Introduction

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Fenfluramine [N-ethyl-a-methyl-m-(trifluoromethyl)-phenethylamine], a ring-substituted amphetamine derivative, is a clinically prescribed appetite suppressant (see Rowland and Carlton, 1986; Derome-Tremblay and Nathan, 1989). As with a number of other amphetamine analogs, e.g., methamphetamine, MDMA, p-chloroamphetamine, fenfluramine has toxic potential toward brain 5-HT neurons in animals (Harvey and McMaster, 1975; Sanders-Bush et al., 1975; Harvey et al., 1977; Clineschmidt et al., 1978; Schuster et al., 1986; Kleven et al., 1988; Kleven and Seiden, 1989; Appel et al., 1990; Johnson and Nichols,1990; Molliver and Molliver, 1990; Zaczek et al., 1990; Caccia et al., 1993; Westphalen and Dodd, 1995). Although the mechanisms underlying the neurotoxicity of fenfluramine and structurally related drugs (e.g., p-chloroamphetamine, MDMA) are not known, they appear to involve an interaction with the 5-HT reuptake site, because treatment with 5-HT reuptake blockers has been shown to prevent the 5-HT neurotoxic effect of these drugs (Fuller and Molloy, 1974; Harvey et al., 1977; Clineschmidt et al., 1978; Schmidt, 1987; Schmidt et al., 1987; Hekmatpanah and Peroutka, 1990).

Reports from individuals who use MDMA, an amphetamine analog that is chemically related to fenfluramine, indicate the subjective effects of MDMA are not substantially altered by concomitant ingestion of the 5-HT reuptake inhibitor fluoxetine (McCann and Ricaurte, 1993). Since, in addition to blocking 5-HT reuptake (see Fuller, 1992), SSRIs such as fluoxetine prevent MDMA-induced 5-HT release (Fitzgerald and Reid, 1990; Schmidt et al., 1987), these reports suggest that MDMA's subjective effects may not depend on 5-HT release, and that MDMA's subjective and neurotoxic effects may be separable. Because direct measures of 5-HT neurotoxicity are not yet feasible in humans, and because the unique subjective effects of MDMA are not readily quantified, an alternative paradigm was needed to explore the possibility that the 5-HT neurotoxic effects of amphetamine analogs could be distinguished from their pharmacological effects. To this end, an initial study was conducted in rats, using fenfluramine, a neurotoxic amphetamine analog with easily measured behavioral and neurotoxic effects (appetite suppression with consequent weight loss, and prolonged reductions in 5-HT axon terminal markers, respectively). Results from that initial study indicated that fluoxetine (5 mg/kg) prevented the neurotoxic effects of fenfluramine (5 mg/kg) without blocking fenfluramine-induced appetite suppression (McCann et al., 1995).

While it is generally accepted that fenfluramine's anorectic effects are indirect and mediated by 5-HT release (Fuxe et al., 1975; Garattini et al., 1975; Trulson and Jacobs, 1976; Garattini et al., 1979; for review see Rowland and Carlton, 1986), some evidence supporting a direct action on 5-HT receptors has been presented (Gobbi et al., 1993). Moreover, using in vivo microdialysis, Raiteri and colleagues (1995) have recently demonstrated that doses of fluoxetine that prevent fenfluramine-induced 5-HT release do not prevent fenfluramine-induced anorexia. These findings, coupled with the recent observation that fenfluramine's anorectic and 5-HT neurotoxic effects are separable (McCann et al., 1995), prompted our series of studies that sought: 1) To determine if the neuroprotective effects of fluoxetine were dose-related; 2) To assess if findings with fluoxetine generalized to other SSRIs; 3) To determine if findings in rats generalized to another species (mice) and 4) To examine the possibility that the anorectic effect of the fenfluramine/fluoxetine combination was secondary to nonspecific behavioral suppression. As indicated above, parts of this research (portions of experiment 1) have been reported in a rapid communication format (McCann et al., 1995).

	Materials and Methods

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Subjects. Male Sprague-Dawley rats (Harlan, Madison, WI) weighing 200 to 225 g and male Swiss-Webster mice (Taconic Farms, Germantown, NY) weighing 35 to 40 g at the beginning of the study were used throughout. Rats were housed individually in suspended wire-mesh cages in a temperature controlled room (22 ± 1^OC) on a 12:12 hr light/dark cycle (light from 6 A.M. to 6 P.M.), with free access to food PMI Feeds Inc. (St. Louis, MO) and water. Mice were housed under similar environmental conditions in acrylic cages, also with free access to food and water. The facility for housing and care of the animals is accredited by the American Association for the Accreditation of Laboratory Animal Care and all studies were performed in accordance with accepted standards.

