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NEUROPHARMACOLOGY

Chronic Fluoxetine Differentially Affects 5-Hydroxytryptamine_2A Receptor Signaling in Frontal Cortex, Oxytocin- and Corticotropin-Releasing Factor-Containing Neurons in Rat Paraventricular Nucleus

Center for Serotonin Disorders Research and Department of Pharmacology and Experimental Therapeutics, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois

Received February 17, 2003; accepted April 16, 2003.

	Abstract

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Differential adaptive changes in serotonin_2A [5-hydroxytryptamine (5-HT)_2A] receptor signaling during treatment may be one mechanism involved in the latency of therapeutic improvement with antidepressants, such as fluoxetine. We examined the effects of fluoxetine (2, 3, 7, 21, or 42 days) on hypothalamic 5-HT_2A receptor signaling. The hormone responses to an injection of the 5-HT_2A receptor agonist (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane HCl (DOI) were used as an index of hypothalamic 5-HT_2A receptor function. Treatment with fluoxetine for 21 or 42 days produced diminished adrenocorticotropic hormone (ACTH) and oxytocin (but not corticosterone) responses to DOI injections (2.5 mg/kg i.p.; 15 min postinjection). Regulators of G protein signaling 4 and G_q protein levels in the hypothalamic paraventricular nucleus were not altered during fluoxetine treatment. Because previous studies indicate that treatment with fluoxetine for 21 days resulted in increased hormone responses to DOI when measured at 30 min after injection, we examined the effect of fluoxetine (21 days) on DOI-induced increase hormone levels at 15, 30, and 60 min after DOI injection. Fluoxetine decreased the oxytocin response at 15 but not at 30 min post-DOI injection, and potentiated the ACTH and corticosterone responses at 30 min post-DOI injection. For comparison, we examined the effect of fluoxetine on 5-HT_2A receptor-mediated increase in phospholipase C (PLC) activity in the frontal cortex. 5-HT-stimulated, but not guanosine 5'-O-(3-thio)triphosphate-stimulated PLC activity was increased after 21 days of fluoxetine-treatment. Overall, these results indicate that chronic fluoxetine treatment can potentiate 5-HT_2A receptor signaling in frontal cortex but differentially alters 5-HT_2A receptor signaling in oxytocin-containing neurons and corticotropin-releasing factor-containing neurons in the paraventricular nucleus.

Previous investigations have shown that treatment with fluoxetine for 21 days results in a supersensitivity of 5-HT_2A receptor signaling in the cortex and hypothalamus of rats and in cortex of guinea pigs (Cadogan et al., 1993; Li et al., 1993; Tilakaratne et al., 1995). In the frontal cortex, 5-HT_2A receptors are coupled to G_q/11 proteins (Roth et al., 1998), which in turn activate phospholipase C (PLC). PLC catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate into diacylglycerol and inositol 1,4,5-trisphosphate. We measured PLC activity in the frontal cortex by monitoring its stimulation by 5-HT. Activation of PLC by 5-HT represents the overall activation of the signaling system, from 5-HT_2A receptor, through G_q/11 protein to PLC. On the other hand, GTPS activates PLC via G proteins without involvement of the 5-HT_2A receptor. In our current study, 5-HT-mediated PLC activity was used to examine 5-HT_2A receptor responsivity in the frontal cortex.

Hormone responses to an injection of the 5-HT_2A receptor agonist DOI were used to examine 5-HT_2A receptor responsivity in the hypothalamus. Plasma hormone concentrations are known to be regulated by neurons in the hypothalamus (Van de Kar, 1991). ACTH is secreted from the anterior pituitary gland, under hypothalamic control by corticotropin-releasing factor (CRF) secreted from parvocellular cells in paraventricular nucleus. ACTH then activates the secretion of corticosterone from the adrenal gland. Oxytocin is secreted from nerve terminals located in the posterior lobe of the pituitary gland. The cell bodies that synthesize oxytocin are located in the hypothalamic supraoptic nucleus and magnocellular cells in the paraventricular nucleus.

DOI is a selective 5-HT₂ receptor agonist (5-HT_2A receptor, pK_D = 8.89; 5-HT_2C receptor, pK_D = 8.19) with a low affinity for the other 5-HT receptor subtypes (5-HT_1A receptor, pK_D = 5.16; 5-HT_1B receptor, pK_D = 5.69) (Van Wijngaarden et al., 1990; Zifa and Fillion, 1992). Administration of DOI produces a reliable increase in the secretion of ACTH, corticosterone, and oxytocin through activation of 5-HT₂ receptors. Several 5-HT₂ receptor antagonists inhibit the effects of DOI on plasma hormone levels (Van de Kar, 1991). In the case of corticosterone, DOI can induce increases in its levels by both central and peripheral mechanisms (Alper, 1990; Rittenhouse et al., 1991). Recent evidence indicates that the effect of DOI on plasma levels of ACTH, corticosterone, oxytocin, prolactin, and renin is mediated by 5-HT_2A receptors in the paraventricular nucleus of the hypothalamus, because intraparaventricular injections of the 5-HT_2A receptor antagonist MDL 100,907 dose dependently blocked the DOI-induced increase in hormone levels (Zhang et al., 2002). Thus, the measurement of plasma ACTH and oxytocin after a peripheral injection of DOI is a useful marker of the functioning of 5-HT_2A receptors in the paraventricular hypothalamic nucleus.

