D. K. RAAP, S. EVANS, F. GARCIA, Q. LI, N. A. MUMA, W. A. WOLF, G. BATTAGLIA and L. D. VAN DE KAR

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1 /99/ $03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 288, No. 1 Copyright 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 288:98 106, 1999 Daily Injections of Fluoxetine Induce Dose-Dependent Desensitization of Hypothalamic 5-HT 1A Receptors: Reductions in Neuroendocrine Responses to 8-OH-DPAT and in Levels of G z and G i Proteins D. K. RAAP, S. EVANS, F. GARCIA, Q. LI, N. A. MUMA, W. A. WOLF, G. BATTAGLIA and L. D. VAN DE KAR Department of Pharmacology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois Accepted for publication July 28, 1998 This paper is available online at ABSTRACT The present studies examined the dose-response relationship of fluoxetine-induced desensitization of hypothalamic postsynaptic 5-HT 1A receptors, as measured from the reduced neuroendocrine responses to a 5-HT 1A agonist. Because hypothalamic G z proteins mediate the ACTH and oxytocin responses to 5-HT 1A receptor activation, we also determined the effect of fluoxetine on the levels of G z proteins in the hypothalamus. Rats were injected daily for 14 days with saline or with fluoxetine doses of 0.3, 1, 3, 5, 7.5, or 10 mg/kg/day. Fluoxetine produced a dose-dependent reduction in the oxytocin, ACTH, and corticosterone responses to the 5-HT 1A agonist 8-hydroxy- 2-(dipropylamino)tetralin (8-OH-DPAT, 50 g/kg, s.c.). The lowest fluoxetine dose that significantly, although incompletely, reduced the neuroendocrine responses to 8-OH-DPAT was 5 The use of selective 5-hydroxytryptamine (serotonin) (5- HT) ) reuptake inhibitors (SSRIs), such as fluoxetine, in the treatment of neuropsychiatric disorders has become increasingly widespread (Goldberg, 1995; Fuller, 1996). Inhibition of 5-HT reuptake increases 5-HT levels in the synaptic cleft, thereby prolonging the activation of postsynaptic 5-HT receptors. Therapeutic effects, however, are not attained in patients until 2 to 3 weeks after the beginning of treatment (Artigas et al., 1996; Quitkin et al., 1996). Adaptive changes in 5-HT receptors may underlie the therapeutic effectiveness of SSRIs (Le Poul et al., 1995; Blier and de Montigny, 1996; Li et al., 1996; Li et al., 1997a, 1997b). Repeated exposure to SSRIs induces desensitization of somatodendritic 5-HT 1A autoreceptors in the raphe region Received for publication March 19, Supported in part by United States Public Health Service Grant NS34153 (LDVdK). A portion of this research was previously presented at the Society for Neuroscience 27th Annual Meeting (1997; 385.4). mg/kg/day. The 10 mg/kg/day dose of fluoxetine maximally inhibited all neuroendocrine responses to 8-OH-DPAT. Hypothalamic levels of G z protein were reduced by both the 7.5 and 10 mg/kg/day doses of fluoxetine, whereas G i1 protein levels were reduced only after the highest dose (10 mg/kg/day) of fluoxetine. G i2,g i3, and G o levels were not reduced by any fluoxetine dose. Cytosolic levels of G i1 and G z proteins were unaltered, indicating that reductions in G z and G i1 proteins are not caused by a redistribution of the proteins from the membrane into the cytosol. The results from the present study indicate that fluoxetine-induced desensitization of hypothalamic postsynaptic 5-HT 1A receptor systems is dose-dependent and may be caused in part by reductions in the hypothalamic levels of G z proteins. within 3 days (Le Poul et al., 1995). Because these autoreceptors serve as feedback inhibitors of serotonergic cell firing, their desensitization leads to increased serotonergic neurotransmission. In vivo microdialysis studies indicate that chronic treatment with fluoxetine increases the levels of 5-HT in the forebrain (Rutter et al., 1994). Higher levels of 5-HT in the synaptic cleft lead to a subsequent desensitization of the postsynaptic 5-HT 1A receptors in forebrain regions like the hypothalamus (Lesch et al., 1991; Li et al., 1993b, 1994, 1996; Lerer et al., 1997; Li et al., 1997b). Hence, neuroadaptive changes produced by repeated exposure to SSRIs include 5-HT 1A receptors. We have consistently shown in rats that daily injections of 10 mg/kg/day fluoxetine or paroxetine for 14 to 21 days completely inhibit the oxytocin, ACTH, and corticosterone responses to the 5-HT 1A agonists 8-OH-DPAT or ipsapirone (Li et al., 1993b, 1994, 1996, 1997b). This fluoxetine dose is about 10 times higher than the clinically relevant doses. For example, a reduced ACTH response to ipsapirone is observed ABBREVIATIONS: 5-HT, 5-hydroxytryptamine (serotonin); 8-OH-DPAT, 8-hydroxy-2-(dipropylamino)tetralin; ACTH, adrenocorticotropic hormone; ANOVA, analysis of variance; DOI, ( )-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl; integrated optical density, IOD; SDS, sodium dodecyl sulfate; SSRI, selective serotonin reuptake inhibitor. 98

