Introduction

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The treatment of mood disorders exhibits two problems, a 2- to 6-week delay in onset of therapeutic improvement after administration of antidepressants and the lack of consistent response to treatment of depression for ~30% of the patients (Ananth, 1998). Although the desensitization of postsynaptic 5-hydroxytryptamine (5-HT)_1A receptors may not contribute to the treatment of depression (Sargent et al., 2000), it may underlie the therapeutic effects of selective serotonin reuptake inhibitors (SSRIs) for other disorders such as anxiety, aggression, and obsessive compulsive disorders (Li et al., 1996, 1997; Sargent et al., 1997; Lerer et al., 1999). For example, SSRI treatment of patients with obsessive compulsive disorders results in a desensitization of the adrenal corticotrophic hormone (corticotropin; ACTH) response to the 5-HT_1A agonist ipsapirone (Lesch et al., 1991).

Although pindolol, a 5-HT_1A/1B/-adrenoceptor antagonist has been reported to decrease the delay in the onset of antidepressant effects when taken in combination with SSRIs, the effectiveness of this treatment remains controversial (Blier and Bergeron, 1995; Berman et al., 1999; Pérez et al., 1999). It also is unclear whether pindolol can accelerate the therapeutic effects of SSRIs in other disorders such as anxiety, obsessive-compulsive, and eating disorders. Hence, an in-depth analysis of the combination of 5-HT_1A antagonists with SSRIs is needed to evaluate the possible role of somatodendritic versus postsynaptic 5-HT_1A receptors in mediating the effects of SSRIs.

The effect of pindolol has been attributed to its antagonistic effect on somatodendritic 5-HT_1A autoreceptors, which inhibit the firing rate of serotonergic neurons. SSRIs are thought to have a delayed onset partly because of the need to overcome the inhibitory influence of these somatodendritic 5-HT_1A autoreceptors (Artigas, 1993). Microdialysis studies have shown that pindolol potentiates the SSRI-induced increase in extracellular levels of 5-HT in forebrain regions (Gardier et al., 1996; Gartside et al., 1999). This effect of pindolol is mimicked by systemic pretreatment with the selective 5-HT_1A antagonist WAY-100635 in combination with a SSRI (Invernizzi et al., 1996). Thus, selective 5-HT_1A antagonists could potentiate the fluoxetine-induced increase in levels of 5-HT in the synaptic cleft.

The sensitivity of postsynaptic receptors in the hypothalamus can be evaluated from the magnitude of elevation in plasma ACTH and oxytocin levels in response to a challenge injection with the 5-HT_1A receptor agonist (±)-8-hydroxy-2-dipropylaminoetetralin (8-OH-DPAT; Cowen, 1998). Activation of 5-HT_1A receptors by 5-HT_1A agonists increases the secretion of ACTH and oxytocin (Pan and Gilbert, 1992; Bagdy, 1996). This effect of 5-HT_1A agonists is antagonized by several 5-HT_1A antagonists, indicating that the hormone responses are 5-HT_1A receptor mediated (Cowen, 1998; Vicentic et al., 1998). Measuring plasma levels of ACTH and oxytocin, therefore, provides an indicator of hypothalamic postsynaptic 5-HT_1A receptor function.

Gi/o proteins are known to couple 5-HT_1A receptors to adenylyl cyclase and to K⁺ channels (Albert et al., 1996). Gz proteins are members of the Gi protein family that have a high affinity for 5-HT_1A receptors in cell culture (Butkerait et al., 1995). The coupling of 5-HT_1A receptors in the hypothalamic paraventricular nucleus to the secretion of ACTH and oxytocin was shown to be mediated by Gz proteins (Serres et al., 2000). In addition, treatment with fluoxetine produces a dose-dependent reduction in the levels of Gz proteins in the hypothalamus (Raap et al., 1999). To examine the possible role of Gz proteins in the onset of fluoxetine-induced desensitization of hypothalamic 5-HT_1A receptors, the levels of Gz protein in the hypothalamus were examined.

