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NEUROPHARMACOLOGY

Single Exposure to a Serotonin 1A Receptor Agonist, (+)8-Hydroxy-2-(di-n-propylamino)-tetralin, Produces a Prolonged Heterologous Desensitization of Serotonin 2A Receptors in Neuroendocrine Neurons in Vivo

Department of Pharmacology (G.A.C., L.D.V.d.K., H.X., Z.C., R.C., F.G., N.A.M., G.B.), Neuroscience Institute (G.A.C., L.D.V.d.K., C.J., N.A.M., G.B.), Loyola University Chicago, Maywood, Illinois

Received October 26, 2006; accepted December 8, 2006.

	Abstract

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We previously demonstrated colocalization of serotonin 1A (5-HT_1A) and serotonin 2A (5-HT_2A) receptors in oxytocin and corticotropin-releasing factor neurons in the hypothalamic paraventricular nucleus (PVN). Because a functional imbalance between hypothalamic 5-HT_1A and 5-HT_2A receptors has been implicated in several neuropsychiatric disorders, in this study we investigated whether acute in vivo activation of 5-HT_1A receptors in the PVN results in desensitization of 5-HT_2A receptor signaling. Functional desensitization of hypothalamic 5-HT_2A receptors was assessed via a reduction in oxytocin and adrenocorticotropin (ACTH) responses to the 5-HT_2A/2C receptor agonist (-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl [(-)DOI]. We report here that a single systemic injection of the 5-HT_1A receptor agonist (+)-8-hydroxy-2-(di-n-propylamino)-tetralin [(+)8-OH-DPAT] (200 µg/kg) significantly reduced the 5-HT_2A receptor-mediated oxytocin responses for at least 72 h. Direct intraparaventricular injection of (+)8-OH-DPAT (0.2 nmol) 24 h before a submaximal dose of (-)DOI (0.35 mg/kg) significantly inhibited the 5-HT_2A receptor-mediated increases in both oxytocin and ACTH (-39 and -16%, respectively). In addition, the (+)8-OH-DPAT-induced desensitization of the 5-HT_2A receptor-mediated oxytocin but not the ACTH response was inhibited in rats pretreated with either a systemic (0.1 mg/kg) or intraparaventricular (10 nmol) injection of the 5-HT_1A receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride (WAY100635). This is the first in vivo demonstration of a prolonged heterologous intracellular desensitization of 5-HT_2A receptors after acute activation of 5-HT_1A receptors. These findings may provide insight into the long-term heterologous interactions between 5-HT_1A and 5-HT_2A receptor signaling that could occur in response to antidepressants, antipsychotics, or drugs of abuse that target these receptor subtypes.

5-HT_1A and 5-HT_2A receptors mediate opposing or compensatory functions in a variety of cellular and behavioral events (Araneda and Andrade, 1991; Hensler and Truett, 1998; Valdez et al., 2002; Amargos-Bosch et al., 2004). Indeed, several in vivo studies suggested that a two-way interaction exists between 5-HT_1A and 5-HT_2A receptors (Darmani et al., 1990; Eison and Mullins, 1995; Maswood et al., 1996; Hensler and Truett, 1998; Krebs-Thomson and Geyer, 1998; Valdez et al., 2002). For example, 5-HT_1A receptor-mediated behaviors, such as flattened posture and reciprocal forepaw treading, and hypothermia are attenuated by prior 5-HT_2A receptor activation (Berendsen and Broekkamp, 1990; Hensler and Truett, 1998). On the other hand, chronic or subchronic treatment with full or partial 5-HT_1A receptor agonists can reduce 5-HT_2A receptor-mediated behaviors (Eison and Yocca, 1985; Yocca et al., 1990; Ootsuka and Blessing, 2006) and reduce 5-HT_2A receptor density (Eison and Yocca, 1985; Schechter et al., 1990; Taylor and Hyslop, 1991). However, whether this effect is due to a direct interaction of 5-HT_1A receptors with 5-HT_2A receptors in cells that colocalize both receptors or is due to stimulation of somatodendritic 5-HT_1A autoreceptors producing a general reduction of serotonergic neurotransmission remains unknown.

5-HT_1A receptors and 5-HT_2A receptors exhibit overlapping distributions in various brain regions (Araneda and Andrade, 1991; Burnet et al., 1996; Amargos-Bosch et al., 2004). Colocalization of 5-HT_1A and 5-HT_2A receptors has been demonstrated in frontal and prefrontal cortex (Araneda and Andrade, 1991; Amargos-Bosch et al., 2004). Likewise, we previously reported varying degrees of colocalization of 5-HT_1A and 5-HT_2A receptors in oxytocin and corticotropin-releasing hormone (CRF) neurons in the hypothalamic paraventricular nucleus (PVN) (Zhang et al., 2004). Furthermore, we demonstrated that activation of 5-HT_2A receptors in the PVN produced a delayed and reversible heterologous reduction in the ACTH and oxytocin responses to a 5-HT_1A receptor agonist, (+)-8-hydroxy-2-(di-n-propylamino)-tetralin [(+)8-OH-DPAT] (Zhang et al., 2001, 2004). The maximal desensitization that occurred at 2 to 4 h appeared to recover by 24 h after the (-)DOI injection (Zhang et al., 2001, 2004).