2 HT 1A Receptors and Dose Response of Fluoxetine 99 in normal humans and in patients with obsessive compulsive disorder taking 20 to 60 mg/day fluoxetine (approximately equivalent to mg/kg/day) for 4 weeks to 3 months (Lesch et al., 1991; Lerer et al., 1997). The seemingly high dose of fluoxetine (10 mg/kg/day) in rats is the minimum required to produce a complete inhibition of 5-HT uptake (Fuller et al., 1978); it is unknown what dose is required in humans. If the degree of desensitization of the postsynaptic 5-HT 1A receptors is dependent on the degree of blockade of 5-HT transporters, then it should depend on the dose of SSRI administered. The present study investigated chronic exposure to lower doses of fluoxetine to determine if the degree of desensitization of hypothalamic 5-HT 1A receptors is dosedependent. We have previously shown that exposure to fluoxetine does not affect the density or the affinity of hypothalamic 5-HT 1A receptors, suggesting that the mechanism of desensitization involves alterations in signal transduction (Li et al., 1993b). 5-HT 1A receptors are coupled to the G i family of G proteins, which include G i1,g i2,g i3,g o, and G z proteins (Raymond et al., 1993; Barr et al., 1997). Hypothalamic G z proteins mediate the ACTH and oxytocin responses to activation of 5-HT 1A receptors (Serres et al., 1998; Van de Kar et al., 1998). Therefore, to understand the mechanism of fluoxetine-induced desensitization of 5-HT 1A receptor systems, we investigated changes in hypothalamic G z protein levels as a likely component of the 5-HT 1A signaling system to be altered by fluoxetine. Our previous time course study indicates that daily injections of fluoxetine produce a gradual reduction in hypothalamic G i1 and G i3 protein levels that matches the time course of desensitization of hypothalamic 5-HT 1A receptors (as measured from the reduction in the neuroendocrine responses to 8-OH-DPAT) (Li et al., 1996). Levels of G z protein in the hypothalamus have not been examined. Therefore, we compared the dose-dependent changes in G i proteins with the G z protein levels in the hypothalamus and with the neuroendocrine responses to 8-OH-DPAT. To begin to determine the mechanisms underlying the reduction in the levels of membrane-associated G z and G i1 proteins, we investigated whether there was a redistribution of membrane-bound G proteins to the cytosol. Materials and Methods Animals Male Sprague-Dawley rats ( g) were purchased from Harlan Laboratories (Indianapolis, IN). Animals were housed 2 per cage in a room controlled for temperature, humidity, and lighting (lights on ). Food and water were available ad libitum at all times. 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 and Co. (Indianapolis, IN). A fresh fluoxetine solution for the highest dose in each experiment was made daily by dissolving fluoxetine in the vehicle (0.9% saline). Serial dilutions of the fluoxetine solution were then prepared to correspond to the lower doses in each experiment, allowing all animals to receive injections (i.p.) in a volume of 2 ml/kg. 8-OH- DPAT was purchased from Research Biochemical Inc. (Natick, MA); it was dissolved in saline and injected (s.c.) in a volume of 1 ml/kg. Animal Drug Treatments In experiment 1, rats were injected (i.p.) once daily for 14 days with either 0 (saline), 0.3, 1, 3, or 10 mg/kg fluoxetine. In experiment 2, rats were injected (i.p.) once daily for 14 days with either 0 (saline), 5, 7.5, or 10 mg/kg fluoxetine. Doses of fluoxetine were chosen in experiment 1 to cover two logarithmic scales. In experiment 2, the choice of doses was based on the results obtained in experiment 1. All other procedures were similar in experiments 1 and 2. Body weight was recorded throughout the injection period. Rats were then challenged 18 h after the last fluoxetine injection with either saline or 8-OH-DPAT (50 g/kg, s.c.) and decapitated 15 min later. 