Drug treatment. Animals received drugs (or vehicle) orogastrically by means of gavage twice daily (0900 and 1700 h) for 4-6 consecutive days. In each of the five experiments described, treatment duration was the same for all groups. For all experiments (except the immunocytochemical studies, where the fluoxetine alone group was excluded), there were four treatment groups: 1) Fenfluramine alone; 2) SSRI alone; 3) Fenfluramine plus SSRI or; 4) Equal volumes of the vehicle. In experiment 1, the 5-mg/kg dose of fenfluramine was selected on the basis that it is known to produce both anorectic and neurotoxic effects in the rat (Rowland and Carlton, 1986); the fluoxetine dose selection was based on the fact that a 5-mg/kg dose of fluoxetine was likely to produce neuroprotective effects (Sabol et al., 1992), but not produce significant anorectic effects, making it easier to determine whether reduced food intake in fenfluramine/fluoxetine-treated rats was related to preservation of fenfluramine's anorectic effect or to an anorectic effect of fluoxetine. In experiment 2, which sought to explore the importance of relative dosages of fenfluramine and fluoxetine required to observe neuroprotection, dose selection was based on the results of experiment 1, and on previous results showing that rats could tolerate 4-fold higher doses of fenfluramine or fluoxetine. In experiment 3, doses of the various SSRIs were selected on the basis of in vivo results demonstrating that 5-mg/kg doses of the various SSRIs effectively blocked binding to the 5-HT transporter (Scheffel et al., 1994). In experiment 5, doses were selected on the basis of previous findings in mice (McCann et al., 1994). All drugs were dissolved in distilled water and administered on an mg/kg basis. Control animals received an equivalent volume of saline. Drugs were administered sequentially. Dose was expressed as the salt. Animals were tested in groups of five to nine in all studies except in the binding and immunocytochemical studies, where three animals per group were used.

Food and water intake measurements. Food intake was measured by weighing the food on a daily basis. The difference in weight on sequential days was taken as an index of food intake. Water intake was only measured in experiment 5, also on a daily basis. An electronic scale (Mettler-Toledo PR5002DR, Greifensee, Switzerland) with a 0.1 g accuracy was used for both food and water measurements.

Weight determinations. Animals were weighed daily (0900 hr) before, during and for 2 wk after drug treatment, also using an electronic scale with a 0.1 g accuracy.

Monoamine and metabolite determinations. Two weeks after the last drug treatment, the effects of various treatments on regional brain content of 5-HT and 5-HIAA were determined as previously described (Ricaurte et al., 1992). Briefly, frozen tissue samples were weighed, placed in 10 to 15 volumes (wt/vol) 0.4 N perchloric acid and homogenized using a Brinkman (Westbury, NY) Polytron Homogenizer (setting of 5 for 12 sec). The homogenates were then centrifuged for 20 minutes at 25,000 × g in a refrigerated Sorval RC2B Newton, CT centrifuge at 0 to 4°C. The supernatant was removed and 0.3-ml aliquots were placed in polypropylene tubes, which were then stored in liquid nitrogen until assay. Monoamines and their metabolites were separated using a reverse phase C-18 column from Brownlee Applied Systems (Norwalk, CT) using a mobile phase that was 100% aqueous and contained citric acid (125 mM), sodium phosphate (125 mM), EDTA (0.27 mM), sodium octyl sulfate (0.12 mM) and had a pH of 3.0-3.5. The flow rate was 1 ml/min. The column was housed at 40°C in a Shimadzu (Columbia, MD) CTO-6A column oven. A Shimadzu amperometric L-ECD-6A detector containing a glassy carbon working electrode and a silver/silver chloride reference electrode with a potential difference of 0.70 V was used. Shimadzu Chromatopacs C-R 4A and 7A were used to quantify the electrochemical response by measuring the area under a given curve and comparing it to that of a standard processed identically.

