Administration of the agonist to the 5-HT1A receptor subtype with estrogens elicited a clear antidepressant-like action (p<0.05), and promoted a slight increase in both swimming and climbing behaviors, without reaching statistical significance. For one-way ANOVA test, see Table 2.

Table 2 Effect of the Simultaneous Administration of 8-OH-DPAT and E2 or EE2 48 h before the FST
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DISCUSSION

In the present study, the 5-HT1A agonist 8-OH-DPAT produced an antidepressant-like action in ovariectomized female rats in the FST, characterized by a decrease in immobility behavior and an increase in swimming behavior. A similar behavioral profile has been reported for male rats treated with the SSRIs, indorenate (a full agonist to the 5-HT1A receptor) and buspirone (a partial agonist to 5-HT1A receptors) (Detke et al, 1995; Martínez-Mota et al, 2002). However, female rats seem more sensitive than males to the actions of 8-OH-DPAT, since lower doses of this compound were needed in the present study to induce an antidepressant-like action in the FST. Hence, the behavioral profile found with 8-OH-DPAT by other groups in male rats can be extended to ovariectomized female rats. In the present study, the antidepressant-like action of 8-OH-DPAT was completely blocked by the selective antagonist to the 5-HT1A receptor, WAY 100635 (Forster et al, 1995), a result that confirms the involvement of this receptor subtype in the antidepressant-like effect of 8-OH-DPAT in female rats.

As mentioned before, the behavioral profile of drugs acting at the serotonergic system, including agonists to 5-HT1A receptors, is a decrease in inmmobility and a concomitant increase in swimming behavior in the FST (Detke et al, 1995; Cryan and Lucki, 2000; Martínez-Mota et al, 2002; Page et al, 1999). In relation to the effects of estrogens, in the present study, it was observed that the antidepressant-like action of E2 was characterized by an increase in swimming, whereas that of EE2 was distinguished by an increase in both swimming and climbing behaviors. Hence, it is reasonable to consider that the serotonergic system contributes to the antidepressant-like actions of both estrogenic compounds. In the present series of experiments, it was also found that the administration of suboptimal doses of 8-OH-DPAT combined with noneffective doses of E2 or EE2 resulted in clear antidepressant-like effects. These results suggest that both compounds, steroids and the 5-HT1A receptor agonist, could act together to promote the antidepressant-like action observed. Jackson and Uphouse (1998) reported a similar result when showing that low doses of E2 combined with low doses of 8-OH-DPAT synergized to inhibit lordosis behavior. Notwithstanding, it is possible that the interaction between estrogens and the 5-HT1A receptor subtype may be different for each type of estrogen (vide infra).

Present results showed that E2: (1) produced a behavioral profile similar to that induced by an agonist to the 5-HT1A receptor, 8-OH-DPAT, in the FST; (2) synergized with 8-OH-DPAT to induce an antidepressant-like action and, finally, estradiol’s antidepressant-like effect was totally cancelled by the simultaneous administration of the selective 5-HT1A antagonist, WAY 100635. Therefore, it can be concluded that the 5-HT1A receptor subtype is a mediator of the antidepressant-like action of E2. Evidences in favor of this hypothesis are the results derived from in vitro and in vivo studies, showing that the 5-HT1A receptor subtype is a target of estrogens and some estrogen-like compounds (Bethea et al, 1998; Biegon et al, 1983; Mize and Alper, 2000; Mize et al, 2001; Raap et al, 2000; Uphouse, 2000). Indeed, Lu and Bethea (2002) demonstrated that E2 induces the desensitization of 5-HT1A receptors in the DRN of monkeys. Thus, it is possible that the antidepressant-like actions of E2 are mediated by its action on these presynaptic receptors. Interestingly, it has also been reported that E2 may induce changes at postsynaptic 5-HT1A receptors. In this sense, in ovariectomized Flinders rats (a genetic model of depression), the characteristic increased mRNA expression of 5-HT1A receptors in some limbic areas such as amygdala, hypothalamus, and hippocampus, is reduced by a single injection of E2 (Österlund et al, 1999). Furthermore, in Wistar female rats, ovariectomy induces an increase in the sensitivity of 5-HT1A receptors in the hypothalamus that is abolished by E2 treatment (Raap et al, 2002). Taken together, these results support the notion that the actions of E2 on 5-HT1A receptors may promote adaptive changes similar to those induced by antidepressants to exert their effects. Interestingly, the occurrence of these adaptive changes may take less time for estrogens than for antidepressants (Raap et al, 2000).

At least two mechanisms have been proposed to explain the actions of estrogens. The first one involves the activation of intracellular steroid receptors, while the second one implies the activation of membrane receptors, with the subsequent activation of second messenger systems (for a review, see Lee and McEwen, 2001). Considering this, it could be expected that E2 stimulates the 5-HT1A receptor subtype in two different manners: one mechanism, underlying the effects that last from several hours to days, that could involve the activation of intracellular estrogen receptors and the subsequent transcription of the gene that codifies for the 5-HT1A receptor subtype. The other mechanism, underlying those estrogenic effects that appear in the order of minutes, that implies the activation of the 5-HT1A receptor subtype and its respective second messenger cascade (Mize and Alper, 2000). The fact that the antidepressant-like actions of E2 were blocked by WAY 100635 when both compounds were administered simultaneously (48 h before the test) indicates that the 5-HT1A receptor subtype has a significant participation in the anti-immobility effect induced by this steroid, suggesting a direct interaction with 5-HT1A receptors. This hypothesis could find support in the fact that the application of WAY 100635, 30 min before the test (or 47.5 h after E2), did not block the antidepressant-like action of the steroid. Taken together, these results suggest that under these circumstances E2 may compete with the antagonist for the occupancy of the 5-HT1A receptor, and in this case the action of E2 would not be intracellularly mediated. Another possibility is that E2 activates other type of receptors, indirectly promoting the activation of the 5-HT1A receptor subtype like it has been suggested by in vitro studies (Mize and Alper, 2002). In this case, the process could require several minutes before the 5-HT1A receptor is activated by a heterologous process, a time sufficient for WAY 100635 to occupy 5-HT1A receptors and cancel its activation. Notwithstanding, specific experiments are necessary to explore both possibilities, the present results are in favor of a direct interaction between E2 and 5-HT1A receptors, that is not mediated by the intracellular estrogen receptor.