8-OH- DPAT is a 5-HT 1A agonist with a high affinity for 5-HT 1A receptors and at least a 10-fold lower affinity for other 5-HT receptor subtypes (Boess and Martin, 1994). By activating hypothalamic 5-HT 1A receptors, 8-OH-DPAT stimulates the secretion of oxytocin, ACTH, and corticosterone (Bagdy, 1996). Maximal stimulation of the secretion of oxytocin and ACTH is reached by 8-OH-DPAT doses of 200 to 500 g/kg (s.c.) (Li et al., 1993b). A submaximal dose of 8-OH-DPAT was used in the present study (50 g/kg, s.c.) to prevent activation of other 5-HT receptor subtypes. This dose of 8-OH-DPAT was previously shown to be the most effective dose in demonstrating desensitization of 5-HT 1A receptors (Li et al., 1996). Trunk blood was collected in centrifuge tubes containing 0.5 ml of a 0.3 M ethylenediamine tetraacetic acid (EDTA; ph 7.4) solution. The plasma was separated and stored at 70 C until measurement of hormone levels. The hypothalamus was removed from the brain immediately after decapitation and frozen in liquid nitrogen, then stored at 70 C for the measurement of G protein levels. Radioimmunoassay of Hormones Plasma oxytocin, ACTH, and corticosterone concentrations were determined in all animals from both experiments by radioimmunoassays as previously described in detail (Li et al., 1993b, 1997b). Immunoblots of G Proteins Cytosolic and Membrane-Bound Protein Fractionation. G protein levels were measured in hypothalamic tissue from animals in experiment 2 that received a saline challenge. All procedures were conducted at 4 C unless otherwise indicated. Briefly, the tissues were homogenized in 0.5 ml of a 50 mm Tris buffer, ph 7.4, containing 150 mm NaCl, 10% sucrose, and 0.5 mm phenylmethanesulfonyl fluoride (PMSF). After centrifugation at 20,000 g for 60 min, the supernatant was collected and stored for determination of cytosolic G protein levels. The pellets (containing the membrane-bound proteins) were solubilized in a 20 mm Tris buffer, ph 8 (containing 1 mm EDTA, 100 mm NaCl, 1% sodium cholate, and 1 mm dithiothreitol) in a ratio of 3 l buffer/mg tissue. The resuspended homogenates were incubated and shaken for 1 h, followed by centrifugation at 100,000 g for 60 min. The supernatant (containing the membranebound G proteins) was collected and stored for the determination of G protein levels. Protein concentrations were measured using bovine serum albumin as a standard. The total-protein concentrations were between 3.6 to 5.4 g/ l. Quantification of G proteins. The cytosolic and membranebound solubilized proteins were resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, using 0.75 mm thick Tris- Glycine denaturing reducing gels, containing 0.1% SDS, 12.5% acrylamide/bisacrylamide (30:0.2), 4.6 M urea and 375 mm Tris, ph 8.7 (Mullaney and Miligan, 1990). Three samples from each treatment group and three randomly selected controls were loaded on each gel. Each sample was measured on three independent gels. The proteins were then electrophoretically transferred for 2 hours to nitrocellulose membranes. After drying, the membranes were incubated in a blocking solution containing 5% nonfat dry milk, 0.05% NP40, 50 mm Tris, and 150 mm NaCl, ph 7.4 for 1 h, washed and incubated overnight at 4 C with polyclonal antisera for G i1/2 (AS/7, Du- Pont NEN, Boston, MA, 1:2,500 dilution), G i3 (Anti-G i3, Upstate

3 100 Raap et al. Vol. 288 Biotechnology Inc., Lake Placid, NY, 1:2,000 dilution), G o (GC/2, Du-pont NEN, 1:10,000 dilution), or G z (I-20, Santa Cruz Biotechnology Inc., Santa Cruz, CA, 1:6,000 dilution) at 4 C overnight. The membranes were then incubated with a secondary antibody (goat anti-rabbit serum, Cappell, Organon Teknika Corp, Durham, NC, 1:5,000 dilution), followed by an incubation with rabbit peroxidaseantiperoxidase (Cappell, Organon Teknika, 1:10,000). The membranes were incubated with the ECL chemiluminescence substrate solution (Amersham, Arlington Heights, IL) and then exposed to Kodak X-ray film. Data Analysis. Films were analyzed densitometrically using NIH Image (v. 1.57) for Macintosh computers. The gray-scale density readings were calibrated using a transmission step wedge standard. The integrated optical density (IOD) of each band was calculated as the sum of optical densities of all the pixels within the area of the band outlined. An area adjacent to the G protein bands was used to calculate the background optical density of the film. The IOD for the film background was subtracted from the IOD for each band. The resulting IOD for each G protein band was then divided by the amount of protein loaded on the corresponding lane. The IOD/ g protein values obtained from treated rats were divided by the mean IOD/ g protein values obtained from control rats in each gel to determine the relative amounts of the G proteins. The data for each rat were the means obtained from the three gels. High-Pressure Liquid Chromatography (HPLC) of Fluoxetine and Norfluoxetine Plasma Preparation. Fluoxetine and norfluoxetine levels were measured in plasma from treated animals that received a saline challenge before sacrifice. Extraction of fluoxetine and norfluoxetine from plasma was carried out at room temperature. In a 2-ml polypropylene microfuge tube, 5 l ofa100 M fluvoxamine solution in 0.01 MH 3 PO 4 (internal standard) was added to 500 l of thawed plasma; 500 l of 1 M NaOH was added and tubes were mixed; 600 l of chloroform was then added and tubes were shaken vigorously for 3 min. Tubes were then