5-HT transporter measurements. [³H] Paroxetine was used to label and quantify 5-HT transporters as described previously (Ricaurte et al., 1992). Briefly, the tissue was placed in 50 volumes of ice cold 50 mM Tris-HCl buffer containing 120 mM NaCl, 5 mM KCl, (pH 7.4) and homogenized with a Brinkman Polytron (setting of 5 for 30 sec). The homogenate was centrifuged in a Sorvall RC2B centrifuge at 42,400 × g with a subsequent resuspension and wash of the pellet. The resulting pellet was resuspended in the same buffer at a final concentration of 6 mg/ml. Tissue aliquots (0.5 ml) were then placed in test tubes containing 4.5 ml buffer and [³H] paroxetine at 1 of 4 concentrations: 0.24, 0.12, 0.03 or 0.015 nM. Of six tubes used for each concentration, half contained citalopram (1 M) to determine nonspecific binding. Tubes were incubated (22-24°C; 60 min) and membranes were harvested by filtration through Whatman (Maidstone England) GF/B filters previously soaked in 0.05% polyethylenimine. Filters were collected and their radioactivity was measured using a Packard (Downers Grove, IL) 1500 scintillation counter. Kinetic constants were computed with the aid of the computer program entitled "EBDA" (GraphPAD Software, San Diego, CA).

Immunocytochemical studies. Morphological studies of 5-HT-containing axons were performed using a rabbit antiserum directed at 5-HT as detailed elsewhere (Fischer et al., 1995). Briefly, 1 to 2 hr before death, animals (n = 3 for each treatment group) received injections with 10 mg/kg of the monoamine oxidase inhibitor trans-2-phenylcyclopropylamine (i.p.). Under deep chloral hydrate anesthesia (400 mg/kg, i.p.), the animals were then perfused by an intracardiac route. After an initial rinse with ice-cold phosphate-buffered saline, perfusion was continued using 4% paraformaldehyde and 0.1% glutaraldehyde (pH 7.4). Tissue blocks were placed in buffered 4% paraformaldehyde for 4 to 6 hr and then in 10% dimethylsulfoxide in PBS overnight. Frozen sections (30 micrometer) were incubated in an anti-5-HT antisera diluted 1:14,000 in phosphate buffered saline with 0.2% Triton X-100 and 1% normal serum at 4°C for 3 days. The antibody was visualized with the Vectastain ABC-peroxidase method (Vector Laboratories, Inc., Burlingame, CA), and staining was enhanced with the osmiophilic reaction sequence of Gerfen (1985).

Data analysis. Food intake, body weight and water intake data were analyzed by two-way ANOVA for repeated measures, with treatment as the between-subjects factor and time as the within-subjects factor. When appropriate, group means at individual time points were compared by a one-way ANOVA, and post hoc comparisons were performed by the Duncan's multiple range test. Regional brain 5-HT data were analyzed by one-way ANOVA, followed by Duncan's multiple range post hoc comparisons. Results were considered significant when P < .05 using a two-tailed test. Data analysis was done using the Statistical Program for the Social Sciences (SPSS for Windows, Release 6).

Drugs and chemicals. Fenfluramine hydrochloride, fluoxetine hydrochloride, serotonin creatinine sulfate, 5-hydroxyindoleacetic acid and trans-2-phenylcyclopropylamine were purchased from the Sigma Chemical Co., St Louis, MO. The antibody directed at 5-HT was purchased from Incstar Corp., Stillwater, MN. [³H]Paroxetine was purchased from New England Nuclear, Boston, MA. Citalopram hydrobromide was obtained from H. Lundbeck, Copenhagen, Denmark. Paroxetine hydrochloride was kindly provided by SmithKline Pharmaceuticals, Worthing, England. Sertraline was obtained from Pfizer Pharmaceuticals, Groton, CT.

	Results

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Experiment 1: Fenfluramine 5 mg/kg and Fluoxetine 5 mg/kg

Food intake. As previously reported in a rapid communication format (McCann et al., 1995), repeated measures ANOVA revealed a significant main effect of time (F_19,570 = 43.5, P < .001) and a significant group x time interaction (F_57,570 = 7.53, P < .001), with post hoc analyses showing that treatment with fenfluramine (5 mg/kg), as well as treatment with fenfluramine (5 mg/kg) plus fluoxetine (5 mg/kg), produced significant decreases in food intake early in the course of treatment (days 1 and 2) (see fig. 1A in McCann et al., 1995). Food intake returned to control levels by day 5 of drug treatment in both rats treated with fenfluramine alone and in rats treated with fenfluramine plus fluoxetine. After drugs were discontinued on day 6, all drug-treated groups exhibited transient (1-7 day) increases in food intake which returned to control levels by day 15 (fig.1A, McCann et al., 1995).