In relation to the antidepressant-like actions of EE2, the participation of the 5-HT1A receptor seems to be different from that of E2. In this case, it was found that: (1) the behavioral profile of EE2 was different from that induced by 8-OH-DPAT and E2 in the FST; (2) the combination of subthreshold doses of both 8-OH-DPAT and EE2 promoted an antidepressant-like action in the FST; and (3) WAY 100635 was unable to cancel the antidepressant-like effects of EE2 when simultaneously applied (48 h before the test), but blocked it when applied 47.5 h after the steroid (30 min before the test). The present results suggest the activation of the 5-HT1A receptor subtype. It is possible that the stimulation of this serotonergic receptor by EE2 is secondary to the activation of other unknown sites. In this point, it is important to mention that the behavioral profile induced by EE2 alone was similar to that produced by mixed reuptake inhibitors that act at both the noradrenergic and serotonergic systems, such as venlafaxine and duloxetine in the FST, that is, these compounds produce a decrease in immobility with a concomitant increase in both swimming and climbing behaviors (Rénéric and Lucki, 1998). Furthermore, in vitro studies have shown that EE2 inhibits both serotonin and noradrenaline transporters with a similar potency (Michel et al, 1987; Ghraf et al, 1983). At present, data on the mechanism of action of EE2 on neurotransmitter systems are derived only from in vitro studies and there are no data from behavioral studies. The reasons why the noradrenergic actions are not as evident as the serotonergic ones are unknown; however, it is important to mention that there is a marked tendency of EE2 to increase climbing behavior when compared with the control group, a datum pointing towards the participation of the noradrenergic system. At present, no data in relation to this point exist; therefore, future studies should be undertaken to analyze the possible sites of action other than estrogenic receptors for EE2.

The administration of a subthreshold dose of E2 or EE2 together with a subacute 8-OH-DPAT treatment (three injections between pre-test and test sessions) promotes an antidepressant-like action characterized by a decrease in immobility, accompanied by an increase in swimming behavior. Since E2 promotes a behavioral profile similar to that induced by 8-OH-DPAT itself, it is possible to suggest that both compounds act at the 5-HT1A receptor subtype. In the case of the combination of EE2 and 8-OH-DPAT, it is possible that the specific action of 8-OH-DPAT at the 5-HT1A receptor subtype, after a subacute treatment, masked the behavioral profile induced by EE2, for example, an increase in swimming instead of an increase in both swimming and climbing behaviors. Moreover, when an injection of EE2 was combined with one of 8-OH-DPAT (48 h before the test), the antidepressant-like action observed was characterized by a decrease in immobility behavior, accompanied by an increase in both swimming and climbing behaviors. Hence, it is possible to consider that, under this experimental schedule, the behavioral profile of EE2 prevails over that induced by 8-OH-DPAT.

Changes in the sensitivity of the 5-HT1A receptor have been associated with the antidepressant action of several compounds in clinical and preclinical studies (for reviews, see Blier and Ward, 2003; Cryan and Leonard, 2000; Duman et al, 1997). Recently, it was shown that the application of a high dose of 8-OH-DPAT (0.5 mg/kg) or FLX (10 mg/kg) desensitized presynaptic 5-HT1A receptors in a short time (Kennett et al, 1987; Riad et al, 2001, 2004). These results suggest that the overstimulation of the 5-HT1A receptor could accelerate its desensitization and an antidepressant action would follow with a shorter latency. In the present study, it was observed that a low dose of 8-OH-DPAT by itself was insufficient to induce an antidepressant-like action; however, when this agonist was combined with a noneffective dose of E2 or EE2, administered 48 h before the FST, a clear antidepressant-like action was observed. Hence, it is possible that the combined treatment of compounds which act (directly or indirectly) on the 5-HT1A receptor subtype could synergize to promote changes in its sensitivity that result in antidepressant-like effects.

In conclusion, the 5-HT1A receptor seems to be a target of E2 and EE2 in their antidepressant-like actions. This receptor subtype has been considered to play a role in the physiology of depression and in the mechanism of action of several types of antidepressants (Blier and Ward, 2003; Duman, 1999; Stahl, 1998a). Recently, some groups proposed that combining antidepressants with other compounds that promote the desensitization of 5-HT1A receptors could shorten the latency for the onset of SSRI’s therapeutic effects (Artigas et al, 1994, 1996; Blier and de Montigny, 1994; Cryan and Leonard, 2000). From this perspective, the administration of E2, an estrogenic compound that acts at the 5-HT1A receptor subtype, could be an alternative to improve the action of antidepressants. Indeed, some clinical studies have combined estrogens with FLX, sertraline or citalopram, with positive results (Stahl, 1998b; Schneider et al, 1997, 2001; Soares et al, 2001).