centrifuged at 9,000g for 1 min, and the aqueous phase was aspirated and discarded. Compounds were backextracted from the chloroform phase by adding 400 l of 0.1 M H 3 PO 4, shaking for 3 min and centrifuging at 9,000 g for 1 min; 375 l of the aqueous phase was collected, placed in new tubes, and evaporated at 43 C in a SpeedVac (Savant Instruments; Holbrook, NY). The dried residue was reconstituted in 100 l of M H 3 PO 4 and centrifuged at 9,000 g for 1 min. The clear extract was placed in autosampler vials in a refrigerated autosampler (SIL-10A; Shimadzu Scientific Instrument; Wood Dale, IL) for injection of 50 l into the HPLC. Preliminary studies using spiked control plasma demonstrated that the recovery of fluvoxamine, fluoxetine, and norfluoxetine were similar and averaged 66 2%. HPLC Conditions. Fluoxetine and norfluoxetine were separated by reversed-phase HPLC. The mobile phase consisted of 0.02 M NaH 2 PO 4, 0.02 M sodium citrate, 200 mm EDTA, 35% acetonitrile, 4.4 mm tetraethylammonium (hydrogen sulfate salt), and was brought to an apparent ph of 4.5 using H 3 PO 4. The stationary phase consisted of a 7.5 cm 4.6 mm (i.d.) Ultrasphere ODS 3 mm reversed-phase column (Beckman Instruments, CA). Flow rate was adjusted to 0.9 ml/min. Under these conditions, the retention times of fluvoxamine, norfluoxetine, and fluoxetine were 3.0, 3.8, and 4.5 min, respectively. Compounds were detected by ultraviolet absorbance at 230 nm using a UV detector (SPD-10A; Shimadzu Scientific Instruments). Quantitation was achieved by comparing peak heights of samples with peak heights of standards. The limit of sensitivity was 75 nm for fluoxetine and 25 nm for norfluoxetine. Statistical Analyses Hormone data were analyzed by two-way analysis of variance (ANOVA). G protein levels were analyzed by one-way ANOVA. Group means were compared by Newman-Keuls multiple range test. Linear correlation analysis also was carried out for within-group relationships between plasma hormone and hypothalamic G protein levels in untreated rats (Steel and Torrie, 1960). GB-STAT software (Dynamic Microsystems, Inc., Silver Spring, MD) was used for all statistical analyses. Results Fluoxetine and Norfluoxetine in Plasma Fluoxetine and norfluoxetine concentrations were determined in the plasma of rats 18 h after the last injection. The data are presented in Table 1. Consistent with the short half-life (7.7 h) of fluoxetine in rats (Caccia et al., 1990), no detectable levels of fluoxetine were observed in plasma even after treatment with the highest fluoxetine dose (10 mg/kg/ day). The levels of norfluoxetine in plasma were elevated in a dose-dependent manner (Table 1). The minimum dose of fluoxetine that resulted in detectable levels of norfluoxetine in plasma was 5 mg/kg/day. Body Weight Changes Daily injections of 10 mg/kg/day fluoxetine significantly inhibited weight gain during the 14 days of injections (Fig. 1). Doses of fluoxetine between 0.3 and 3 mg/kg/day had no effect on weight gain (Fig. 1A), whereas fluoxetine doses of 5, 7.5, and 10 mg/kg/day inhibited weight gain in a dose-dependent manner (Fig. 1B). Hormone Responses to 8-OH-DPAT 8-OH-DPAT significantly increased plasma oxytocin (Fig. 2), ACTH (Fig. 3), and corticosterone (Fig. 4) levels. Consistent with our previous results, chronic injections of 10 mg/ kg/day fluoxetine completely blocked plasma oxytocin (Fig. 2), ACTH (Fig. 3), and corticosterone (Fig. 4) responses to 8-OH-DPAT without altering basal hormone levels. Doses of fluoxetine between 0.3 and 3 mg/kg/day did not alter the effect of 8-OH-DPAT on plasma hormones (Figs. 2A, 3A, and 4A). However, fluoxetine doses between 5 and 10 mg/kg/day produced a dose-dependent decrease in the effect of 8-OH-DPAT on plasma oxytocin (Fig. 2B), ACTH (Fig. 3B), and corticosterone (Fig. 4B), without altering basal hormone levels. The minimum effective dose of fluoxetine that reduced the neuroendocrine TABLE 1 Plasma levels of fluoxetine and norfluoxetine in rats treated with saline or fluoxetine Fluoxetine Dose (mg/kg/day) Fluoxetine (nm) Plasma Levels a Norfluoxetine (nm) 0 (Saline) ND ND (n 8) 0.3 ND ND (n 8) 1 ND ND (n 8) 3 ND ND (n 8) 5 ND (n 5) 7.5 ND (n 6)* 10 ND (n 12)** The data represent the mean S.E.M. The number of rats per group is represented in parentheses. ND, undetectable. a The detection limit for fluoxetine was 75 nm and for norfluoxetine 25 nm. * Significant difference from the group treated with 5 mg/kg/day dose of fluoxetine P.01 (1-way ANOVA: F(2,20) and Newman-Keuls multiple-range test). ** Significant difference from the group treated with 7.5 mg/kg/day dose of fluoxetine, P.01 (1-way ANOVA and Newman-Keuls multiple-range test). The rats with no detectable levels of norfluoxetine were not included in the ANOVA.