Heterologous desensitization occurs between different receptor systems in which the responsiveness of one receptor system is regulated negatively by activation of another receptor system (Berg et al., 1998). However, homologous or heterologous receptor desensitization typically occurs after sustained exposure to agonists. The present study demonstrates a prolonged heterologous desensitization of 5-HT_2A receptors by acute in vivo activation of 5-HT_1A receptors in neuroendocrine neurons in the PVN. Functional desensitization of 5-HT_2A receptors was assessed from reductions in the magnitude of increases in the plasma levels of oxytocin and ACTH after an injection of the 5-HT_2A/2C receptor agonist (-)DOI (Van de Kar et al., 2001; Zhang et al., 2002). These findings, demonstrating cross-talk between 5-HT_1A and 5-HT_2A receptors, may provide insight into the etiology of mood disorders or the effects of drugs of abuse and could provide novel strategies for their treatment.

	Materials and Methods

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Animals
Male Sprague-Dawley rats (225–275 g) were purchased from Harlan (Indianapolis, IN). The rats were housed two per cage in a temperature-, humidity-, and light-controlled room (12 h light/dark cycle, lights on at 7:00 AM to 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 (NIH publication 85-23, revised 1996) as approved by Loyola University Institutional Animal Care and Use Committee. Every effort was made to minimize suffering and discomfort to the animals. The number of rats per group was the minimum needed for statistically meaningful evaluation of the data.

Drugs
(+)8-OH-DPAT was purchased from Tocris Cookson Inc. (Ellisville, MO). (-)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane HCl [(-)DOI] and N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride (WAY100635) were purchased from Sigma-Aldrich (St. Louis, MO). (+)8-OH-DPAT, WAY100635, and (-)DOI were dissolved in saline (0.9% NaCl). All solutions were made fresh before injections and injected at a volume of 1 ml/kg.

Experimental Protocols
After arrival, the rats were housed two per cage for at least 1 week, followed by 4 days of handling. Rats were randomly assigned to different experimental groups (n = 8/group) to receive drug treatments. Cage-mates were assigned to the same treatment group. After receiving different drug treatments, rats were sacrificed by decapitation. The trunk blood was collected in centrifuge tubes containing a 0.5-ml solution of 0.3 M EDTA (pH 7.4). After centrifugation, the plasma was aliquoted and stored at -30°C for radioimmunoassays of plasma hormone concentrations.

Surgery and Cannula Implantation. Cannula implantation and intraparaventricular injection were performed according to the procedures described previously in detail (Zhang et al., 2004). Rats were anesthetized with a mixture of ketamine and xylazine (100 mg/kg ketamine plus 7 mg/kg xylazine, 1.4 ml/kg i.p.). A double-barreled guide cannula (26 gauge, 1.2 mm center-to-center distance) with its corresponding dummy cannula inserted inside (Plastic One, Roanoke, VA) was implanted into the brain above both sides of the PVN according to the following stereotaxic coordinates: 1.8 mm caudal, ±0.6 mm lateral with respect to bregma, and 6.4 mm ventral from the skull surface. The cannulas were kept in place by dental cement attached to microscrews embedded in the skull. Postsurgery dehydration was prevented by injecting 1 ml of saline (s.c.) after surgery. To prevent postsurgical infection, all rats received ampicillin (50 mg/kg s.c.) immediately after surgery, followed by 3 days of administration of sulfamethoxazole and trimethoprim suspended in the drinking water. Twelve days after cannula implantation, the rats were handled for 4 consecutive days and then randomly assigned to different experimental groups (10 rats/group). After removal of the dummy cannula, an injection cannula (33 gauge, 1.2 mm center-to-center distance, 0.5 mm projection from the tip of the double guide cannula) was inserted through the implanted guide cannula to the top of the PVN.

Radioimmunoassay of Plasma Oxytocin and ACTH
Plasma oxytocin and ACTH were determined by radioimmunoassays as described in detail previously (Li et al., 1997). ¹²⁵I-Oxytocin was purchased from PerkinElmer Life and Analytical Sciences (Wellesley, MA), and ¹²⁵I-ACTH was purchased from DiaSorin (Stillwater, MN).

Experiment 1: Time Course of the Effect of (+)8-OH-DPAT on Hormone Responses to a Subsequent (-)DOI Challenge. Adult male Sprague-Dawley rats were injected with either saline (1 ml/kg s.c.) or (+)8-OH-DPAT (200 µg/kg s.c.) at 1, 2, 4, 24, 48, or 72 h before a subsequent injection of either saline (1 ml/kg s.c.) or (-)DOI (1 mg/kg s.c.). The rats were sacrificed 30 min after the (-)DOI injection.

Experiment 2: Effects of One or Two Systemic Injections of (+)8-OH-DPAT on 5-HT_2A Receptor-Mediated Hormone Responses. Adult male Sprague-Dawley rats (eight per group) were randomly assigned to each of the following groups. 1) Saline 24 h/saline 12 h: rats were injected with saline (1 ml/kg s.c.) 24 and 12 h before a subsequent challenge injection of either saline (1 ml/kg s.c.) or (-)DOI (0.35 mg/kg s.c.). (2) (+)8-OH-DPAT 24 h/saline 12 h: rats were injected with (+)8-OH-DPAT (200 µg/kg s.c.) 24 h and with saline (1 ml/kg s.c.) 12 h before a subsequent challenge injection of either saline (1 ml/kg s.c.) or (-)DOI (0.35 mg/kg s.c.). (3) (+)8-OH-DPAT 24 h/(+)8-OH-DPAT 12 h: rats were injected with (+)8-OH-DPAT (200 µg/kg s.c.) 24 and 12 h before a subsequent challenge injection with either saline (1 ml/kg s.c.) or (-)DOI (0.35 mg/kg s.c.). The rats were sacrificed by decapitation 30 min after the (-)DOI injection.