View larger version (168K): [in this window] [in a new window]   Fig. 1.   Photomicrograph of serotonin-immunoreactive axons in the frontal cortex (top panels) and hippocampus (bottom panels) of a control rat (A and D), a rat treated with fenfluramine 2 wk previously (B and E), and a rat treated with a combination of fenfluramine + fluoxetine 2 wk previously (C and F). Drugs were administered orogastrically by gavage at a dose of 10 mg/kg twice daily for 4 consecutive days. The axon density is noticeably reduced after fenfluramine treatment, but there is no apparent effect after treatment with the combination of fenfluramine + fluoxetine. Data shown are the results from one representative animal in each group of three. Bar scale represents 100 µm.

Body weight. As with food intake, a main effect of time (F_20,600 = 908.7, P < .001) and a group x time interaction (F_60,600 = 2.89, P < .01) were observed, with post hoc tests revealing significant decreases in body weight in rats treated with fenfluramine or fenfluramine plus fluoxetine beginning on day 2 of drug treatment (see fig.1B, McCann et al., 1995). Animals treated with the combination of fenfluramine plus fluoxetine had a more sustained weight loss than those treated with fenfluramine alone, although neither group maintained weight loss beyond 3 days after drug discontinuation.

Regional brain markers of 5-HT axons. ANOVA revealed a significant effects of treatment on 5-HT and 5-HIAA in the hippocampus (F_3,30 = 38.9, P < .001; F_3,30 = 94.1, P < .001), hypothalamus (F_3,30 = 34.6, P < .001; F_3,30 = 77.0, P < .001), striatum (F_3,30 = 28.8, P < .001; F_3,30 = 54.8, P < .001) and cerebral cortex (F_3,30 = 3.4, P < .028; F_3,30 = 87.3, P < .001). Post hoc tests showed that animals treated with fenfluramine alone had significant decreases in regional brain 5-HT (fig.1C, McCann et al., 1995) and 5-HIAA (not shown) 2 wk after drug discontinuation, although animals treated with the combination of fenfluramine plus fluoxetine had regional brain 5-HT and 5-HIAA levels that were not different than those in controls or in animals treated with fluoxetine alone.

Experiment 2: Influence of Dose

Fenfluramine 20 mg/kg and fluoxetine 5 mg/kg. Food intake. Repeated measures ANOVA revealed main effects of group (F_3,20 = 4.96, P = .01), time (F_15,300 = 94.3, P < .001) and a group x time interaction (F_45,300 = 20.9, P < .001). Post hoc testing revealed that rats treated with fenfluramine alone (20 mg/kg) or fenfluramine (20 mg/kg) plus fluoxetine (5 mg/kg) had significant decreases in food intake (fig. 2A). As before, animals treated with fluoxetine alone had similar food intake to controls. Animals treated with 20 mg/kg of fenfluramine had greater reductions in food intake than previously observed in rats treated with 5 mg/kg of fenfluramine (compare fig. 2A with fig. 1A in McCann et al., 1995).

View larger version (41K): [in this window] [in a new window]   Fig. 2.   A to D depict the comparative effects of treatment with an increased dose of fenfluramine (FEN) (20 mg/kg), with the serotonin uptake inhibitor fluoxetine (FLU) (5 mg/kg), or with a combination of fenfluramine (20 mg/kg) + fluoxetine (5 mg/kg), on rat food intake (A), body weight (B), regional brain 5-HT levels (C) and regional brain 5-HIAA levels (D). Drugs were administered orogastrically by gavage twice daily for 4 consecutive days (as indicated by the bar overlying the abscissa). Results are the means ± S.E.M. (n = 5-9 rats/group). 1 Designates significant difference from control group; 2 designates significant difference from FLU group; 3 designates significant difference from FEN group; 4 designates significant difference from FEN/FLU group. Significance level was set at P < .05. Note incomplete protection when dose of fenfluramine is increased to 20 mg/kg but dose of fluoxetine is maintained at 5 mg/kg (compare with fig. 1, McCann et al., 1995).