4 HT 1A Receptors and Dose Response of Fluoxetine 101 Fig. 1. Dose-response effect of fluoxetine on body weight gain. A, experiment 1 using fluoxetine doses of 0.3, 1, 3, and 10 mg/kg/day; B, experiment 2 using fluoxetine doses of 5, 7.5 and 10 mg/kg/day. The data represent the mean S.E.M. of 20 rats per group. A, the two-way ANOVA indicated the following: main effect of days F(8,693) , p 0.01; main effect of fluoxetine dose F(4,693) 64.83, p 0.01; days fluoxetine dose F(32,693) 3.79, p.01. B, the two-way ANOVA indicated the following: main effect of days F(7,608) 142.7, p.01; main effect of fluoxetine dose F(3,608) 77.27, p.01; days fluoxetine dose F(21, 608) 2.80, p The post hoc Newman-Keuls tests indicated the following: significant difference compared with the saline group, p 0.05; *significant difference compared with the saline group, p responses to 8-OH-DPAT was 5 mg/kg/day. Exposure to 7.5 mg/kg/day fluoxetine produced a greater, although incomplete, reduction in the oxytocin, ACTH, and corticosterone responses to 8-OH-DPAT. In both experiments, a complete blockade of the oxytocin, ACTH, and corticosterone responses to 8-OH-DPAT was attained after exposure to 10 mg/kg/day fluoxetine (see Table 2 for reduction percentages). G Proteins in Hypothalamus Examples of immunoblots of membrane-bound and cytosolic G i1,g i2, and G z proteins in the hypothalamus are presented in Fig. 5 (A D). The specificity of the antibodies for G i1,g i2,g i3, and G o proteins has been verified in previous studies (Li et al., 1996). The specificity of the G z antiserum was confirmed by showing that preabsorption of the antibody with a G z blocking peptide prevented the antiserum from binding to G z protein on immunoblots (Fig. 5E). Membrane Proteins. Hypothalamic levels of G z protein (Fig. 6A) were significantly reduced after exposure to the 7.5 and the 10 mg/kg/day doses of fluoxetine. Consistent with previously reported results (Li et al., 1996), repeated daily injections of 10 mg/kg/day fluoxetine produced a significant decrease in G i1 protein levels (Fig. 6B). G i3 protein levels Fig. 2. Dose-response effect of fluoxetine on the oxytocin response to 8-OH-DPAT (50 g/kg s.c.). A, experiment 1 using fluoxetine doses of 0.3, 1, 3, and 10 mg/kg/day; B, experiment 2 with fluoxetine doses of 5, 7.5, and 10 mg/kg/day. The data represent the mean S.E.M. of 8 to 12 rats per group. A, the two-way ANOVA indicated the following: main effect of fluoxetine dose F(4,60) 4.75, p 0.01; main effect of 8-OH-DPAT F(1,60) 48.60, p 0.01; fluoxetine dose 8-OH-DPAT, F(4,60) 4.86, p B, the two-way ANOVA indicated the following: main effect of fluoxetine dose F(3,66) 16.36, p 0.01; main effect of 8-OH-DPAT F(1,66) 86.30, p 0.01; fluoxetine dose 8-OH-DPAT, F(3,66) 17.44, p The posthoc Newman-Keuls tests indicated the following: *significant difference from the rats challenged with saline (0 dose of 8-OH-DPAT), p 0.01; #significant difference from the vehicle rats (0 dose of fluoxetine) challenged with 8-OH-DPAT, p were not significantly reduced after chronic exposure to 10 mg/kg/day fluoxetine. However, exposure to 5 and 7.5 mg/kg/ day fluoxetine significantly increased the levels of G i3 proteins in the hypothalamus (Fig. 6C). The levels of G i2 protein (Fig. 6D) and G o protein (Fig. 6E) were not altered by chronic exposure to any fluoxetine doses. Cytosolic Proteins. Levels of G z,g i1,g i3,g i2, and G o proteins in the cytosol were unaltered by exposure to fluoxetine (Fig. 6 F J). Comparison of Hormone Responses to Hypothalamic G Proteins. Table 2 presents the percent reduction as compared to controls for the hormone responses to 8-OH-DPAT and hypothalamic G protein levels for the 5, 7.5, and 10 mg/kg/day doses of fluoxetine. Overall, reductions in the magnitudes of hormone responses to 8-OH-DPAT followed a similar pattern for all three hormones (i.e., partial and significant reduction after 5 mg/kg/day fluoxetine, further reduction after 7.5 mg/kg/ day, complete reduction after 10 mg/kg/day). In contrast, the effect of fluoxetine on G protein subunits were variable. Reductions in G z protein levels were the most similar to hormone responses, while reductions in G i1 proteins only occurred with the highest fluoxetine dose.