This study determined whether coadministration of low doses (0.03 and 0.3 mg/kg/day s.c.) and a high dose (1 mg/kg/day s.c.) of the 5-HT_1A antagonist WAY-100635, combined with fluoxetine (10 mg/kg/day i.p.), would induce an earlier and/or more pronounced desensitization of hypothalamic postsynaptic 5-HT_1A receptors. WAY-100635 doses between 0.01 and 0.3 mg/kg block the somatodendritic 5-HT_1A autoreceptors in the raphe, thereby potentiating the effects of SSRIs on extracellular levels of 5-HT (Invernizzi et al., 1996; Hjorth et al., 1997). Although these WAY-100635 doses also might produce a partial occupancy of postsynaptic 5-HT_1A receptors, much higher doses (1 mg/kg) are needed to completely block hypothalamic postsynaptic 5-HT_1A receptors (Vicentic et al., 1998). One dose of WAY-100635 (0.3 mg/kg/day s.c.) also was used in combination with fluoxetine to investigate whether the desensitization occurs after only 2 daily injections. This dose of WAY-100635 was previously shown to maximally potentiate the effects of SSRIs on the extracellular levels of 5-HT (Hjorth et al., 1997).

	Materials and Methods

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Animals. Male Sprague-Dawley rats (225-250 g) were purchased from Harlan Laboratories (Indianapolis, IN). The rats were housed two per cage in a room controlled for temperature, humidity, and lighting (lights on 7:00 AM-7:00 PM). Food and water were available ad libitum. All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals as approved by the Loyola University Institutional Animal Care and Use Committee.

Drugs. Fluoxetine HCl was donated by Eli Lilly (Indianapolis, IN). A fresh fluoxetine solution was prepared daily by dissolving fluoxetine in the vehicle (0.9% saline). Fluoxetine was injected i.p. in a volume of 2 ml/kg. WAY-100635 was donated by Wyeth-Ayerst (Princeton, NJ). It was prepared fresh daily by dissolving it in saline. WAY-100635 was injected s.c. in a volume of 1 ml/kg. (±)8-OH-DPAT HBr was purchased from Research Biochemicals International (Natick, MA) and was dissolved in saline. (±)8-OH-DPAT was injected s.c. in a volume of 1 ml/kg.

Animal Drug Treatment. In experiment 1, the rats received an injection of either saline or WAY-100635 (0.3 mg/kg/day s.c.), followed 15 min later by an injection of either saline or fluoxetine (10 mg/kg/day i.p.). This procedure was repeated for 2 consecutive days. On day 3, the rats were challenged with 8-OH-DPAT (50 µg/kg s.c.), 15 min before decapitation. Trunk blood was collected in centrifuge tubes containing 0.5 ml of a 0.3 M EDTA (pH 7.4) solution. The plasma was separated and stored at 70°C for subsequent measurement of hormone levels.

Radioimmunoassays of Hormones and Immunoblots of Gz Protein. Plasma oxytocin and ACTH concentrations were determined in all animals by radioimmunoassays as previously described in detail (Li et al., 1993, 1997). Procedures used to determine the levels of Gz protein were described by us in detail in a previous article (Raap et al., 1999). Briefly, Gz protein levels were measured in hypothalamic tissue from animals that received a saline challenge. All procedures were conducted at 4°C. Tissues were homogenized, the membrane-bound proteins were solubilized, and their concentration was determined with BSA as a standard.

Statistical Analyses. All data are expressed as the mean ± S.E. Hormone data were analyzed by a three-way ANOVA. G-protein data were analyzed with a two-way ANOVA. Group means were compared by Newman-Keuls multiple range test (Steel and Torrie, 1960). GB-STAT software (Dynamic Microsystems, Inc., Silver Spring, MD) was used for all statistical analyses.