Fenfluramine 20 mg/kg and fluoxetine 20 mg/kg. Food intake. Repeated measures ANOVA revealed significant main effects of group (F_3,28 = 5.35, P = .005) and time (F_12,336 = 194.5, P < .001) and a group x time interaction (F_36,336 = 29.4, P < .001). As before, post hoc testing revealed significant decreases in food intake in animals receiving fenfluramine alone or fenfluramine plus fluoxetine (fig. 3A), with the most dramatic decreases occurring early in the course of treatment and gradually dissipating during the course of treatment and after drug discontinuation. In contrast to the lower dose of fluoxetine, fluoxetine at a dose of 20 mg/kg led to significant decreases in food intake, although these decreases were less than those seen in animals treated with fenfluramine alone or fenfluramine plus fluoxetine (fig. 3A). After drugs were discontinued on day four, all drug-treated groups exhibited transient (1-9 day) increases in food intake which returned to control levels by day 14. 

View larger version (44K): [in this window] [in a new window]   Fig. 3.   A to D depict the comparative effects of treatment with a higher dose of fenfluramine (FLU) (20 mg/kg), with a higher dose of fluoxetine (FEN) (20 mg/kg) or with a combination of fenfluramine (20 mg/kg) + fluoxetine (20 mg/kg), on rat food intake (A), body weight (B), regional brain 5-HT levels (C) and regional brain 5-HIAA levels (D). Drugs were administered orogastrically by gavage twice daily for 4 consecutive days (as indicated by the bar overlying the abscissa). Results are the means ± S.E.M. (n = 5-9 rats/group). 1 Designates significant difference from control group; 2 designates significant difference from FLU group; 3 designates significant difference from FEN group; 4 designates significant difference from FEN/FLU group. Significance level was set at P < .05. Note complete protection when dose of fluoxetine is also increased.

Experiment 3: Effects of other SSRIs (Citalopram, Paroxetine and Sertraline)

Food intake. Repeated measures ANOVA revealed significant main effects of group, time and a group x time interaction for all SSRIs (citalopram, paroxetine and sertraline) tested (see table 1 for F values). Post hoc tests indicated that these effects reflected similar phenomena as were observed in studies with fluoxetine. In particular, regardless of the SSRI, animals treated with fenfluramine or fenfluramine plus an SSRI had significant decreases in food intake during the course of treatment. In contrast, treatment with an SSRI alone did not lead to significant decreases in food intake at the doses of SSRIs tested.

Body weight. As with food intake, repeated measures ANOVA revealed significant main effects of group, time and a group x time interaction for all SSRIs tested (citalopram, paroxetine and sertraline) (see table 1 for F values). Similar to the results obtained with fenfluramine and fluoxetine, rats treated with fenfluramine alone or fenfluramine plus an SSRI developed weight loss that was most prominent during the early days of drug treatment, and that dissipated during the course of treatment and after drug discontinuation. Animals treated with an SSRI alone did not differ from controls on measures of body weight.

Regional brain markers of 5-HT axons. For all three SSRIs tested (citalopram, paroxetine and sertraline), ANOVA showed a significant effect of treatment on 5-HT and 5-HIAA in the hippocampus (F_7,32 = 7.39, P < .001; F_7,32 = 16.7, P < .001), hypothalamus (F_7,32 = 5.5, P < .001; F_7,32 = 11.4, P < .001), striatum (F_7,32 = 15.4, P < .001; F_7,32 = 9.1, P < .001) and cerebral cortex (F_7,32 = 4.4, P < .002; F_7,32 = 21.7, P < .001) (see figs. 4, 5, 6). In all three studies, fenfluramine alone led to significant decreases in 5-HT and 5-HIAA concentrations in every brain region examined (figs. 4, 5, 6, C and D). Regardless of the SSRI, rats treated with fenfluramine plus an SSRI had no decreases in brain 5-HT in any brain region examined. Similarly, rats treated with an SSRI alone had no decrements in brain 5-HT, when compared to rats treated with vehicle (figs. 4, 5, 6, C and D).