Regulators of G protein signaling (RGS) 4 proteins are involved in modulating the 5-HT_2A receptor signaling cascade (Berman et al., 1996). RGS proteins potentiate the native ability of the G subunit of G proteins to hydrolyze the GTP bound to it and thereby cause a reduction in the activation of the second messenger system. A decrease in RGS4 protein levels could induce a supersensitization response, whereas an increase in RGS4 protein levels could induce desensitization of 5-HT_2A receptor signaling. We determined whether the levels of RGS4 protein in the paraventricular nucleus of the hypothalamus are altered at any time throughout chronic fluoxetine treatment. The levels of G_q protein also were measured in the paraventricular nucleus of the hypothalamus to determine a possible molecular mechanism underlying adaptive changes in 5-HT_2A receptor signaling induced by chronic fluoxetine treatment. An increased amount of G_q protein would be expected to be associated with a potentiation of the hormone response.

	Materials and Methods

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Animals
Male Sprague-Dawley rats (225–250 g) were purchased from Harlan (Indianapolis, IN). The animals were housed two per cage in an environment controlled for lighting (7:00 AM–7:00 PM), temperature, and humidity. Food and water were available ad libitum. Eight to 12 rats were used per experimental group (12 being used in the saline-challenged groups). 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 was donated by Eli Lilly & Co. (Indianapolis, IN). (±)-DOI [(±)-1-(2,5-dimethoxy-4-lodophenyl)-2-aminopropane HCl] was purchased from Sigma/RBI (Natick, MA). Both drugs were dissolved in 0.9% saline. DOI was injected at doses of 0.5, 2.5, 5.0 mg/kg i.p. or at a dose of 2.5 mg/kg s.c. Fluoxetine was injected at a dose of 10 mg/kg/day i.p.

Experimental Procedures
All rats were handled for at least 7 days before the challenge injection and acclimated to a quiet environment to minimize stress. Rats were randomly assigned to the various experimental groups and cage mates received the same drug treatment. Weights were recorded every other day throughout the entire experiment with the final weight being taken the evening before the challenge injections.

Time Course of Fluoxetine Treatment. Fluoxetine (10 mg/kg/day i.p.) was injected daily for 2, 3, 7, 21, or 42 days. The initiation of these injections was staggered so that the animals were all given the challenge injection of DOI on the same day. Saline treatment (2 ml/kg i.p.) consisted of one group injected daily for 42 days. Challenge administration of DOI (0.5, 2.5, or 5.0 mg/kg i.p.) or saline (2 ml/kg i.p.) occurred 1 day after the last fluoxetine injection and 15 min before decapitation. The trunk blood was collected in centrifuge tubes containing 0.5 ml of a 0.3 M EDTA (pH 7.4) solution. The plasma samples were stored at –80°C until used for radioimmunoassays. Brains were removed, frozen on dry ice, and stored at –80°C until used for Western blot analyses.

Effect of Fluoxetine Treatment on the Time Course of Hormone Responses to DOI. Fluoxetine (10 mg/kg/day) or saline (2 ml/kg) was injected daily for 21 days. One day after the last fluoxetine injection, DOI (2.5 mg/kg or saline 2 ml/kg) was administered either by intraperitoneal injection 15, 30, or 60 min before decapitation or by subcutaneous injection 15 or 30 min before decapitation. Control rats received saline injection. The trunk blood was collected and plasma was stored as mentioned previously.

Radioimmunoassay of Hormones. Plasma ACTH, corticosterone (Li et al., 1993), and oxytocin (Li et al., 1997a) concentrations were determined by radioimmunoassays as described previously. Intra-assay variability is 4.2% for ACTH, 4.5% for corticosterone, and 5% for oxytocin. Interassay variability is 14.6% for ACTH, 11.9% for corticosterone, and 9% for oxytocin.

Immunoblot Analysis of G_q and RGS4 Proteins
Tissue Preparation. The hypothalamic paraventricular nuclei from the 0.5 mg/kg DOI challenge group across all the various fluoxetine treatments were used for RGS4 and G_q protein measurements. The paraventricular nuclei used in these analyses were dissected from a 700-µm coronal section obtained using a cryostat (–10°C). Punches of paraventricular nuclei were sonicated in 100 µl of 10 mM Tris buffer containing 0.1 M NaCl, 0.1 M EDTA, and protease inhibitor cocktail (1:1000) from Sigma-Aldrich (St. Louis, MO). The frontal cortex from the saline-challenged group receiving 21-day fluoxetine and 21-day saline treatment was used for RGS4 and G_q protein measurements. The tissue was prepared as described above, except that the frontal cortex was homogenized in 600 µl of 10 mM Tris buffer. Protein concentrations in these whole homogenates were measured using the bicinchoninic acid protein assay kit (Pierce Chemical, Rockford, IL).

Quantification of RGS4 Proteins. Solubilized proteins (5 µg/lane for RGS4) were resolved by SDS-polyacrylamide gel electrophoresis containing 0.1% SDS, 12.5% acrylamide/bisacrylamide (30: 0.2), 4.6 M urea, and 375 mM Tris, pH 8.7 (Blank and Exton, 1994). Gels for paraventricular tissue were designed to contain two samples from each fluoxetine treatment time group and two aliquots from a pooled frontal cortex homogenate. Gels for tissue from the frontal cortex contained six samples of 21-day fluoxetine treatment and six samples of 21-day saline treatment. Actin was examined to verify equal loading of protein in each lane. The proteins were electrophoretically transferred for 2 h to nitrocellulose membranes. Membranes were incubated with a blocking buffer for 1 h at room temperature (5% nonfat dry milk and 2% Triton X detergent in Trisbuffered saline buffer). The membrane was incubated overnight at 4°C with primary polyclonal RGS4 antibody, (N-16, 1:2000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Next, the membrane was washed in Tris-buffered saline and incubated for 1 h at room temperature with an anti-goat antibody (IgG1, 1:2000; Sigma-Aldrich, St. Louis, MO). The membrane was washed again and was incubated for 1 h in goat peroxidase-antiperoxidase (1:10,000 dilution; ICN Pharmaceuticals Biochemicals Division, Aurora, OH). The membranes were incubated with enhanced chemiluminescence chemiluminescence substrate solution (Amersham Biosciences Inc., Piscataway, NJ) before exposure to X-ray film (Eastman Kodak, Rochester, NY).