	Results

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Experiment 1. Administration of 8-OH-DPAT (50 µg/kg s.c.) produced a significant increase in plasma levels of oxytocin (1306%; Fig. 1) and ACTH (962%; Fig. 1) compared with saline-challenged rats. Two daily injections of fluoxetine (10 mg/kg/day) did not induce a significant reduction in the 8-OH-DPAT-induced increase in oxytocin or ACTH (Fig. 1). WAY-100635 administered alone for 2 days (at a dose of 0.3 mg/kg s.c.) induced a significant potentiation of the oxytocin response to 8-OH-DPAT compared with control (saline-pretreated) rats. However, WAY-100635 did not significantly change the ACTH response to 8-OH-DPAT (Fig. 1). The oxytocin response to 8-OH-DPAT was significantly decreased after the combination treatment of fluoxetine plus WAY-100635, compared with treatment with WAY-100635 alone, but this was not significantly different from control (saline-treated) animals (Fig. 1). The combination of WAY-100635 (0.3 mg/kg s.c.) and fluoxetine (10 mg/kg i.p.) administered daily for 2 days did not induce a significant change in plasma levels of ACTH after a challenge injection of 8-OH-DPAT. For oxytocin, the three-way ANOVA revealed no significant main effect of fluoxetine or WAY-100635 but a significant main effect of 8-OH-DPAT (F_1,53 = 312.98, P < .001). A significant fluoxetine × WAY-100635 interaction (F_1,53 = 4.32, P < .01) was observed, but no significant interaction was observed between fluoxetine × 8-OH-DPAT or WAY-100635 × 8-OH-DPAT. The three-way ANOVA for ACTH revealed a significant main effect of WAY-100635 (F_1,53 = 5.30, P < .01) and a significant main effect of 8-OH-DPAT (F_1,53 = 304.73, P < .0001) but no significant main effect of fluoxetine (F_1,53 = 1.55, NS). Also, there was no significant fluoxetine × 8-OH-DPAT, WAY-100635 × 8-OH-DPAT, or fluoxetine × WAY-100635 interaction.

View larger version (37K): [in this window] [in a new window]   Fig. 1.   Pretreatment with WAY-100635 (0.3 mg/kg/day s.c., two daily injections) combined with fluoxetine (10 mg/kg/day i.p., two daily injections) does not desensitize oxytocin (A) and ACTH (B) responses to 8-OH-DPAT (50 µg/kg s.c.). The data represent the mean ± S. E. of eight rats per group. *, significant difference from control (saline-challenged) rats; P < .05 (three-way ANOVA and Newman-Keuls multiple range test). #, significant difference from saline-treated rats challenged with 8-OH-DPAT, P < .05 (three-way ANOVA and Newman-Keuls multiple range test).

Experiment 2. An injection of 8-OH-DPAT (50 µg/kg s.c.) produced a significant increase in plasma levels of oxytocin (13,421%) and ACTH (1,615%) compared with saline-challenged rats (Fig. 2). Three daily injections of fluoxetine (10 mg/kg/day i.p.) significantly reduced the oxytocin (by 67.7%) and ACTH (by 39.5%) responses to 8-OH-DPAT. Pretreatment of the rats with increasing doses of WAY-100635 produced a dose-dependent reversal of the fluoxetine-induced desensitization of the oxytocin and ACTH responses to 8-OH-DPAT (Fig. 2). Pretreatment with the highest dose of WAY-100635 (1 mg/kg/day) completely reversed the effect of fluoxetine on both oxytocin and ACTH responses to 8-OH-DPAT (Fig. 2). Daily injections of WAY-100635 alone did not alter the effect of 8-OH-DPAT on plasma oxytocin or ACTH (Fig. 2). None of the treatments altered the basal levels of ACTH or oxytocin. For oxytocin, the three-way ANOVA revealed a significant main effect of fluoxetine (F_1,107 = 19.18, P < .0001), a significant main effect of 8-OH-DPAT (F_1,107 = 167.79, P < .001), but no significant main effect for WAY-100635 (F_1,107 = 2.08, P > .1). In addition, the ANOVA revealed a significant interaction between fluoxetine and 8-OH-DPAT (F_1,107 = 17.37, P < .001) but no significant interaction between fluoxetine and WAY-100635. The three-way ANOVA revealed for ACTH a significant main effect of WAY-100635 (F_3,109 = 4.42, P < .01), a significant main effect of fluoxetine (F_1,109 = 7.58, P < .01), and a significant main effect of 8-OH-DPAT (F_1,109 = 284.44, P < .001). There was also a significant fluoxetine × 8-OH-DPAT interaction (F_1,109 = 7.88, P < .01), and a significant WAY-100635 × 8-OH-DPAT interaction (F_3,109 = 4.47, P < .01). However, no significant interaction between fluoxetine × WAY-100635 was observed.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Pretreatment with WAY-100635 (0.03, 0.3, and 1 mg/kg/day s.c., three daily injections) reverses the desensitization by fluoxetine (10 mg/kg/day i.p., three daily injections) of oxytocin (A) and ACTH (B) responses to 8-OH-DPAT (50 µg/kg s.c.). The data represent mean ± S.E. of eight rats per group. *, significant difference from the control (saline-challenged) rats, P < .01 (three-way ANOVA and Newman-Keuls multiple range test). #, significant fluoxetine effect, difference from saline-treated rats challenged with 8-OH-DPAT, P < .01 (three-way ANOVA and Newman-Keuls multiple range test).