View larger version (40K): [in this window] [in a new window]   Fig. 4.   A to D depict the comparative effects of treatment with fenfluramine (FEN) (5 mg/kg), with the serotonin uptake inhibitor citalopram (CIT) (5 mg/kg) or with a combination of fenfluramine (5 mg/kg) + citalopram (5 mg/kg), on rat food intake (A), body weight (B), regional brain 5-HT levels (C) and regional brain 5-HIAA levels (D). Drugs were administered orogastrically by gavage twice daily for 5 consecutive days (as indicated by the bar overlying the abscissa). Results are the means ± S..E.M (n = 5-9 rats/group). 1 Designates significant difference from control group; 2 designates significant difference from CIT group; 3 designates significant difference from FEN group; 4 designates significant difference from FEN/CIT group. Significance level was set at P < .05.

View larger version (41K): [in this window] [in a new window]   Fig. 5.   A to D depict the comparative effects of treatment with fenfluramine (FEN) (5 mg/kg), with the serotonin uptake inhibitor paroxetine (PAROX) (5 mg/kg) or with a combination of fenfluramine (5 mg/kg) + paroxetine (5 mg/kg), on rat food intake (A), body weight (B), regional brain serotonin levels (C) and regional brain 5-HIAA levels (D). Drugs were administered orogastrically by gavage twice daily for 5 consecutive days (as indicated by the bar overlying the abscissa). Results are the means ± S.E.M (n = 5-9 rats/group). 1 Designates significant difference from control group; 2 designates significant difference from PAROX group; 3 designates significant difference from FEN group; 4 designates significant difference from FEN/PAROX group. Significance level was set at P < .05.

View larger version (39K): [in this window] [in a new window]   Fig. 6.   A to D depict the comparative effects of treatment with fenfluramine (FEN) (5 mg/kg), with the serotonin uptake inhibitor sertraline (SERT) (5 mg/kg) or with a combination of fenfluramine (5 mg/kg) + sertraline (5 mg/kg), on rat food intake (A), body weight (B), regional brain serotonin levels (C) and regional brain 5-HIAA levels (D). Drugs were administered orogastrically by gavage twice daily for 5 consecutive days (as indicated by the bar overlying the abscissa). Results are the means ± S.E.M (n = 5-9 rats/group). 1 Designates significant difference from control group; 2 designates significant difference from SERT group; 3 designates significant difference from FEN group; 4 designates significant difference from FEN/SERT group. Significance level was set at P < .05.

Experiment 4: Species GeneralityStudies in Mice

Food intake. Repeated measures ANOVA revealed a main effect of time (F_14,392 = 17.7, P < .001). Post hoc tests indicated that treatment with fenfluramine alone or treatment with fenfluramine plus fluoxetine led to decreases in food intake early in the course of drug treatment but that only the latter achieved statistical significance (fig. 7A).

View larger version (40K): [in this window] [in a new window]   Fig. 7.   A to D depict the comparative effects of treatment with fenfluramine (FEN) (10 mg/kg), with fluoxetine (FLU) (10 mg/kg) or with a combination of fenfluramine (10 mg/kg) + fluoxetine (10 mg/kg), on food intake (A), body weight (B), regional brain serotonin levels (C) and 5-HIAA levels (D) in mice. Drugs were administered orogastrically by gavage twice daily for 4 consecutive days (as indicated by the bar overlying the abscissa). Results are the means ± S.E.M. (n = 6 mice/group). 1 Designates significant difference from control group; 2 designates significant difference from FLU group; 3 designates significant difference from FEN group; 4 designates significant difference from FEN/FLU group. Significance level was set at P < .05.

Body weight. As with food intake, a main effect of time (F_14,392 = 19.4, P < .001) was observed. Post hoc tests indicated that there were no significant differences in body weight among the four treatment groups (fig. 7B)

Regional brain markers of 5-HT axons. Analysis showed a significant treatment effects on 5-HT and 5-HIAA in the hippocampus (F_3,28 = 6.1, P < .002; F_3,28 = 9.4, P < .001), hypothalamus (F_3,28 = 4.1, P < .016; F_3,28 = 2.9.0, P < .047), striatum (F_3,28 = 6.0, P < .003; F_3,30 = 5.6, P < .004) and cerebral cortex (F_3,28 = 8.1, P < .001; F_3,28 = 5.8, P < .003). Post hoc analyses indicated that mice treated with fenfluramine alone had significant decreases in 5-HT and 5-HIAA concentrations 2 wk after drug discontinuation in all brain regions examined. Mice treated with fenfluramine plus fluoxetine or fluoxetine alone did not differ from vehicle-treated controls on either measure (fig. 7, C and D).