Experiment 3: Effect of a Direct Injection of (+)8-OH-DPAT into the PVN on 5-HT_2A Receptor-Mediated Neuroendocrine Responses. Cannula implantation and intraparaventricular injection were performed according to the procedures described above. Saline (0.5 µl/side) or (+)8-OH-DPAT (0.2 nmol or 2 nmol, 0.5 µl/side) was injected directly into the PVN. Twenty-four hours after the intraparaventricular injections, the rats received a challenge injection of either (-)DOI (0.35 mg/kg s.c.) or saline (1 ml/kg s.c.). The rats were sacrificed by decapitation 30 min after the injection of (-)DOI.

Experiment 4: Effect of a Systemic Injection of WAY100635 on the (+)8-OH-DPAT-Induced Desensitization of 5-HT_2A Receptor-Mediated Neuroendocrine Responses. Cannula implantation and intraparaventricular injection were performed according to the procedures described above. Rats were injected with either saline or WAY100635 (0.1 mg/kg s.c.), a highly specific 5-HT_1A receptor antagonist (Forster et al., 1995; Chemel et al., 2006), 30 min before an intraparaventricular injection of either saline (0.5 µl/side) or (+)8-OH-DPAT (0.2 nmol, 0.5 µl/side). Twenty-four hours after the intraparaventricular injections, the rats received an injection of (-)DOI (0.35 mg/kg s.c.) or saline. The rats were sacrificed by decapitation 30 min after the challenge injection of (-)DOI.

Experiment 5: Effect of an Intraparaventricular Injection of WAY100635 on the (+)8-OH-DPAT-Induced Desensitization of 5-HT_2A Receptor Signaling. Cannula implantation and intraparaventricular injection were performed according to the procedures described above. Rats received an intraparaventricular injection of either saline (0.5 µl/side) or WAY100635 (10 nmol, 0.5 µl/side), and 30 min later the rats received an injection of either saline (1 ml/kg s.c.) or (+)8-OH-DPAT (200 µg/kg s.c.). Twenty-four hours after the intraparaventricular injections, the rats received a challenge injection of (-)DOI (0.35 mg/kg s.c.) or saline and were sacrificed by decapitation 30 min later.

Statistics
All data are expressed as means ± S.E.M., where n indicates the number of rats per group. Hormone data were analyzed by two- or three-way analyses of variance (ANOVAs). Group means were compared by a 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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Time Course of the Effect of (+)8-OH-DPAT on Hormone Responses to a Subsequent (-)DOI Challenge
Figure 1 illustrates the effects of a single injection of either saline or (+)8-OH-DPAT on the basal and (-)DOI-stimulated increases in plasma levels of oxytocin. We previously demonstrated that the sensitivity of 5-HT_2A receptors in hypothalamus can be measured from the magnitude of increases in the plasma levels of oxytocin and ACTH, after an injection of (-)DOI (Van de Kar et al., 2001; Zhang et al., 2002). Saline or the 5-HT_1A receptor agonist (+)8-OH-DPAT (200 µg/kg s.c.) was injected at different time points (1, 2, 4, 24, 48, and 72 h) before a subsequent saline or (-)DOI challenge (1 mg/kg s.c.). Saline or (+)8-OH-DPAT pretreatment did not significantly alter plasma oxytocin levels to a subsequent saline injection at any of these time points. (-)DOI significantly increased plasma oxytocin levels in rats pretreated with either saline or (+)8-OH-DPAT (Fig. 1), but the magnitude of hormone responses to the subsequent challenge with (-)DOI was attenuated in rats pretreated with (+)8-OH-DPAT.

View larger version (42K): [in this window] [in a new window]   Fig. 1. Time course of (+)8-OH-DPAT (200 µg/kg s.c.)-induced reduction of the oxytocin response to a (-)DOI (1.0 mg/kg s.c.) challenge. The data represent the means ± S.E.M. of six to eight rats per group. , significant difference from the respective saline-challenged group (p < 0.01); ##, significant difference (p < 0.01) from the saline/(-)DOI group (three-way ANOVA and Newman-Keuls multiple-range test).

The three-way ANOVA for oxytocin indicated significant main effects of (+)8-OH-DPAT pretreatment [F_1,149 = 173.38, p < 0.0001]) and the (-)DOI challenge [F_1,149 = 750.86, p < 0.0001] but no significant main effect of time [F_5,149 = 0.56974, p > 0.72]. There was a significant interaction between (+)8-OH-DPAT pretreatment and the (-)DOI challenge [F_1,149 = 152.97, p < 0.0001], but there was no main interaction between (+)8-OH-DPAT pretreatment and time [F_5,149 = 1.230, p > 0.29] and between the (-)DOI challenge and time [F_5,149 = 0.30635, p > 0.90]. There was no significant interaction between (+)8-OH-DPAT pretreatment, time, and the (-)DOI challenge [F_5,149 = 1.16399, p < 0.3296]. The Newman-Keuls test indicated that (+)8-OH-DPAT pretreatment significantly decreased plasma oxytocin responses to the (-)DOI challenge at all the points examined, 1, 2, 4, 24, 48, and 72 h, with a maximal effect between 24 and 72 h (70% inhibition). However, there were no significant (p > 0.01) differences in the (+)8-OH-DPAT-induced desensitization of 5-HT_2A-mediated oxytocin release at any of the time points studied. These data indicate that a single injection of (+)8-OH-DPAT produces a long-lasting inhibition of 5-HT_2A receptor-mediated neuroendocrine responses.