5 102 Raap et al. Vol. 288 Fig. 3. Dose-response effect of fluoxetine on the ACTH response to 8-OH- DPAT (50 g/kg s.c.). A, experiment 1 using fluoxetine doses of 0.3, 1, 3, and 10 mg/kg/day; B, experiment 2 with fluoxetine doses of 5, 7.5, and 10 mg/kg/day. The data represent the mean S.E.M. of 8 to 12 rats per group. A, the two-way ANOVA indicated the following: main effect of fluoxetine F(4,70) 5.86, p 0.01; main effect of 8-OH-DPAT F(1,70) 89.83, p 0.01; fluoxetine 8-OH-DPAT, F(4,70) 5.93, p B, the two-way ANOVA indicated the following: main effect of fluoxetine F(3,68) 13.64, p 0.01; main effect of 8-OH-DPAT F(1,68) 75.90, p 0.01; fluoxetine X 8-OH-DPAT, F(3,68) 12.15, p The post hoc Newman-Keuls tests indicated the following: *significant difference from the rats challenged with saline (0 dose of 8-OH-DPAT), p 0.01; #significant difference from the vehicle rats (0 dose of fluoxetine) challenged with 8-OH-DPAT, p A within-group correlation analysis examined the hypothalamic levels of G proteins with the corresponding hormone levels in the untreated rats that were challenged with saline. Only G z protein levels significantly correlated with plasma levels of ACTH and oxytocin (Table 3). Discussion The most important and novel findings in these studies are: 1) fluoxetine dose-dependently reduces the neuroendocrine response to a 5-HT 1A agonist, suggesting a dose-dependent desensitization of hypothalamic postsynaptic 5-HT 1A receptors; 2) fluoxetine is more effective in reducing the hypothalamic levels of G z proteins, which couple 5-HT 1A receptors to the secretion of ACTH and oxytocin, than it is in reducing the levels of other G i and G o proteins; 3) fluoxetine does not induce a translocation of G proteins from the membranes into the cytosol; and 4) to our knowledge, this is the first in vivo study showing changes in G z protein levels in the brain. This study provides a systematic examination of fluoxetine s dose-response effect on postsynaptic 5-HT 1A receptor systems in the hypothalamus of rats. Although many studies Fig. 4. Dose-response effect of fluoxetine on the corticosterone response to 8-OH-DPAT (50 g/kg s.c.). A, experiment 1 using fluoxetine doses of 0.3, 1, 3, and 10 mg/kg/day; B, experiment 2 with fluoxetine doses of 5, 7.5, and 10 mg/kg/day. The data represent the mean S.E.M. of 8 to 12 rats per group. A, the two-way ANOVA indicated the following: main effect of fluoxetine F(4,67) 3.22, p 0.05; main effect of 8-OH-DPAT F(1,67) 51.60, p 0.01; fluoxetine X 8-OH-DPAT, F(4,67) 3.23, p B, the two-way ANOVA indicated the following: main effect of fluoxetine F(3,66) 10.23, p 0.01; main effect of 8-OH-DPAT F(1,66) 72.86, p 0.01; fluoxetine 8-OH-DPAT, F(3,66) 7.28, p The posthoc Newman- Keuls tests indicated the following: * significant difference from the rats challenged with saline (0 dose of 8-OH-DPAT), p 0.01; # significant difference from the vehicle rats (0 dose of fluoxetine) challenged with 8-OH-DPAT, p using fluoxetine in rats have used doses of 10 to 30 mg/kg/day (Nestler et al., 1990; Trouvin et al., 1993; Gardier et al., 1994), others have used lower doses of 2.5 to 5 mg/kg/day (Lesch and Manji, 1992; Nibuya et al., 1996). Because of this large variability in daily fluoxetine doses, there could be disparities in the effects of fluoxetine on serotonergic function. Our studies provide information on the minimum dose that will produce adaptive changes in postsynaptic 5-HT 1A receptors in rats. Consistent with previously reported results in rats (Li et al., 1993b, 1996), the present study demonstrates that repeated daily injections of a 10 mg/kg/day dose of fluoxetine produces a complete inhibition of the neuroendocrine responses to a submaximal dose of 8-OH-DPAT, suggesting a desensitization of hypothalamic 5-HT 1A receptors. Furthermore, the current findings indicate a dose-dependent effect of fluoxetine on the hormone responses to the 5-HT 1A agonist. The lowest dose of fluoxetine necessary to produce desensitization of hypothalamic postsynaptic 5-HT 1A receptors after 14 days of exposure is 5 mg/kg. This dose in rats was reported to produce a 77% inhibition of 5-HT uptake in vivo, whereas a fluoxetine dose of 3 mg/kg only produced a 27% inhibition of 5-HT uptake (Fuller et al., 1978). These observations suggest