Experiment 5: Evaluation of Possible Nonspecific Behavioral Suppression

Food intake. Repeated measures ANOVA revealed a significant main effect of time (F_13,260 = 33.7, P < .001) and a group x time interaction (F_39,260 = 9.0, P < .001). As before, animals treated with fenfluramine alone or fenfluramine plus fluoxetine had significant decreases in food intake that were most prominent early in the course of treatment, whereas animals treated with fluoxetine alone were no different than controls (fig. 8A).

View larger version (27K): [in this window] [in a new window]   Fig. 8.   A to C depict the comparative effects of treatment with fenfluramine (FEN) (5 mg/kg), with fluoxetine (FLU) (5 mg/kg) or with a combination of fenfluramine (5 mg/kg) + fluoxetine (5 mg/kg), on rat food intake (A), water intake (B), cortical 5-HT and 5-HIAA levels (C). Drugs were administered orogastrically by gavage twice daily for 4 consecutive days (as indicated by the bar overlying the abscissa). Results are the means ± S.E.M. (n = 5-9 rats/group). 1 Designates significant difference from control group; 2 designates significant difference from FLU group; 3 designates significant difference from FEN group; 4 designates significant difference from FEN/FLU group. Significance level was set at P < .05.

Water intake. As with food intake, a main effect of time (F_13,260 = 10.6, P = .001) and a group x time interaction (F_39,260 = 3.3, P = .001) were observed. However, in contrast to what was observed for food intake, animals treated with fenfluramine alone or fluoxetine plus fenfluramine increased their water intake relative to the other two treatment groups (fig. 8B). Further, animals in the fenfluramine plus fluoxetine group had greater increases in water intake than those in the fenfluramine alone group. As was observed with decreases in food intake, increases in water intake did not persist beyond the period of drug treatment.

Regional brain markers of 5-HT axons. As before, ANOVA revealed main effects of treatment on brain 5-HIAA and 5-HIAA concentrations in the cerebral cortex (F_3,20 = 12.1, P < .0001; F_3,20 = 22.6, P < 000). Post hoc analyses indicated that rats treated with fenfluramine alone had significant decreases in cortical 5-HT and 5-HIAA content 2 wk after drug discontinuation (fig. 8C).

	Discussion

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Our results provide further evidence that the anorectic and neurotoxic effects of fenfluramine are separable. In particular, doses of 5-HT reuptake inhibitors that prevent fenfluramine-induced 5-HT neurotoxicity do not prevent fenfluramine-induced hypophagia or weight loss. These results, when considered with data demonstrating that 5-HT reuptake inhibitors prevent fenfluramine-induced 5-HT release (Maura et al., 1982; Kreiss and Lucki, 1993; Sabol et al., 1992; Raiteri et al., 1995), lend additional support to the view that the appetite suppressant effect of fenfluramine may not be solely mediated by 5-HT release.

Our studies confirm previous findings using fluoxetine in rats (McCann et al., 1995), and extend them to other SSRIs (citalopram, paroxetine, sertraline). Further, the observation in mice that fluoxetine prevents fenfluramine-induced 5-HT neurotoxicity without affecting fenfluramine-induced anorexia extends previous findings to another animal species. If, as proposed by some (Kalia et al., 1992), the mouse provides the best model for predicting fenfluramine's effects in humans based on similar metabolite ratios in the two species (Baumgarten et al., 1992), our results suggest that separation of fenfluramine's anorectic and neurotoxic actions may also be feasible in humans. Of course, this assumes that humans, like every other species examined to date (see Schuster et al., 1986), develop fenfluramine-induced 5-HT neurotoxicity, an assumption that awaits empiric validation.

Although the ability for SSRIs to prevent fenfluramine-induced neurotoxicity is clear, based on our findings, this ability is a dose-related phenomenon. In particular, if the SSRI/fenfluramine ratio is not sufficiently high, blockade of fenfluramine-induced 5-HT neurotoxicity is not complete. However, even when fenfluramine is given at doses as high as 20 mg/kg (a dose at least 4-fold higher than that necessary for fenfluramine's acute anorectic effects in rats), fluoxetine can completely prevent fenfluramine-induced 5-HT neurotoxicity, as long as it is given at sufficiently high doses (20 mg/kg). This suggests that the neuroprotective effect of fluoxetine is related to an interaction between fluoxetine and fenfluramine at the level of the 5-HT transporter, an interaction that appears to strongly influence the 5-HT releasing effect of fenfluramine in rat (Sabol et al., 1992; Raiteri et al., 1995) as well as human brain (Bonanno et al., 1994).