Quantification of G_q Proteins. Solubilized proteins (7.5 µg/lane) were resolved by SDS-polyacrylamide gradient gel (4–15% Tris-HCl) (Criterion precast gel; Bio-Rad, Hercules, CA). Similar Western blot procedures were used as described above for RGS4 detection. The G_q protein was detected using a rabbit polyclonal primary G_q antibody (1:1000 dilution; Santa Cruz Biotechnology, Inc.), followed by a goat anti-rabbit secondary antibody (1:10,000 dilution; ICN Pharmaceuticals Biochemicals Division) and rabbit peroxidase-antiperoxidase (1:10,000 dilution; ICN Pharmaceuticals Biochemicals Division).

Western Blot Data Analysis. Films were analyzed densitometrically using the Image program (Scion, Frederick, Maryland). Gray scale density readings were calibrated using a transmission step wedge standard. The integrated optical densities (IODs) of each band were calculated as the sum of the densities of all of the pixels within the area of the band outlined. An area adjacent to the band was used to calculate the background density of the band. The background IOD was subtracted from the IOD of each band. For normalization, the IOD of protein bands on each blot were divided by the mean IOD of pooled cortex homogenates loaded on each gel in duplicate. Each experimental sample was measured in triplicate.

PLC Activity
Tissue from the treatment groups that received the saline-challenges for 15 min was used for the PLC activity measurements. PLC activity was determined as described previously (Wolf and Schutz, 1997). Briefly, 3 µg/µl membrane homogenates of frontal cortex were diluted into 100 µl of total volume. The reaction was carried out in a buffer containing 25 mM HEPES-Tris, 3 mM EGTA, 10 mM LiCl, 12 mM MgCl₂, 1.44 mM sodium deoxycholate with 1 µM GTPS, 300 nM free Ca²⁺, 3 µM 5-HT, and 1.1 mM unlabeled phosphatidyl inositol. The assay tubes were kept on ice until the incubation was started by addition of 100 µM [³H]phosphatidyl inositol. The tubes were subsequently incubated for 20 min at 37°C. The reaction was stopped by adding 0.9 ml of CHCl₂/MeOH (1:2) and 0.3 ml of chloroform. The assay tubes were shaken vigorously for 90 s and centrifuged at 9000 rpm at room temperature for 90 s. Then 0.3 ml of the upper aqueous phase was mixed with 6 ml of scintillation cocktail and counted on a scintillation counter for 7 min.

Statistical Analyses
All data are represented as group mean ± S.E.M. Body weights were analyzed using a one-way analyses of variance (ANOVA) and two-way repeated measures ANOVA followed by a Newman-Keuls post hoc analysis. Hormone data from the fluoxetine time-course experiment were analyzed by two-way ANOVA, whereas the hormone data from the DOI challenge time-course experiments were analyzed by one-way ANOVA. Newman-Keuls post hoc analysis was used as a follow-up test for all hormone data. G_q and RGS4 protein levels were analyzed using a one-way ANOVA for the fluoxetine time-course and a Student's t test for the 21-day fluoxetine treatment. PLC activity was analyzed by a Student's t test. GB-STAT software (Dynamic Microsystems, Inc., Silver Spring, MD) was used for all statistical analyses.

	Results

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Body Weight
All rats showed a steady increase in body weight during the drug treatment period. Chronic fluoxetine (10 mg/kg i.p.) significantly inhibited this growth rate during the 42 days of the experiment compared with their saline (2 ml/kg i.p.)-injected controls as shown in Fig. 1A [F_(1,1422) = 710.66, p < 0.0001]. In rats treated for 42 days, the difference in mean daily weight between the fluoxetine-treated group and the saline-treated group was significant within 8 days (Fig. 1A) [F_(19,1422) = 5.77, p < 0.0001]. The magnitude of this difference in body weight increased as the duration of fluoxetine injections increased.

View larger version (20K): [in this window] [in a new window]   Fig. 1. Effect of daily injections of fluoxetine on body weight. A, average body weight across a 42-day fluoxetine (10 mg/kg/day i.p.) (open circles) and saline (2 ml/kg) (closed circles) treatment regime, **, p < 0.01. B, average body weight of rats in the saline treatment group and fluoxetine treatment groups (2, 3, 7, 21, and 42 days) on the last day of treatment. The data represent the mean ± S.E.M. (n = 36). Two-way repeated measures ANOVA was used for data in A and one-way ANOVA for data in B., Newman-Keuls multiple range test for both (**, p < 0.01).

The mean body weights on the final day of the fluoxetine or saline injections for each treatment group (i.e., rats treated with fluoxetine for 2, 3, 7, 21, and 42 days) are depicted in Fig. 1B. The difference in the final body weight was dependent on the duration of fluoxetine injection [F_(5,210) = 18.20, p < 0.001]. The body weights were significantly different in the rats treated with fluoxetine for 7 days or longer (7, 21, and 42 days) compared with saline controls.