Effects of One or Two Systemic Injections of (+)8-OH-DPAT on 5-HT_2A Receptor-Mediated Hormone Responses
Oxytocin. Figure 2 illustrates basal and (-)DOI-stimulated increases in plasma levels of oxytocin (Fig. 2A) and ACTH (Fig. 2B) in rats pretreated 12 and 24 h previously with saline or (+)8-OH-DPAT. (+)8-OH-DPAT (200 µg/kg s.c.) pretreatment was administered once (24 h) or twice (24 and 12 h) before an injection of a submaximal dose of (-)DOI (0.35 mg/kg). Pretreatment with either one or two injections of (+)8-OH-DPAT did not significantly alter basal oxytocin levels. (-)DOI significantly increased oxytocin plasma levels in rats pretreated with saline and (+)8-OH-DPAT. The 5-HT_2A receptor-mediated oxytocin (Fig. 2A) responses were inhibited (-66%) when (+)8-OH-DPAT was injected 24 h before the subsequent challenge with (-)DOI. A similar degree of inhibition (-72%) was observed in rats pretreated with (+)8-OH-DPAT 24 and 12 h before the subsequent challenge with (-)DOI.

View larger version (16K): [in this window] [in a new window]   Fig. 2. Inhibition of the (-)DOI (0.35 mg/kg s.c.)-induced (A) oxytocin and (B) ACTH neuroendocrine responses by one (12 h) or two (24 and 12 h) preadministrations of the 5-HT1A receptor agonist (+)8-OH-DPAT (200 µg/kg s.c.). The data represent the means ± S.E.M. of six to eight rats per group. , significant difference (p < 0.01) from the respective saline-challenged groups; ##, significant difference (p < 0.01) from the saline 24 h/saline 12 h/(-)DOI group; #, significant difference (p < 0.05) from the saline 24 h/saline 12 h/(-)DOI group; &&, significant difference (p < 0.01) from the DPAT 24 h/saline 12 h/(-)DOI group (two-way ANOVA and Newman-Keuls multiple-range test).

The two-way ANOVA for oxytocin indicated a significant effect for the (-)DOI challenge [F_1,50 = 177.009, p < 0.0001] and for (+)8-OH-DPAT pretreatment [F_2,50 = 47.0975, p < 0.0001]. There was a significant interaction between the (-)DOI challenge and (+)8-OH-DPAT pretreatment [F_2,50 = 46.2009, p < 0.0001]. The Newman-Keuls test indicated that (+)8-OH-DPAT administered once or twice significantly (p < 0.01) decreased the plasma oxytocin responses to (-)DOI. There were no significant differences in the 5-HT_2A receptor-mediated release of oxytocin between rats pretreated with one or two injections of (+)8-OH-DPAT.

ACTH. Pretreatment with either one or two injections of (+)8-OH-DPAT did not significantly alter basal ACTH levels. (-)DOI significantly increased ACTH plasma levels in rats pretreated with either saline or (+)8-OH-DPAT. The 5-HT_2A receptor-mediated ACTH (Fig. 2B) responses were inhibited (-17%) when (+)8-OH-DPAT was injected 24 h before the subsequent challenge with (-)DOI. This inhibition was significantly greater (-44%) in rats pretreated with (+)8-OH-DPAT at both 24 and 12 h before the subsequent injection of (-)DOI.

The two-way ANOVA for ACTH indicated a significant effect of (-)DOI [F_1,49 = 258.979, p < 0.0001] and for (+)8-OH-DPAT pretreatment [F_2,49 = 8.22037, p < 0.0009]. There was a significant interaction between the (-)DOI challenge and (+)8-OH-DPAT pretreatment [F_2,49 = 7.98, p < 0.001]. The Newman-Keuls test indicated that (+)8-OH-DPAT administered once (24 h) significantly (p < 0.05) decreased plasma ACTH responses to (-)DOI. The inhibition of the (-)DOI-induced hormone responses was greater (p < 0.01) in rats pretreated with two injections (24 and 12 h) of (+)8-OH-DPAT than in rats that received a single administration. These data indicate a cumulative effect on the (+)8-OH-DPAT-mediated inhibition of 5-HT_2A receptor-associated ACTH release.

Effect of a Direct Injection of (+)8-OH-DPAT into the PVN on the 5-HT_2A Receptor-Mediated Neuroendocrine Responses
Oxytocin. Figure 3 illustrates the effect of the intraparaventricular administration of the 5-HT_1A receptor agonist (+)8-OH-DPAT (0.2 and 2 nmol) on the 5-HT_2A receptor-mediated neuroendocrine responses. (+)8-OH-DPAT was injected 24 h before the subsequent systemic injection of (-)DOI. (+)8-OH-DPAT (0.2 nmol) significantly inhibited (-39%) the (-)DOI-mediated release of oxytocin (Fig. 3A). A comparable attenuation (-43%) of the (-)DOI-mediated release of oxytocin was produced by a 10-fold higher dose (2 nmol) of (+)8-OH-DPAT (Fig. 3A).

View larger version (15K): [in this window] [in a new window]   Fig. 3. Inhibition of (-)DOI (0.35 mg/kg s.c.)-induced (A) oxytocin, and (B) ACTH neuroendocrine responses by a direct injection of (+)8-OH-DPAT (0.2 or 2 nmol, 0.5 µl/side) into the hypothalamic paraventricular nucleus. The data represent the means ± S.E.M. of 8 to 10 rats/group. , significant difference (p < 0.01) from the respective saline-challenged group; ##, significant difference (p < 0.01) from the saline/(-)DOI group; #, significant difference (p < 0.05) from the saline/(-)DOI group (two-way ANOVA and Newman-Keuls multiple-range test).