Of note is the fact that, rather than preventing fenfluramine-induced appetite suppression, 5-HT reuptake inhibitors might actually enhance fenfluramine's anorectic effects. This phenomenon is observed even at doses of SSRI that are not anorectic themselves (e.g., 5 mg/kg fluoxetine) (fig. 1), although, at higher doses (i.e., 20 mg/kg), fluoxetine alone led to decreases in food intake and body weight (fig 5). It is also of note that the appetite suppressant effect of the fenfluramine/fluoxetine combination is not secondary to nonspecific behavioral suppressant effects, because water intake in the same animals increased, rather than decreased (fig. 8).

As has been previously reported, the appetite suppressant effects of fenfluramine do not persist for substantial time periods beyond the period of drug administration. Concomitant administration of an SSRI did not prolong fenfluramine's anorectic properties substantially. This indicates, as has been shown previously (see Rowland and Carlton, 1986), that fenfluramine's anorectic effects are not a direct consequence of 5-HT depletion, because animals with fenfluramine-induced 5-HT damage and prolonged 5-HT depletion have similar levels of food intake as control animals.

As in previous studies (see Introduction), fenfluramine-induced neurotoxicity was documented using both neurochemical and anatomical methods. Data collected using regional brain 5-HT and 5-HIAA as indexes of 5-HT axon terminal injury had reasonable correspondence with a structural index of terminal damage (i.e., loss of 5-HT transporter sites). Further, 5-HT neurochemical deficits were associated with a reduced density of 5-HT-immunoreactive axons, and both the neurochemical and immunocytochemical deficits induced by fenfluramine were prevented by fluoxetine. As before, all measures of 5-HT neurotoxicity were collected at least 2 wk after the last drug treatment, to eliminate the possibility that changes in 5-HT measures might be secondary to lingering pharmacological effects of drug.

The finding that SSRIs do not the attenuate the anorectic effect of fenfluramine (and, indeed, may actually enhance this effect while protecting against fenfluramine-induced 5-HT neurotoxicity) may have implications for humans. Specifically, this finding suggests that humans treated with an appropriate combination of fenfluramine and an SSRI might benefit from the desired anorectic effects of fenfluramine without exposing themselves to the risk of fenfluramine-induced 5-HT neurotoxicity. Raiteri (1995) has also suggested that the combination of fenfluramine and SSRI antidepressants might be desirable, particularly in obese patients suffering from depression or as a means to reduce untoward secondary effects secondary to a excesses release of serotonin. Interestingly, manufacturers of fenfluramine and dexfenfluramine contraindicate the concomitant use of SSRIs and dexfenfluramine because of the possible development of the serotonin syndrome. However, because SSRIs prevent serotonin release (Maura et al., 1982; Kreiss and Lucki, 1993; Sabol et al., 1992; Raiteri et al., 1995), the combination might actually reduce, rather than increase, the likelihood of the serotonin syndrome. In any case, it would be premature to generalize results from our studies to humans. Because our studies were conducted in rodents using dosage regimens different than those used clinically, it is not known whether the combination of fenfluramine and SSRIs would be safe or efficacious in humans.

In conclusion, our studies indicate that in rats and mice, fenfluramine-induced anorexia and fenfluramine-induced 5-HT neurotoxicity are separable. Rats treated with fenfluramine plus an SSRI (fluoxetine, paroxetine, sertraline or citalopram) exhibit significant decreases in food intake, yet do not have evidence of 5-HT axonal damage. Decreases in food intake seen in animals treated with fenfluramine plus an SSRI are not secondary to nonspecific behavioral suppressant effects of the drug combination, because another related behavior (e.g., water intake) was increased rather than decreased in the same animals. Finally, because SSRIs are known to prevent 5-HT release in addition to 5-HT reuptake, these findings add to growing evidence that fenfluramine-induced anorexia and fenfluramine-induced 5-HT release may not be as closely related as has heretofore been suspected.