Plasma Hormone Levels
Time Course of Fluoxetine. Injection of DOI (2.5 and 5.0 mg/kg) significantly increased the plasma levels of ACTH [F_(3,170) = 94.46, p < 0.0001] and oxytocin [F_(3,170) = 121.09, p < 0.0001] in the saline-treated group (Fig. 2, A–C, white columns). Fluoxetine treatment for 21 or 42 days inhibited the effect of DOI on ACTH [F_(15,170) = 2.75, p < 0.001] and oxytocin [F_(15,170) = 4.08, p < 0.0001] levels (Fig. 2, A and C). This desensitization was evident at the 2.5-mg/kg dose of DOI for ACTH and at the 2.5- and 5.0-mg/kg doses for oxytocin. Fluoxetine treatment for 3 and 7 days also reduced the effect of the 5.0 mg/kg dose of DOI on plasma oxytocin levels. No change was seen in the levels of corticosterone during fluoxetine treatment (Fig. 2B) [F_(15,169) = 1.58, p = 0.083].

View larger version (28K): [in this window] [in a new window]   Fig. 2. Effect of chronic fluoxetine (10 mg/kg/day i.p. at 2, 3, 7, 21, or 42 days of treatment) or saline (2 ml/kg, 42 days) on DOI-induced increases in plasma ACTH (A), corticosterone (B), and oxytocin (C) levels. DOI was injected at doses of 0.5, 2.5, and 5.0 mg/kg i.p. with a 15-min challenge interval. The 0-mg/kg dose was the saline challenge. The data represent the mean ± S.E.M. (n = 6–12). A significant effect of fluoxetine compared with saline pretreatment is indicated by *, p < 0.05; **, p < 0.01. A significant effect of DOI is indicated by , p < 0.01 (2-way ANOVA and Newman-Keuls multiple range test).

Effect of Fluoxetine (21 Days) on Hormone Time-Course Responses to DOI (i.p.). Rats received daily injections of saline or fluoxetine (10 mg/kg/day i.p.) for 21 days. Administration of DOI (2.5 mg/kg i.p.) to saline-pretreated rats produced a significant increase in plasma ACTH [F_(9,64) = 18.49, p < 0.0001], corticosterone [F_(9,65) = 42.36, p < 0.0001] and oxytocin [F_(9,64) = 19.93, p < 0.0001] levels 15, 30, and 60 min postinjection. The DOI-induced increase in plasma ACTH and oxytocin levels was maximal at 15 min postinjection. The oxytocin response to DOI was still maximal at 30 min postinjection and subsided by 60 min (Fig. 3C). The corticosterone response was more delayed and reached a maximum at 60 min postinjection (Fig. 3B).

View larger version (21K): [in this window] [in a new window]   Fig. 3. Time course of hormone responses to intraperitoneal DOI (2.5 mg/kg i.p.) (15-, 30-, and 60-min challenge intervals) after 21 days of saline (2 ml/kg) or fluoxetine (10 mg/kg/day i.p.) treatment. The data represent the mean ± S.E.M. of ACTH (A), corticosterone (B), and oxytocin (C) levels (n = 6–8). A significant effect of fluoxetine compared with saline treatment is indicated by *, p < 0.05 and **, p < 0.01. A significant effect of DOI is indicated by , p < 0.05 and , p < 0.01 (one-way ANOVA and Newman-Keuls' multiple range test).

Treatment with fluoxetine resulted in a reduced ACTH response at 15 min after injection of DOI, but a potentiation at 30 min post-DOI injection (Fig. 3A). At 60 min after DOI challenge, there were no significant differences between fluoxetine- and saline-treated rats. Fluoxetine potentiated the effect of DOI on plasma corticosterone at 30 min after DOI injection, but did not potentiate the effect of DOI at 15 or 60 min after injection (Fig. 3B). Treatment with fluoxetine for 21 days resulted in a reduced oxytocin response at 15 min after DOI injection, but the oxytocin response to DOI at 30 min postinjection was not different from the response in saline-treated rats (Fig. 3C).

Effect of Fluoxetine (21 Days) on Hormone Time-Course Responses to DOI (s.c.). Hormone responses to subcutaneous DOI injections in saline- and fluoxetine-treated rats were examined to determine whether the route of administration of DOI impacts on the hormone responses. Administration of DOI (2.5 mg/kg s.c.) produced a significant increase in plasma ACTH [F_(5,39) = 21.38, p < 0.0001], corticosterone [F_(5,39) = 27.27, p < 0.0001], and oxytocin [F_(5,39) = 12.38, p < 0.0001] levels both at 15 and 30 min postinjection (Fig. 4). These results in saline-pretreated rats, with s.c. injections of DOI, are similar to those seen with the i.p. administration of DOI. Fluoxetine treatment produced no desensitization of the ACTH response to DOI at 15 min postinjection, but a potentiated ACTH response at 30 min after subcutaneous injections of DOI (p < 0.05 by Newman-Keuls test; Fig. 4A). Note that the potentiation of the ACTH response to DOI was observed at 30 min post-DOI injection with both routes of DOI administration (i.p. and s.c.) (Figs. 3A and 4A). Consistent with the previous experiment, fluoxetine treatment potentiated the corticosterone response to DOI when measured at 30 min post-DOI injection (p < 0.05 by Newman-Keuls test; Fig. 4B). Fluoxetine treatment reduced the oxytocin response 15 min after a DOI injection (p < 0.05 by Newman-Keuls test; Fig. 4C). However, the oxytocin response at 30 min after DOI injection was not different from the oxytocin response in the saline pretreatment controls (Fig. 4C). These hormone responses in the fluoxetine-treated rats given subcutaneous injections of DOI are similar to those seen in fluoxetine-treated rats given intraperitoneal injections of DOI.