View larger version (40K): [in this window] [in a new window]   Fig. 3.   Hypothalamic levels of Gz protein are not altered in rats treated with WAY-100635 (0.03, 0.3, and 1 mg/kg/day s.c.) alone, fluoxetine (10 mg/kg/day i.p.) alone, or their combination for 3 days. The data represent mean ± S.E. of eight rats per group. Filled columns, saline; gray columns, fluoxetine (10 mg/kg/day).

	Discussion

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This study examined some of the mechanisms through which chronic exposure to fluoxetine produces a desensitization of postsynaptic 5-HT_1A receptors in the hypothalamus. Several conclusions can be drawn from this study. First, in rats, the minimum treatment required for fluoxetine (10 mg/kg) to produce a (partial) desensitization of hypothalamic postsynaptic 5-HT_1A receptors is three daily injections. Second, fluoxetine produces a homologous desensitization of postsynaptic 5-HT_1A receptors by inducing their overstimulation. And third, a reduction in the levels of Gz proteins in the hypothalamus is not the only mechanism mediating the desensitization of hypothalamic 5-HT_1A receptors. The inability of low doses of WAY-100635 to accelerate the fluoxetine-induced desensitization of hypothalamic 5-HT_1A receptors could be interpreted either as a lack of involvement of somatodendritic 5-HT_1A autoreceptors in the time course of fluoxetine-induced desensitization of hypothalamic 5-HT_1A receptors, or as a possible interference of low doses of WAY-100635 with the homologous desensitization induced by elevated 5-HT levels in the synaptic cleft.

WAY-100635 is a relatively selective 5-HT_1A antagonist with a low affinity for other 5-HT receptor subtypes (Routledge, 1995). WAY-100635 has a relatively short half-life of 3 to 4 h in vivo with complete loss of antagonistic activity at 8 h postadministration (T. Andree and P. McGonigle, personal communication). The short half-life of WAY-100635 is also apparent from the fact that 18 h after its last injection, WAY-100635 did not produce an inhibition of the effects of 8-OH-DPAT on plasma ACTH and oxytocin levels, at any dose. In contrast with WAY-100635, fluoxetine produces a long-lasting inhibition (up to 16 h postinjection) of 5-HT reuptake (Fuller et al., 1978). Therefore, it is likely that antagonism of postsynaptic 5-HT_1A receptors for a few hours each day is sufficient to prevent their desensitization by fluoxetine.

Neuroendocrine studies indicate that doses of WAY-100635 <0.1 mg/kg may produce a very limited occupancy of hypothalamic postsynaptic 5-HT_1A receptors. This conclusion was based on the fact that a dose of 0.1 mg/kg s.c. injected 45 min before 8-OH-DPAT did not inhibit the effect of 8-OH-DPAT on plasma levels of ACTH and produced a 22% inhibition of the oxytocin response to 8-OH-DPAT (Vicentic et al., 1998). In contrast, the dose of 1 mg/kg WAY-100635 completely blocked the effect of 8-OH-DPAT on plasma levels of ACTH and oxytocin, suggesting complete antagonism of hypothalamic 5-HT_1A receptors (Vicentic et al., 1998). The fact that, in this study, this dose of WAY-100635 (1 mg/kg) also prevented the fluoxetine-induced desensitization of hypothalamic 5-HT_1A receptors suggests that increased levels of 5-HT in the synaptic cleft, due to the blockade of its reuptake mechanism, leads to a sustained overactivation of these postsynaptic 5-HT_1A receptors and to their homologous desensitization.