6 HT 1A Receptors and Dose Response of Fluoxetine 103 TABLE 2 Comparison of degree of reduction of hormone responses to 8-OH-DPAT with reductions in the levels of G proteins in the hypothalamus in rats treated with various doses of fluoxetine Fluoxetine Dose (mg/kg/day, ip) Responses to 8-OH-DPAT (% of Control) a % of Control Group Oxytocin ACTH Corticosterone G z G i1 G i3 0 (Saline) a The hormone responses to 8-OH-DPAT (minus baseline levels) in rats treated with no fluoxetine (i.e., saline) were set as 100% and are for oxytocin, 35.6 pg/ml; for ACTH, pg/ml; and for corticosterone, 15.9 g/dl. Fig. 5. A D, examples of immunoblots of membrane-bound (left) and cytosolic (right) G proteins in the hypothalamus of rats treated with increasing doses (0, 5, 7.5, and 10 mg/kg/day, i.p.) of fluoxetine for 14 days. A, membrane-bound G i1 and G i2 proteins; B, cytosolic G i1 and G i2 proteins; C, membrane-bound G z proteins; D, cytosolic G z proteins; E, a preabsorption control, indicating that a G z blocking protein prevents the antibody from binding to the G z protein on the blot. that a high percentage of 5-HT uptake sites need to be blocked in order to produce desensitization of postsynaptic 5-HT 1A receptors. Furthermore, as inhibition of 5-HT uptake sites becomes more complete, the degree of 5-HT 1A receptor desensitization increases. As mentioned in the introduction, the doses necessary to achieve desensitization of 5-HT 1A receptors in rats are about 5- to 10-fold higher than the doses necessary in humans. The reason for this difference in dose effectiveness is not clear. It is possible that the difference in doses is caused by differences in pharmacokinetics of fluoxetine between rats and humans. In rats, the half-life after a 10 mg/kg dose of fluoxetine is 7.7 h for the parent drug and 15.8 h for its pharmacologically active metabolite, norfluoxetine (Caccia et al., 1990). In contrast, the half-life of fluoxetine in humans is 4 to 6 days, and of norfluoxetine is 4 to 16 days (DeVane, 1994). Because humans take fluoxetine (20 60 mg) daily, the accumulation of fluoxetine and norfluoxetine in their brain could be as high or even higher than it would be with higher doses in rats. Examination of plasma levels of fluoxetine 18 h after the last fluoxetine injection revealed no detectable levels of fluoxetine. This observation is consistent with data reported by Caccia et al. (1990). Fluoxetine treatment resulted in a dose-dependent increase in the levels of norfluoxetine in plasma. The levels of norfluoxetine in plasma correspond to the changes in body weight and the desensitization of hypothalamic 5-HT 1A receptor signaling. Thus, the data suggest that fluoxetine doses below 5 mg/kg/day do not produce a sufficient level of fluoxetine and norfluoxetine that would produce a desensitization of hypothalamic 5-HT 1A receptors. This study focused on signal transduction mechanisms as possible targets of SSRI action because repeated injections of fluoxetine and other SSRIs do not alter the density or the affinity of hypothalamic postsynaptic 5-HT 1A receptors (Hensler et al., 1991; Li et al., 1993b). A novel finding in the present study is the fluoxetine-induced reduction in the levels of membrane-associated G z protein at both the 7.5 and the 10 mg/kg/day doses. G z proteins are pertussis toxin insensitive members of the G i protein family that have a high affinity for 5-HT 1A receptors in vitro (Barr et al., 1997). Our studies using pertussis toxin and G z antisense oligodeoxynucleotides indicate that hypothalamic G z proteins mediate 5-HT 1A receptor-stimulated ACTH and oxytocin secretion (Serres et al., 1998; Van de Kar et al., 1998). Therefore, G z proteins might play a role in fluoxetine-induced desensitization of hypothalamic postsynaptic 5-HT 1A receptors. Consistent with our previous findings (Li et al., 1996), membrane-associated G i1 protein levels in the hypothalamus are also reduced after exposure to the 10 mg/kg/day dose of fluoxetine. This is a pertussis toxin-sensitive G protein that probably couples 5-HT 1A receptors in the raphe and hippocampus to hyperpolarization of neurons and possibly to other physiological responses (Clarke et al., 1987; Blier et al., 1993; Penington et al., 1993). Hence, the fluoxetine-induced reduction in G i1 proteins in the hypothalamus might be relevant to other physiological phenomena linked to 5-HT 1A receptor desensitization. In contrast with our previous observation (Li et al., 1996), fluoxetine did not produce a significant reduction in G i3 proteins. The inconsistency in effects of fluoxetine on the levels of G i3 proteins suggest that G i3 proteins are not significant to the desensitization of 5-HT 1A receptor systems in the hypothalamus. In the present study, levels of G i2 and G o proteins in the hypothalamus also were not altered after exposure to any dose of fluoxetine, further confirming the lack of involvement of G i2 or G o proteins in SSRI-induced desensitization of postsynaptic 5-HT 1A receptors in the hypothalamus (Li et al., 1996, 1997b). Several mechanisms could underlie the fluoxetine-induced reduction in the levels of G proteins in the hypothalamus. These mechanisms could include reduced synthesis or increased degradation of G proteins. An additional mechanism responsible for the apparent reduction in hypothalamic levels