View larger version (19K): [in this window] [in a new window]   Fig. 4. Time course of hormone responses to subcutaneous DOI (2.5 mg/kg) (15- and 30-min challenge intervals) after 21 days of fluoxetine (10 mg/kg/day i.p.) or saline (2 ml/kg i.p.) treatment. The data represent the mean ± S.E.M. of ACTH (A), corticosterone (B), and oxytocin (C) levels (n = 6–8). A significant effect of fluoxetine compared with saline treatment is indicated by *, p < 0.05 and **, p < 0.01. A significant effect of DOI is indicated by , p < 0.05 and , p < 0.01 (one-way ANOVA and Newman-Keuls multiple range test).

G_q and RGS4 Protein Levels
Chronic treatment with fluoxetine did not alter the levels of G_q or RGS4 proteins in the "punches" of the hypothalamic paraventricular nucleus or in the frontal cortex. The one-way ANOVA indicated no significant difference in RGS4 [F_(5,42) = 0.175, p > 0.05] or G_q protein [F_(5,42) = 0.573, p > 0.05] concentrations across the time periods of treatment with fluoxetine, including 2, 3, 7, 21 and 42 days of injections in the hypothalamic paraventricular nucleus (Fig. 5, B and C). When comparing 21-day fluoxetine treatment against the 21-day saline treatment, the Student's t test indicated no significant difference in RGS4 (p = 0.52) or G_q protein (p = 0.14) levels in the frontal cortex. We found no difference in RGS4 protein levels after DOI challenge compared with saline-challenged rats (data not shown).

View larger version (34K): [in this window] [in a new window]   Fig. 5. A, representative Western blots of Gq and RGS4 proteins in paraventricular nuclei of rats treated with saline (0) or fluoxetine for 2, 3, 7, 21, and 42 days. Actin was used to verify the loading of protein in each lane. The mean ± S.E.M. of Gq protein (B) and RGS4 protein (C) levels in the hypothalamic paraventricular nucleus (PVN) and Gq protein (D) and RGS4 protein (E) in frontal cortex indicate that fluoxetine treatment produced no change in the levels of Gq or RGS4 proteins (n = 6).

PLC Activity
Chronic fluoxetine treatment (21 days) potentiated the 5-HT-induced stimulation of PLC activity in the frontal cortex (Fig. 6A). 5-HT-stimulated PLC activity was increased by 18.9% after 21 days of fluoxetine injections (p < 0.05). GTPS-stimulated PLC activity in the frontal cortex (representing direct stimulation of the enzyme by G proteins) was not altered after treatment with fluoxetine (p = 0.88) (Fig. 6B). It was not possible to measure 5-HT-stimulated PLC activity in the punches of the hypothalamic paraventricular nucleus due to the small amount of tissue.

View larger version (22K): [in this window] [in a new window]   Fig. 6. Effect of 21 days of chronic fluoxetine treatment (10 mg/kg/day i.p.) on PLC activity in the frontal cortex. A, 5-HT-stimulated PLC activity. B, GTPS-stimulated PLC activity. The data represent the mean ± S.E.M. (n = 8). A significant effect of fluoxetine compared with saline treatment is indicated by *, p < 0.05 (Student's t test, pooled variances).

	Discussion

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The influence of treatment with fluoxetine on hormone responses to DOI ranged from attenuation of the oxytocin response (at 15 min post-DOI injection), to potentiation for ACTH and corticosterone responses at 30 min after DOI injection. A summary of the impact of fluoxetine treatment on hormone secretion mediated by 5-HT_2A receptors is shown in Table 1. Fluoxetine treatment also produced a decrease in the rate of weight gain. Additionally, our neurochemical data indicate that treatment with fluoxetine also produces a supersensitivity of 5-HT_2A receptors in the frontal cortex. Fluoxetine produced a decrease in the rate of weight gain but not a decrease in body weight. This phenomenon in rats has been published previously (Alper, 1992). Fluoxetine has also successfully been used in humans as a treatment for obesity (Goldstein et al., 1994). Fluoxetine has an anorectic effect that may lead to this decrease in weight gain observed clinically and experimentally (McGuirk and Silverstone, 1990). Fluoxetine-induced hypophagia is thought to be mediated by fluoxetine acting on the 5-HT_2C receptor (Lightowler et al., 1996). The 5-HT_2C receptor is important in feeding as 5-HT_2C receptor knockout mice have increased feeding and obesity (Tecott et al., 1995).

Stimulation of 5-HT_2A receptors increases the secretion of ACTH, corticosterone, and oxytocin and increases Fos expression in CRF and oxytocin neurons in the paraventricular nucleus of the hypothalamus (Van de Kar, 1991). 5-HT_2A receptors in the hypothalamic paraventricular nucleus mediate the DOI-stimulated increases in plasma ACTH and oxytocin levels, because intraparaventricular and peripheral injections of the 5-HT_2A receptor antagonist MDL 100,907 dose dependently inhibit these responses (Van de Kar et al., 2001; Zhang et al., 2002). Hence, the magnitude of DOI-induced increases in plasma ACTH and oxytocin levels can be used as a peripheral marker of 5-HT_2A receptor function in the hypothalamic paraventricular nucleus.