In vivo microdialysis studies indicate that relatively low doses of WAY-100635 (0.01-0.3 mg/kg s.c.) block the somatodendritic 5-HT_1A autoreceptors in the raphe, and potentiate the effects of SSRIs on extracellular levels of 5-HT in several forebrain regions, including the hypothalamus (Hjorth et al., 1997). These doses of WAY-100635 do not completely block postsynaptic 5-HT_1A receptors in the hypothalamus but could produce a partial occupancy of these receptors (Critchley et al., 1994; Vicentic et al., 1998). Our data indicate that antagonism of somatodendritic 5-HT_1A receptors with these low doses of WAY-100635 (0.03 and 0.3 mg/kg) do not significantly alter the degree of fluoxetine-induced desensitization of hypothalamic 5-HT_1A receptors. Based on literature indicating that blockade of somatodendritic 5-HT_1A autoreceptors increases the levels of 5-HT in the synaptic cleft (Invernizzi et al., 1996), one would expect to see a more rapid and/or a more pronounced desensitization of hypothalamic postsynaptic 5-HT_1A receptors after a combination of WAY-100635 and fluoxetine treatment. The fact that treatment with these low doses of WAY-100635 did not potentiate the fluoxetine-induced desensitization of postsynaptic 5-HT_1A receptors in the hypothalamus could be interpreted in one of two ways: 1) somatodendritic 5-HT_1A autoreceptors in the raphe do not play a major role in this phenomenon; or 2) the low doses of WAY-100635 produced a partial antagonism of postsynaptic 5-HT_1A receptors, thereby preventing their overactivation by synaptic 5-HT.

Treatment of rats with fluoxetine for 3, 7, 14, and 22 days induces a gradual desensitization of hypothalamic 5-HT_1A receptor systems. A maximum desensitization was apparent after 7 and 14 days (Li et al., 1996). A similar study with paroxetine also indicated that the maximum desensitization occurs between 7 and 14 days after initiation of paroxetine injections (Li et al., 1997). In both studies, a partial desensitization was already apparent after three daily injections of the respective SSRI (Li et al., 1996, 1997). No studies were conducted to identify the onset of desensitization. In this study, the observation that two daily injections of fluoxetine do not induce a desensitization of hypothalamic 5-HT_1A receptors, but three daily injections induce a partial desensitization, suggests a sharp transition between days 2 and 3 of fluoxetine injections. The nature of this transition and the mechanisms responsible remain to be elucidated.

5-HT_1A receptors are coupled with a high affinity to the Gi family of G-proteins, including Gi1, Gi2, Gi3, Go, and Gz (Butkerait et al., 1995). The G-protein mediating the effects of 5-HT_1A receptors on the secretion of ACTH and oxytocin is the pertussis toxin-insensitive Gz protein (Serres et al., 2000). We have recently observed that treatment with fluoxetine for 14 days produced a dose-dependent reduction in the levels of Gz proteins in the hypothalamus (Raap et al., 1999). No data have been published regarding changes in Gz proteins after only 3 daily injections of fluoxetine or other SSRIs. The present data indicate that daily injections of fluoxetine for 3 days do not produce a reduction in the level of Gz proteins in hypothalamus. This is not entirely surprising considering the fact that members of the Gi protein family have a half-life of ~2 to 3 days (Bahouth, 1995), although no data on the exact half-life of Gz proteins have been published. Therefore, it must be concluded that changes in the levels of Gz proteins are not the initial mechanism underlying the desensitization of hypothalamic 5-HT_1A receptors. Alternative mechanisms could include alterations in relocation of Gz proteins from the membrane to the cytosol; post-translational modifications in Gz proteins (such as changes in phosphorylation or palmitoylation); or an involvement of regulatory G-protein signaling (RGS) proteins such as RGSZ1, which would reduce the effective coupling to 5-HT_1A receptors to their effector mechanisms (Bhamre et al., 1998; Glick et al., 1998; Wang et al., 1998).

In conclusion, a 5-HT_1A antagonist blocked the desensitization of hypothalamic 5-HT_1A receptor systems induced by treatment with fluoxetine. These observations suggest that exposure to fluoxetine gradually increases the levels of 5-HT in the synaptic cleft, resulting in homologous desensitization of postsynaptic 5-HT_1A receptors in the hypothalamus with a delayed onset. The minimum exposure time for fluoxetine to induce desensitization in rats is 3 days; somatodendritic 5-HT_1A autoreceptors in the hindbrain raphe nuclei do not seem to play a critical role in altering the time course of fluoxetine-induced desensitization of postsynaptic 5-HT_1A receptors in the hypothalamus.