ACTH. Intraparaventricular injection of (+)8-OH-DPAT (0.2 nmol) significantly inhibited (-16%) the (-)DOI-mediated release of ACTH (Fig. 3B). A comparable attenuation (-24%) of the (-)DOI-mediated release of ACTH was produced by a 10-fold higher dose (2 nmol) of (+)8-OH-DPAT (Fig. 3B).

The two-way ANOVA for ACTH showed a significant main effect of (+)8-OH-DPAT treatment [F_1,46 = 3.2875, p < 0.05] and the (-)DOI challenge [F_1,46 = 384.063, p < 0.0001]. There was also a significant interaction between (+)8-OH-DPAT treatment and the (-)DOI challenge (F_1,46 = 3.4223, p < 0.05). The Newman-Keuls test indicated that both doses of (+)8-OH-DPAT (0.2 and 2 nmol) significantly (p < 0.05) inhibited the (-)DOI-mediated ACTH release.

Effect of a Systemic Injection of WAY100635 on the (+)8-OH-DPAT-Induced Desensitization of 5-HT_2A Receptor-Mediated Neuroendocrine Responses
Oxytocin. Figure 4 illustrates the effect of a systemic administration of the 5-HT_1A receptor antagonist WAY100635 on the (+)8-OH-DPAT-induced desensitization of the 5-HT_2A receptor-mediated neuroendocrine responses. (+)8-OH-DPAT (0.2 nmol) was injected directly into the PVN 30 min after the WAY100635 pretreatment (0.1 mg/kg s.c.). Twenty-four hours later the rats were challenged with a submaximal dose of (-)DOI (0.35 mg/kg s.c.) and were sacrificed 30 min later. None of the pretreatment paradigms significantly altered basal oxytocin levels. (-)DOI significantly increased plasma oxytocin levels in rats pretreated with either saline or (+)8-OH-DPAT. The oxytocin responses were inhibited (-37%) in rats pretreated with (+)8-OH-DPAT 24 h before the subsequent challenge with (-)DOI (Fig. 4A). WAY100635 did not modify the (-)DOI-induced oxytocin release in saline-pretreated rats, but blocked the (+)8-OH-DPAT-induced inhibition of the 5-HT_2A receptor-mediated oxytocin release (Fig. 4A).

View larger version (17K): [in this window] [in a new window]   Fig. 4. Effect of a systemic injection of WAY100635 (0.1 mg/kg s.c.) on the (+)8-OH-DPAT (0.2 nmol, 0.5 µl/side)-induced desensitization of 5-HT2A receptor-mediated (A) oxytocin and (B) ACTH neuroendocrine responses. The data represent the means ± S.E.M. of 8 to 10 rats/group. , significant difference (p < 0.01) from the respective saline-challenged group; ##, significant difference (p < 0.01) from the saline/saline/(-)DOI group; #, significant difference (p < 0.05) from the saline/saline/(-)DOI group; &&, significant difference (p < 0.01) from the saline/DPAT/(-)DOI group (two-way ANOVA and Newman-Keuls multiple-range test).

The three-way ANOVA for oxytocin showed a significant main effect of (+)8-OH-DPAT treatment [F_1,47 = 18.996, p < 0.0001] and the (-)DOI challenge]F_1,47 = 531.4505, p < 0.0001] but not WAY100635 pretreatment [F_1,47 = 3.1336, p < 0.08]. Significant main interactions were found between WAY100635 pretreatment and (+)8-OH-DPAT treatment [F_1,47 = 3.9483, p < 0.05], between WAY100635 pretreatment and the (-)DOI challenge [F_1,47 = 3.89456, p < 0.05], and between (+)8-OH-DPAT treatment and the (-)DOI challenge [F_1,47 = 17.64105, p < 0.0001]. There was also a main interaction between WAY100635 pretreatment, (+)8-OH-DPAT treatment, and the (-)DOI challenge [F_1,47 = 3.96844, p < 0.05]. The Newman-Keuls test indicated that (+)8-OH-DPAT treatment significantly (p < 0.01) inhibited the (-)DOI-mediated oxytocin release (Fig. 4A) and that in rats pretreated with WAY100635, the magnitude of (+)8-OH-DPAT-induced inhibition of 5-HT_2A receptor stimulation of oxytocin (-19%) was significantly (p < 0.01) different from that of their respective counterparts that did not receive the 5-HT_1A receptor antagonist (Fig. 4A).

ACTH. As shown in Fig. 4B, none of the pretreatment paradigms modified the basal ACTH levels. (-)DOI significantly increased plasma ACTH levels in rats pretreated with saline or (+)8-OH-DPAT. The ACTH responses were inhibited (-19%, p < 0.05) in rats pretreated with (+)8-OH-DPAT 24 h before the subsequent challenge with (-)DOI (Fig. 4B). WAY100635 did not modify the (-)DOI-induced ACTH release in saline-pretreated rats nor did it significantly block the (+)8-OH-DPAT-induced inhibition of the 5-HT_2A receptor-mediated ACTH release (Fig. 4B).