The oxytocin data are probably the most puzzling. We have now repeated this experiment twice, once using a DOI challenge administered intraperitoneally and once by subcutaneous administration. In both experiments, treatment with fluoxetine resulted in an attenuation of the oxytocin response when examined 15 min after DOI injection and no change in the oxytocin response at 30 min after DOI injection. Hence, the data suggest that treatment with fluoxetine produces either a delayed onset or a desensitization of 5-HT_2A receptor signaling in oxytocin-expressing neurons. On the other hand, treatment with fluoxetine resulted in potentiation of the effect of DOI on both ACTH and corticosterone levels, suggesting an increase in 5-HT_2A receptor signaling in CRF neurons. However, the reduced ACTH response to DOI at 15 min post-DOI injection suggests a delay in the onset of receptor signaling of supersensitive 5-HT_2A receptors. Although previous in vitro studies using human embryonic kidney-293 and C6 glioma cells reported cell type-specific 5-HT_2A receptor regulation (Gray et al., 2001), to our knowledge, this is the first demonstration of such a phenomenon in vivo.

Although treatment with fluoxetine produces a delay in both the ACTH and oxytocin responses to DOI. Fluoxetine may cause desensitization in 5-HT_2A signaling in the oxytocin neurons and a supersensitive response in CRF neurons in the paraventricular nucleus. The differences in fluoxetine-induced changes between the ACTH and oxytocin responses to DOI suggest that 5-HT_2A receptors in the oxytocin-containing magnocellular cells and the CRF-containing parvo-cellular cells in the paraventricular nucleus are regulated differently.

A second possible reason for the differences in the time course of hormone responses to DOI is its route of administration and subsequent differences in the rate of absorption of DOI from the site of administration. Fluoxetine was noted to produce tissue necrosis at the site of administration (Fisher et al., 1999) and indeed tissue necrosis was seen in the peritoneal cavities of rats injected with fluoxetine for 21 days in our studies. Tissue necrosis could result in less efficient absorption of DOI and thus explain the attenuation of hormone levels at the 15-min time point compared with saline controls. However, a subcutaneous injection of DOI, which avoided the peritoneum would not have a problem of decreased absorption of DOI. Yet the same phenomenon of delayed hormone response, after fluoxetine treatment, was observed when DOI was injected subcutaneously, suggesting that the delayed oxytocin and ACTH responses are not due to differences in DOI absorption from the site of injection.

Treatment with fluoxetine resulted in a shift in the ACTH time course to the right, thereby E_max is reached at 30 min rather than 15 min post-DOI injection. In contrast to ACTH, treatment with fluoxetine shifts the time course for corticosterone to the left. Thus, E_max is reached at 30 min rather than 60 min post-DOI challenge injection. If corticosterone secretion were solely dependent on central serotonergic control, then we would expect to see the same rightward shift of the corticosterone time course as is seen with the ACTH time course. Plasma corticosterone levels are also regulated by peripheral serotonergic mechanisms (Alper, 1990), which may help to explain why corticosterone is undergoing an opposite shift in its time course compared with ACTH.

The analysis of 5-HT-stimulated PLC activity in the frontal cortex confirms the conclusion that treatment with fluoxetine can lead to supersensitive 5-HT_2A receptors. Our analysis compared the activation of PLC with 5-HT to activation with GTPS, a nonhydrolyzable form of GTP, which irreversibly activates G proteins. The determination of 5-HT-stimulated PLC activity examines the functioning of 5-HT_2A receptor signaling from the receptor, through the G protein to the effector enzyme (PLC). On the other hand, GTPS directly binds to G_q/11 proteins, changing their confirmation. Activated GTPS-G_q/11 proteins then directly activate PLC. 5-HT-stimulated PLC activity is 5-HT_2A receptor mediated in the frontal cortex because the 5-HT_2A receptor antagonists ketanserin, spiperone, and mianserin significantly attenuate the 5-HT-stimulated PLC activity (Wolf and Schutz, 1997). Fluoxetine treatment (21 days) increased 5-HT-stimulated PLC activity without changing GTPS-stimulated PLC activity. This not only shows a supersensitivity of the 5-HT_2A receptors at the cellular level in the frontal cortex, but also shows that the supersensitivity involves a change between 5-HT_2A receptors and G_q/11 proteins and not between G_q/11 proteins and the effector enzyme PLC.

This supersensitivity of 5-HT_2A receptors in the frontal cortex is observed at a cellular level with only one level of amplification (G proteins) between receptor stimulation and effector activation. Unfortunately, the hypothalamic paraventricular nucleus does not provide sufficient tissue to determine PLC activity. But because supersensitivity of 5-HT_2A receptors occurs in both CRF neurons and frontal cortex neurons after prolonged fluoxetine exposure, it is likely that the mechanism of 5-HT_2A receptor supersensitivity is similar in both of these neuronal cell types.

Alterations in G_q/11 or RGS4 proteins could underlie changes in sensitivity of 5-HT_2A receptor signaling. However, no changes were observed in the levels of G_q or RGS4 proteins in the paraventricular nucleus at the time periods of fluoxetine treatment (2, 3, 7, 21, or 42 days) or in the frontal cortex after 21 days of fluoxetine treatment. It is possible that differential regulation of 5-HT_2A receptor signaling in the oxytocin neurons and CRF neurons in the paraventricular nucleus might obscure changes in the 5-HT_2A receptor signaling proteins in the whole punch of the paraventricular nucleus. Just as PLC activity increased by approximately 20% in the frontal cortex after 21 days of fluoxetine treatment, the mean in G_q protein levels were 55% above control levels. However, the changes in the levels of G_q proteins in the frontal cortex were not statistically significant and, therefore, cannot explain the supersensitization of the 5-HT_2A receptor in the frontal cortex.