7 104 Raap et al. Vol. 288 Fig. 6. Dose-response effect of fluoxetine on membrane-bound (left) and cytosolic (right) G proteins. From top to bottom, the G proteins are G z,g i1,g i3,g i2, and G o. The data represent the mean S.E.M. of 5 to 6 rats per group. For membrane G proteins, the one-way ANOVAs indicated the following: G z, F(3,19) 4.13, p 0.05; G i1, F(3,20) 5.40, p 0.01; G i3, F(3,32) 4.464, p 0.01; G i2, F(3,21) 2.21, p 0.10; G o, F(3,23) 0.40, p For cytosolic G proteins, the ANOVAs indicated the following: G z, F(3,19) 0.86, p 0.10; G i1, F(3,17) 2.36, p 0.10; G i3, F(3,18) 0.05, p 0.10; G i2, F(3,20) 3.06, p 0.05; G o, F(3,18) 0.63, p The post hoc Newman-Keuls tests indicated the following: *significant difference from the vehicle rats, p 0.05; **significant difference from the vehicle rats, p of G z and G i1 proteins could be their redistribution from the membrane to the cytosol as has been shown to occur for G z proteins in vitro (Hallak et al., 1994). However, cytosolic levels of the G proteins, including G i1 and G z proteins, were not altered after repeated exposure to any dose of fluoxetine, indicating that fluoxetine-induced reductions in membranebound proteins were not a result of redistribution to the cytosol. Table 2 indicates that no relationship exists between the decrease in G protein levels and reduced hormone responses to 8-OH-DPAT; the best correspondence occurs with the levels of G z proteins. One possible interpretation of these results is that, in addition to reductions in levels of G proteins, other mechanisms are also involved in the desensitization of hypothalamic 5-HT 1A receptors. Such a mechanism could include post-translational modifications of G z proteins that would reduce the coupling efficiency of 5-HT 1A receptors to their second messengers. These possibilities will be investigated in future studies. Table 3 shows that only G z protein levels were significantly correlated with basal plasma levels of ACTH and oxytocin in untreated rats. We do not believe, however, that G z proteins are the sole regulators of basal levels of ACTH and oxytocin because fluoxetine treatment reduced the levels of G z proteins without changing the basal ACTH and oxytocin levels. Furthermore, the resting plasmas levels of hormones normally are maintained by multiple neurotransmitter and feedback mechanisms. Therefore, one would not expect a single factor to correlate with basal levels of hormones.

8 HT 1A Receptors and Dose Response of Fluoxetine 105 TABLE 3 Correlation coefficients for plasma hormone levels versus hypothalamic G protein levels in saline treated/saline challenged control rats G Protein Oxytocin ACTH Corticosterone G z 0.84* 0.90* 0.66 G i G i G i G o * P.05. An alternative explanation for the incongruity between the degree of reduction in G z proteins and hormone responses to 8-OH-DPAT is that the methodology used for quantifying G protein levels is not sensitive enough to detect small changes between groups. The immunoblot technique currently used by most researchers can detect gross changes but may not be sensitive enough to detect smaller changes. Because the maximum reduction in G protein levels after the highest dose of 10 mg/kg fluoxetine is only about a 35% decline, methodological issues would impede detecting small dose-dependent changes if there were indeed some. Therefore, the possibility that changes in G protein levels may be involved with functional desensitization of 5-HT 1A receptors cannot be excluded. The importance of the results on change in body weight should be emphasized in light of the fact that most other classes of antidepressant drugs increase body weight, whereas SSRIs reduce body weight, or at least do not alter body weight substantially (Connolly et al., 1995; Greeno and Wing, 1996). The current results also support findings in rodents that weight gain is inhibited after chronic exposure to fluoxetine (Dryden et al., 1996; Halford and Blundell, 1996) and further indicate that this inhibition is dose-dependent. It could be argued that the inhibition in weight gain may contribute to the decline in neuroendocrine responses to the 5-HT 1A agonist 8-OH-DPAT. However, chronic exposure to fluoxetine produces a heightened hormone responses to the 5-HT 2C agonist MK-212 and the 5-HT 2A/2C agonist ( )-1-(2,5-Dimethoxy-4-lodophenyl)-2-aminopropane HCl (DOI) (Li et al., 1993a). Hence, chronic exposure to fluoxetine does not produces a generalized reduction in neuroendocrine systems. In conclusion, the results of the present study indicate that fluoxetine-induced desensitization of hypothalamic postsynaptic 5-HT 1A receptors is dose-dependent, suggesting that the degree of 5-HT uptake blockade determines the degree of desensitization of postsynaptic 5-HT 1A receptors. Furthermore, changes in hypothalamic levels of G z proteins (that couple 5-HT 1A receptors to the secretion of ACTH and oxytocin) may play a role in this desensitization. Altered synthesis or degradation of G z proteins likely underlies the reduced levels of membrane associated G z protein because G z proteins were not redistributed to the cytosol. 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