The three-way ANOVA for ACTH showed a significant main effect of (+)8-OH-DPAT treatment [F_1,47 = 4.6042, p < 0.03] and the (-)DOI challenge [F_1,47 = 575.0295, p < 0.0001] but not WAY100635 pretreatment [F_1,47 = 0.0782, p < 0.78]. Significant main interactions were found between (+)8-OH-DPAT treatment and the (-)DOI challenge [F_1,47 = 4.6536, p < 0.03]. The Newman-Keuls test indicated that (+)8-OH-DPAT treatment significantly (p < 0.05) inhibited the (-)DOI-mediated ACTH release (Fig. 4B). However, there were no significant differences in the (-)DOI-mediated ACTH release between rats pretreated with saline and (+)8-OH-DPAT versus WAY100635 and (+)8-OH-DPAT.

Effect of an Intraparaventricular Injection of WAY100635 on the (+)8-OH-DPAT-Induced Desensitization of 5-HT_2A Receptor Signaling
Oxytocin. Figure 5 illustrates the effect of intraparaventricular administration of the 5-HT_1A receptor antagonist WAY100635 on the desensitization of the 5-HT_2A receptor-mediated neuroendocrine responses produced by a systemic injection of (+)8-OH-DPAT. WAY100635 (10 nmol) was injected directly into the PVN 30 min before a single injection of (+)8-OH-DPAT (200 µg/kg s.c.). Twenty-four hours later the rats were challenged with (-)DOI (0.35 mg/kg s.c.) and were sacrificed 30 min after that. As shown in Fig. 5A, none of the pretreatment paradigms significantly altered basal oxytocin levels. (-)DOI significantly increased plasma oxytocin levels in rats pretreated with saline or (+)8-OH-DPAT. However, the oxytocin responses were inhibited (-63%) in rats pretreated with (+)8-OH-DPAT 24 h before the subsequent challenge with (-)DOI (Fig. 5A). WAY100635 did not modify the (-)DOI-induced oxytocin response in saline-pretreated rats. In contrast, WAY100635 injected directly into the PVN partially blocked the (+)8-OH-DPAT-induced inhibition of the 5-HT_2A receptor-mediated oxytocin release.

View larger version (19K): [in this window] [in a new window]   Fig. 5. An intraparaventricular injection of WAY100635 (10 nmol, 0.5 µl/side) partially blocked the (+)8-OH-DPAT (200 µg/kg s.c.) induced-desensitization of 5-HT2A receptor signaling. The data represent the means ± S.E.M. of 8 to 10 rats/group. , significant difference (p < 0.01) from the respective saline-challenged group; ##, significant difference (p < 0.01) from the saline/saline/(-)DOI group; #, significant difference (p < 0.05) from the saline/saline/(-)DOI group; &, significant difference (p < 0.05) from the saline/DPAT/(-)DOI group (two-way ANOVA and Newman-Keuls multiple-range test).

ACTH. As shown in Fig. 5B, none of the pretreatment paradigms significantly altered basal ACTH levels. (-)DOI significantly increased ACTH plasma levels in rats pretreated with saline or (+)8-OH-DPAT. The ACTH responses were inhibited (-25%) in rats pretreated with (+)8-OH-DPAT 24 h before the subsequent challenge with (-)DOI. Direct intraparaventricular injections of WAY100635 did not modify the (-)DOI-induced ACTH release in saline-pretreated rats nor did it significantly alter the (+)8-OH-DPAT-induced inhibition of the 5-HT_2A receptor-mediated ACTH release.

The three-way ANOVA for ACTH showed a significant main effect of (+)8-OH-DPAT treatment [F_1,60 = 3.9919, p < 0.05] and the (-)DOI challenge [F_1,60 = 354.0162, p < 0.0001] but not WAY100635 pretreatment [F_1,60 = 0.00416, p < 0.948]. Significant main interactions were found between (+)8-OH-DPAT treatment and the (-)DOI challenge [F_1,60 = 4.7653, p < 0.03]. The Newman-Keuls test indicated that (+)8-OH-DPAT treatment significantly (p < 0.05) inhibited (-25%) the (-)DOI-mediated oxytocin release, and this effect was reduced (-14%) by WAY100635, but the difference was not statistically significant (p > 0.05) (Fig. 5B).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
This study provides the first in vivo demonstration that acute activation of 5-HT_1A receptors produces a prolonged functional heterologous desensitization of 5-HT_2A receptors in neuroendocrine neurons of the PVN. Data from a variety of pharmacological approaches that support the 5-HT_1A receptor-mediated heterologous desensitization of 5-HT_2A receptor function include: 1) the attenuated 5-HT_2A receptor-mediated release of oxytocin from 1 to 72 h after a single injection of (+)8-OH-DPAT (Fig. 1), 2) the reduced 5-HT_2A receptor-mediated increases of both oxytocin and ACTH at 24 h after a single injection of (+)8-OH-DPAT (Fig. 2), 3) the greater reduction in the 5-HT_2A receptor-mediated ACTH responses produced by two injections of (+)8-OH-DPAT (Fig. 2B), 4) the blunted (-)DOI-mediated increases in both oxytocin and ACTH by a prior direct intraparaventricular injection of (+)8-OH-DPAT (Fig. 3), 5) the blockade of intraparaventricular (+)8-OH-DPAT-induced desensitization of the 5-HT_2A receptor-mediated oxytocin responses by pretreatment with WAY100635 (Fig. 4), and 6) the blockade of the (+)8-OH-DPAT-induced desensitization of the 5-HT_2A receptor-mediated oxytocin responses by direct intraparaventricular administration of WAY100635 (Fig. 5).