Treatment with fluoxetine for 7 or 14 days was accompanied by an increase in high-affinity agonist ¹²⁵I-DOI-labeled 5-HT_2A receptor binding but no change in the density of antagonist [³H]ketanserin-labeled 5-HT_2A receptors, or G_q and G₁₁ protein levels in the hypothalamus (Li et al., 1997b). Receptor phosphorylation in other systems has been shown to inhibit the coupling of G proteins to receptors (Pitcher et al., 1998). For 5-HT_2A receptors, rapid agonist-induced desensitization was dependent on phosphorylation by protein kinase C (Roth et al., 1986; Berg et al., 2001). Thus, fluoxetine might induce alterations in posttranslational modifications of 5-HT_2A receptors or G proteins, leading to changes in 5-HT_2A receptor coupling to G proteins rather than a change in the protein levels of the signaling system.

In summary, our data suggest that sustained treatment with fluoxetine produces a differential change in the sensitivity of 5-HT_2A receptor signaling in the frontal cortex and CRF-containing neurons in the paraventricular nucleus compared with oxytocin-containing neurons in the paraventricular nucleus. Fluoxetine treatment for 21 and 42 days increases the sensitivity of 5-HT_2A receptor-mediated responses in the CRF-containing neurons of the hypothalamic paraventricular nucleus and in neurons in the frontal cortex and decreases the sensitivity of 5-HT_2A receptor-mediated responses in hypothalamic oxytocin-containing neurons. These fluoxetine-induced changes in 5-HT_2A receptor signaling occur without changing the levels of G_q or RGS4 proteins. In the frontal cortex, the mechanism is likely at the level of the 5-HT_2A receptor or receptor-G protein interaction.

	Acknowledgements

 
We are grateful to Eli Lilly & Co. for the generous gift of fluoxetine and to Dr. Lanny C. Keil (NASA Ames Research Center, Moffat Field, CA) for the kind donation of oxytocin antiserum.

	Footnotes

 
This study was supported by U.S. Public Health Service NS38509 (to N.A.M.), and NS34153 and DA13669 (to L.D.V.D.K.).

DOI: 10.1124/jpet.103.050534.

ABBREVIATIONS: SSRI, selective serotonin reuptake inhibitor; 5-HT, 5-hydroxytryptamine; PLC, phospholipase C; GTPS, guanosine 5'-O-(3-thio)triphosphate; DOI, (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane HCl; ACTH, adrenocorticotropic hormone; CRF, corticotropin-releasing factor; MDL 100,907, (±)-2,3-dimethoxyphenyl-1-[2-4-(piperidine)-methanol]; RGS, regulators of G protein signaling; IOD, integrated optical density; ANOVA, analysis of variance.

Address correspondence to: Dr. Nancy A. Muma, Department of Pharmacology, Loyola University Chicago, Stritch School of Medicine, 2160 South First Ave., Maywood, IL 60153. E-mail: nmuma{at}lumc.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Alper RH (1990) Evidence for central and peripheral serotonergic control of corticosterone secretion in the conscious rat. Neuroendocrinology 51: 255–260.[Medline]

Alper RH (1992) Effects of the selective serotonin reuptake inhibitor fluoxetine on baroreceptor reflex sensitivity and body weight in young and old rats. J Gerontol 47: B130–B136.[Medline]

Berg KA, Stout BD, Maayani S, and Clarke WP (2001) Differences in rapid desensitization of 5-hydroxytryptamine2A and 5-hydroxytryptamine2C receptor-mediated phospholipase C activation. J Pharmacol Exp Ther 299: 593–602.[Abstract/Free Full Text]

Berman DM, Kozasa T, and Gilman AG (1996) The GTPase-activating protein RGS4 stabilizes the transition state for nucleotide hydrolysis. J Biol Chem 271: 27209–27212.[Abstract/Free Full Text]

Blank JL and Exton JH (1994) Purification of activated and heterotrimeric forms of Gq proteins, in Methods in Enzymology. Heterotrimeric G Proteins (Iyengar R ed) pp 174–181, Academic Press, San Diego.

Cadogan AK, Marsden CA, Tulloch I, and Kendall DA (1993) Evidence that chronic administration of paroxetine or fluoxetine enhances 5-HT₂ receptor function in the brain of the guinea pig. Neuropharmacology 32: 249–256.[CrossRef][Medline]

Fisher A, McLean AJ, Purcell P, Herdson PB, Dahlstrom JE, and Le Couteur DG (1999) Focal necrotising vasculitis with secondary myositis following fluoxetine administration [letter]. Aust NZ J Med 29: 375–376.[Medline]

Goldstein DJ, Rampey AH Jr, Enas GG, Potvin JH, Fludzinski LA, and Levine LR (1994) Fluoxetine: a randomized clinical trial in the treatment of obesity. Int J Obes Relat Metab Disord 18: 129–135.[Medline]

Gray JA, Sheffler DJ, Bhatnagar A, Woods JA, Hufeisen SJ, Benovic JL, and Roth BL (2001) Cell-type specific effects of endocytosis inhibitors on 5-hydroxytryptamine_2A receptor desensitization and resensitization reveal an arrestin-, GRK2- and GRK5-independent mode of regulation in human embryonic kidney 293 cells. Mol Pharmacol 60: 1020–1030.[Abstract/Free Full Text]

Hollander E, DeCaria C, Gully R, Nitescu A, Suckow RF, Gorman JM, Klein DF, and Liebowitz MR (1991) Effects of chronic fluoxetine treatment on behavioral and neuroendocrine responses to meta-chlorophenylpiperazine in obsessive-compulsive disorder. Psychiatry Res 36: 1–17.[CrossRef][Medline]