We previously demonstrated that 5-HT_1A and 5-HT_2A receptors are coexpressed in oxytocin and CRF-containing neurons in the PVN (Zhang et al., 2004). Immunohistochemical double labeling of 5-HT_1A or 5-HT_2A receptors and oxytocin revealed that a high percentage of oxytocin neurons in the PVN were immunopositive for 5-HT_1A and 5-HT_2A receptors (94 and 97%, respectively) (Zhang et al., 2004). CRF-immunoreactive neurons in the PVN also expressed 5-HT_1A and 5-HT_2A receptor immunoreactivity (Zhang et al., 2004), although we were unable to quantify the percentages of CRF neurons that coexpress 5-HT_1A and 5-HT_2A receptors. Combined, these data suggest that 5-HT_2A receptors might directly interact with 5-HT_1A receptors through their signaling proteins to regulate hormone release. Consequently, the in vivo heterologous desensitization of 5-HT_2A receptors after 5-HT_1A receptor activation presumably is mediated via intracellular rather than transsynaptic mechanisms, although additional studies are needed to confirm this hypothesis.

We have previously demonstrated that activation of 5-HT_2A receptors in the PVN induced a heterologous desensitization of 5-HT_1A receptors within individual neuroendocrine cells (Zhang et al., 200, 2004). Activation of 5-HT_2A receptors produced a delayed and reversible reduction of the ACTH and oxytocin responses to a 5-HT_1A receptor agonist (Zhang et al., 2001). The maximal desensitization occurred at 2 to 4 h, with recovery back to control responses by 24 h after the (-)DOI injection. The desensitization was dose-dependent, and it shifted the oxytocin and ACTH dose-response curves of (+)8-OH-DPAT to the right (increased ED₅₀) with no change in their maximal responses (E_max) (Zhang et al., 2001).

In contrast, the present studies indicated that activation of 5-HT_1A receptors in the PVN produced a faster and more persistent inhibition of 5-HT_2A receptor-mediated neuroendocrine responses. The heterologous desensitization of 5-HT_2A receptors was evident at least 1 h after a single (+)8-OH-DPAT injection and persisted for at least 72 h, the last time point measured in this study. It is unlikely that the reduction of (-)DOI-mediated hormone responses after a (+)8-OH-DPAT injection is due to the hormone-depleting effect of 5-HT_1A receptor activation as the desensitization of the 5-HT_2A receptor-mediated neuroendocrine responses was found even 3 days after a single (+)8-OH-DPAT injection (Fig. 1). If the pretreatment with (+)8-OH-DPAT depleted the oxytocin, CRF, or ACTH stores, these would have been recovered during the first few hours after the single (+)8-OH-DPAT injection. Thus, the long-lasting (+)8-OH-DPAT-induced attenuation of hormone responses to the (-)DOI challenge is likely to be due to a persistent desensitization of hypothalamic 5-HT_2A receptor signal transduction.

In the present studies, respective changes in 5-HT_2A receptor-mediated oxytocin and ACTH neuroendocrine responses were determined as these are mediated by different neurons and use different postreceptor regulatory mechanisms and signaling cascades. The oxytocin cells located in the PVN send their axons directly to the posterior lobe of the pituitary and release oxytocin into the bloodstream (Carrasco and Van de Kar, 2003). Therefore, oxytocin is the most direct indicator of events occurring in the hypothalamus. On the other hand, the ACTH response represents a functional index that is amplified subsequent to hypothalamic activation of 5-HT receptors. ACTH is released by corticotrophs in the anterior lobe of the pituitary (Carrasco and Van de Kar, 2003). This difference in amplification may explain our observation that (+)8-OH-DPAT produced a greater attenuation of the oxytocin response to (-)DOI (Figs. 2 and 3), whereas the inhibition of the ACTH response to (-)DOI was less marked (Figs. 2 and 3). In addition, differences in the degree of 5-HT_1A and 5-HT_2A colocalization on oxytocin- versus CRF-containing cells may be responsible for differences in the magnitude of reduction in oxytocin and ACTH after single versus repeated doses of (+)8-OH-DPAT. As there is a lower degree of colocalization of 5-HT_1A and 5-HT_2A receptors in CRF-containing neurons of the PVN compared with oxytocin-containing neurons (Zhang et al., 2004), repeated doses of (+)8-OH-DPAT may be needed to produce the greater reduction in the 5-HT_2A receptor-mediated ACTH responses. Alternative explanations include the following: 1) systemic (-)DOI injection may also activate 5-HT_2A receptors in other nuclei that are involved in ACTH release, such as the amygdala (Beaulieu et al., 1986; Feldman et al., 1998), which could mask the effects occurring in hypothalamic CRF neurons; and 2) 5-HT_1A receptor-associated signaling pathways in oxytocin-containing cells in the PVN could be different from those found in CRF-containing cells. This hypothesis is supported by studies demonstrating that 5-HT_1A receptors are coupled to different G-proteins within different brain areas (Lin et al., 2002; La Cour et al., 2006).

As (+)8-OH-DPAT injected directly into the PVN produces 5-HT_2A receptor desensitization (Figs. 3 and 4), the activation of hypothalamic 5-HT_1A receptors is required to produce long-term desensitization of 5-HT_2A receptor-mediated neuroendocrine responses. WAY 100635, which exhibits a high affinity for 5-HT_1A receptors (K_i = 2.2 nM) that is 10 to 100 times higher than its affinity for other 5-HT receptors or dopamine D₁, D₂, and D₄ receptors (Forster et al., 1995; Chemel et al., 2006), was used to demonstrate the specificity of the effect of 5-HT_1A receptors. Accordingly, the heterologous desensitization of the (-)DOI-mediated oxytocin responses was prevented when WAY100635 was injected systemically (Fig. 4) or directly into the PVN (Fig. 5). These observations suggest that 5-HT_1A receptors interact directly with 5-HT_2A receptors in the oxytocin containing-neurons of the hypothalamic PVN. Our previous immunocytochemical observations and the high degree of colocalization of 5-HT_1A and 5-HT_2A receptors in oxytocin-containing cells (Zhang et al., 2004) suggest that a direct intracellular interaction could occur between these receptor signaling systems.