Li Q, Brownfield MS, Battaglia G, Cabrera TM, Levy AD, Rittenhouse PA, and Van de Kar LD (1993) Long-term treatment with the antidepressants fluoxetine and desipramine potentiates endocrine responses to the serotonin agonists 6-chloro-2-[1-piperazinyl]-pyrazine (MK-212) and (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCI (DOI). J Pharmacol Exp Ther 266: 836–844.[Abstract/Free Full Text]

Li Q, Muma NA, Battaglia G, and Van de Kar LD (1997a) A desensitization of hypothalamic 5-HT_1A receptors by repeated injections of paroxetine: reduction in the levels of G_i and G_o proteins and neuroendocrine responses, but not in the density of 5-HT_1A receptors. J Pharmacol Exp Ther 282: 1581–1590.[Abstract/Free Full Text]

Li Q, Muma NA, Battaglia G, and Van de Kar LD (1997b) Fluoxetine gradually increases [¹²⁵I]DOI-labelled 5-HT_2A/2C receptors in the hypothalamus without changing the levels of G_q- and G₁₁-proteins. Brain Res 775: 225–228.[CrossRef][Medline]

Lightowler S, Wood M, Brown T, Glen A, Blackburn T, Tulloch I, and Kennett G (1996) An investigation of the mechanism responsible for fluoxetine-induced hypophagia in rats. Eur J Pharmacol 296: 137–143.[CrossRef][Medline]

Massou JM, Trichard C, Attar-Levy D, Feline A, Corruble E, Beaufils B, and Martinot JL (1997) Frontal 5-HT_2A receptors studied in depressive patients during chronic treatment by selective serotonin reuptake inhibitors. Psychopharmacology 133: 99–101.[CrossRef][Medline]

McGuirk J and Silverstone T (1990) The effect of the 5-HT re-uptake inhibitor fluoxetine on food intake and body weight in healthy male subjects. Int J Obes 14: 361–372.[Medline]

Nierenberg AA, Farabaugh AH, Alpert JE, Gordon J, Worthington JJ, Rosenbaum JF, and Fava M (2000) Timing of onset of antidepressant response with fluoxetine treatment. Am J Psychiatry 157: 1423–1428.[Abstract/Free Full Text]

Pitcher JA, Freedman NJ, and Lefkowitz RJ (1998) G protein-coupled receptor kinases. Annu Rev Biochem 67: 653–692.[CrossRef][Medline]

Rittenhouse PA, Bakkum EA, and Van de Kar LD (1991) Evidence that the serotonin agonist, DOI, increases renin secretion and blood pressure through both central and peripheral 5-HT₂ receptors. J Pharmacol Exp Ther 259: 58–65.[Abstract/Free Full Text]

Roth BL, Nakaki T, Chuang D-M, and Costa E (1986) 5-Hydroxytryptamine₂ receptors coupled to phospholipase C in rat aorta: modulation of phosphoinositide turnover by phorbol ester J Pharmacol Exp Ther 238: 480–485.[Abstract/Free Full Text]

Roth BL, Willins DL, Kristiansen K, and Kroeze WK (1998) 5-Hydroxytryptamine₂-family (hydroxytryptamine_2A, hydroxytryptamine_2B, hydroxytryptamine_2C): where structure meets function. Pharmacol Ther 79: 231–257.[CrossRef][Medline]

Steiner M, Steinberg S, Stewart D, Carter D, Berger C, Reid R, Grover D, and Streiner D (1995) Fluoxetine in the treatment of premenstrual dysphoria. Canadian Fluoxetine/Premenstrual Dysphoria Collaborative Study Group. New Engl J Med 332: 1529–1534.[Abstract/Free Full Text]

Tecott LH, Sun LM, Akana SF, Strack AM, Lowenstein DH, Dallman MF, and Julius D (1995) Eating disorder and epilepsy in mice lacking 5-HT₂ serotonin receptors. Nature (Lond) 374: 542–546.[CrossRef][Medline]

Tilakaratne N, Yang ZL, and Friedman E (1995) Chronic fluoxetine or desmethylimipramine treatment alters 5-HT₂ receptor mediated c-fos gene expression. Eur J Pharmacol Mol Pharmacol 290: 263–266.[CrossRef][Medline]

Van de Kar LD (1991) Neuroendocrine pharmacology of serotonergic (5-HT) neurons. Annu Rev Pharmacol Toxicol 31: 289–320.[Medline]

Van de Kar LD, Javed A, Zhang YH, Serres F, Raap DK, and Gray TS (2001) 5-HT_2A receptors stimulate ACTH, corticosterone, oxytocin, renin and prolactin release and activate hypothalamic CRF and oxytocin-expressing cells. J Neurosci 21: 3572–3579.[Abstract/Free Full Text]

Van Wijngaarden I, Tulp MTM, and Soudijn W (1990) The concept of selectivity in 5-HT receptor research. Eur J Pharmacol 188: 301–312.[CrossRef][Medline]

Wolf WA and Schutz LJ (1997) The serotonin 5-HT_2C receptor is a prominent serotonin receptor in basal ganglia: evidence from functional studies on serotonin-mediated phosphoinositide hydrolysis. J Neurochem 69: 1449–1458.[Medline]

Zhang Y, Damjanoska KJ, Carrasco GA, Dudas B, D'Souza DN, Tetzlaff J, Garcia F, Sullivan Hanley NR, Scripathirathan K, Petersen BR, et al. (2002) Evidence that 5-HT_2A receptors in the hypothalamic paraventricular nucleus mediate neuroendocrine responses to (–)DOI. J Neurosci 22: 9635–9642.[Abstract/Free Full Text]

Zifa E and Fillion G (1992) 5-Hydroxytryptamine receptors. Pharmacol Rev 44: 401–458.[Medline]

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