On the other hand, pretreatment with (+)8-OH-DPAT has a less marked effect on 5-HT_2A receptor-mediated ACTH responses. Furthermore, systemic (Fig. 4) or direct intraparaventricular (Fig. 5) administration of WAY100635 did not block the 5-HT_1A receptor-induced reduction of the (-)DOI-mediated ACTH responses. The ACTH responses were inhibited (19%) (Fig. 4B) in rats pretreated with a direct intraparaventricular injection of (+)8-OH-DPAT 24 h before the subsequent challenge with (-)DOI. However, there were no significant differences in the 5-HT_1A receptor-induced inhibition of the 5-HT_2A receptor-mediated ACTH responses between rats pretreated with a systemic injection of either saline or WAY100635 (Fig. 4B). Likewise, (-)DOI-induced ACTH responses were inhibited (-25%) in rats pretreated with (+)8-OH-DPAT (Fig. 5B). However, there were no differences in the 5-HT_1A receptor-induced inhibition of the 5-HT_2A receptor-mediated ACTH responses between rats that received a direct intraparaventricular injection of either saline or WAY100635. Given the small magnitude of 5-HT_2A receptor desensitization by 5-HT_1A receptor activation and the partial blockade of this effect by WAY100365, the evidence of a direct cross-talk between 5-HT_1A and 5-HT_2A receptors in CRF-containing cells in the PVN is inconclusive.

The mechanisms underlying the heterologous desensitization of 5-HT_1A receptors by 5-HT_2A receptors in PVN await further investigation. Our previous studies in vivo demonstrated that activation of 5-HT_1A receptors in PVN activates mitogen-activated protein kinase signaling, specifically the phosphorylation of p42/44 extracellular signal-regulated kinase (Sullivan et al., 2005). This effect was completely abolished by WAY100635 preadministration (Sullivan et al., 2005). The relatively rapid onset of the 5-HT_1A receptor-mediated desensitization of hypothalamic 5-HT_2A receptors (by 1 h after activation of 5-HT_1A receptors) suggests that post-translational modification mechanisms may be involved. However, the long-lasting effects on 5-HT_2A receptor signaling after activation of 5-HT_1A receptors could involve activation of several transcription factors (Mazzucchelli and Brambilla, 2000; Sweatt, 2001; Milanini-Mongiat et al., 2002). We speculate that 5-HT_1A receptor-induced activation of phosphorylated extracellular signal-regulated kinase could participate in the signal transduction pathways involved in the intracellular regulation of 5-HT_2A receptors.

Desensitization of 5-HT_2A receptors has been proposed as the mechanism underlying the therapeutic efficacy of antipsychotic, anxiolytic, and antidepressant agents (Meltzer et al., 2003; Roth and Xia, 2004). Consequently, drugs with 5-HT_2A receptor antagonist properties have been undergoing clinical evaluation for the treatment of several disorders such as schizophrenia, depression, anxiety, migraine, sleep disorders, and feeding disorders (Meltzer et al., 2003; Amargos-Bosch et al., 2004; Roth and Xia, 2004). If indeed, 5-HT_1A receptor activation can negatively regulate 5-HT_2A receptor function, then novel antidepressants and antipsychotics that stimulate 5-HT_1A receptors may have a greater pharmacological impact in some neurons or brain regions.

In summary, the present studies provide in vivo pharmacological evidence that 5-HT_1A receptors may directly cross-talk with 5-HT_2A receptors to regulate neuroendocrine function. Because a functional imbalance between hypothalamic 5-HT_1A and 5-HT_2A receptors could modify the organism's response to stressors (Berendsen, 1995; Zhang et al., 2004), antidepressants (Carrasco and Van de Kar, 2003), and drugs of abuse (Levy et al., 1994), elucidation of the specific mechanisms mediating interactions between hypothalamic 5-HT_1A and 5-HT_2A receptors may lead to novel targets for the development of psychotherapeutic drugs.

	Footnotes

 
This work was supported in part by United States Public Health Service Grants NS034153, DA013669, and DA07741.

Dr. Louis D. Van de Kar, a good friend, colleague, and internationally recognized scientist, passed away on September 4, 2004. His contribution and memory will endure through all of those whose lives he enriched.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.116004.

ABBREVIATIONS: 5-HT, 5-hydroxytryptamine, serotonin; CRF, corticotropin-releasing factor; PVN, hypothalamic paraventricular nucleus; ACTH, adrenocorticotropic hormone; (+)8-OH-DPAT, (+)-8-hydroxy-2-(di-n-propylamino)-tetralin; (-)-DOI, (-)-1-(2,5 dimethoxy-4-iodophenyl)-2-aminopropane HCl; WAY100635, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride; ANOVA, analysis of variance.

Address correspondence to: Dr. George Battaglia, Department of Pharmacology, Loyola University of Chicago, Stritch School of Medicine, 2160 S. First Ave., Maywood, IL 60153. E-mail: gbattag{at}lumc.edu

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