Introduction

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Existing weight-reducing drugs apparently produce their effects by releasing brain dopamine (e.g., amphetamine; Garattini et al., 1978) or serotonin (e.g., d-fen; Garattini et al., 1987; Samanin et al., 1972) into synapses and blocking the reuptake of these monoamines. Amphetamine has been shown to release DA directly from rat brain synaptosomes (McKenna et al., 1991; Marona-Lewicka et al., 1995; Pettersson, 1995), and to increase DA levels in striatal perfusates, as measured by in vivo microdialysis (Westerink et al., 1987, 1989; Kametani et al., 1995). In contrast, d-fen releases 5-HT but not DA from synaptosomes (Garattini et al., 1992; Gobbi et al., 1993) and increases 5-HT levels in dialysates from cortex, hypothalamus and nucleus accumbens (Laferrere and Wurtman, 1989; Sarkissian et al., 1990; Gardier and Wurtman, 1991; Gardier et al., 1992). The principal metabolite of d-fen, dexnorfenfluramine, also directly activates 5-HT₂ receptors (Spedding et al., 1996).

The behavioral actions of the dopaminergic and serotoninergic weight-reducing drugs differ in that amphetamine suppresses appetite but d-fen enhances satiety. Moreover, amphetamine nonselectively suppresses consumption of all of the macronutrients, whereas d-fen selectively inhibits the overconsumption of carbohydrates (Thurlby et al., 1983; Carruba et al., 1985; Wurtman, 1995).

Very high doses of d-fen can be shown to produce prolonged decreases in brain serotonin levels and serotonin uptake (McCann, 1994). These are not associated with demonstrated behavioral consequences or with histological evidence of neurotoxicity (e.g., loss of cell bodies, gliosis; argyrophilia; Gobbi et al., 1996; Rose et al., 1996). Some investigators have suggested that these neurochemical effects might be mediated by DA, inasmuch as pretreatment with L-DOPA enhanced the long-term depletion of 5-HT produced by the related compound p-chloroamphetamine (Schmidt et al., 1991; Henderson et al., 1993), whereas 5-HTP decreased this response (Iyer et al., 1994; Gudelsky and Nash, 1996).

We examined the ability of various doses of d-fen to elevate DA concentrations in striatal dialysates and the involvement of concurrently released 5-HT in this response. To compare the effects of d-fen on DA release with those of known direct-acting DA releasers, we also measured DA and 5-HT levels in striatal dialysates from animals administered amphetamine or a closely related but previously unstudied anorectic drug, phentermine.

Results of studies on effects of 5-HT on striatal DA synthesis, release and metabolism have been inconsistent. 5-HT inhibited the neuronal activity of the striatum (Yamamoto and Morayama, 1980). On the other hand, dorsal raphe stimulation was able to generate an excitatory postsynaptic potential in striatal cells, using intracellular recording techniques (van der Moelen et al., 1979). Destruction of 5-HT fibers decreased DA turnover and tyrosine hydroxylase activity within the striatum (Giambalvo and Snodgrass, 1978). On the other hand, striatal DA content was increased after the destruction of 5-HT terminals (Osborne et al., 1978).

The effect of local applications of 5-HT agonists and antagonists in vivo implies a stimulatory role for the serotoninergic system on the dopaminergic system in rat striatum with contradictory results on the nature of the receptor type or types involved. 5-HT increased striatal DA release in halothane anesthetized rats, and the 5-HT₄, but not 5-HT₁, 5-HT₂ or 5-HT₃ receptor, subtype was implicated in this effect (Bonhomme et al., 1995). Perfusion with 5-HT facilitated DA release in a dose-dependent manner in the anterior striatum of anesthetized rats, and this effect was reduced by pretreatment with the 5-HT₁ antagonist pindolol (Benloucif and Galloway, 1991; Galloway et al., 1993; Parsons and Justice, 1993). In another study, coperfusion of agonists and antagonists indicated the involvement of 5-HT₁ and 5-HT₃ receptors and the lack of involvement of 5-HT₂ receptors in the 5-HT-induced facilitation of DA release (Benloucif et al., 1993).

Our choice of striatum was based on the known roles of 5-HT and DA as neurotransmitters in this brain region. We hypothesized that d-fen would increase dialysate DA concentrations by enhancing serotoninergic transmission at axo-axonal synapses (e.g., of raphe terminals on nigrostriatal terminals; Soubrie et al., 1984). Studies using immunohistochemical techniques have demonstrated serotonin containing nerve terminals in the striatum, within neurons originating in the dorsal raphe nucleus (Steinbusch et al., 1980; van Bockstaele and Pickel, 1993). Dopamine-containing striatal nerve terminals originate in the substantia nigra, which in turn receives serotoninergic inputs from medial raphe and dorsal raphe nuclei (van der Kooy and Hattori, 1980).

	Experimental Procedures

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Animals. Male Sprague Dawley rats weighing 200 to 300 g were purchased from Taconic Farms (Germantown, NY); housed two per cage; kept on a 12/12-hr light/dark cycle; and given ad libitum access to food and water.

Drug treatments. d-Fen and amphetamine, dissolved in saline, were administered intraperitoneally. Tetrodotoxin and 5-HT were dissolved in aCSF. All pharmacological treatments were performed after stabilization of DA and 5-HT levels in the perfusate, usually after 90 min of perfusion. Tetrodotoxin (1 µM) was added to perfusates 80 min before animals received the d-fen (2.5 mg/kg i.p.) or amphetamine (1 mg/kg s.c.) and was present throughout the experiment. Methiothepin (20 µM) was added to perfusates at the same time the animals received d-fen (2.5 mg/kg i.p.). 5-HT was added to perfusates for only one collection period (20 min). Control animals received only the saline vehicle intraperitoneally or the aCSF perfusate.

Brain microdialysis. Dialysis probes of concentric design (Tossman and Ungerstedt, 1986) were constructed from fused silica capillary tubing (Polymicro Technologies, Phoenix, AZ). Tubing of 140-µm diameter was inserted within wider silica capillary tubing (300 µm) and secured at the inflow site using epoxy resin. A 4-mm length of hollow microdialysis fiber (Spectrum Medical Industries, Los Angeles, CA) was sealed at its tip and secured between the end of the wider silica capillary tubing and the inner tubing, using a cyanoacrylate adhesive. The probes exhibited in vitro recoveries of 10% to 20% for both DA and 5-HT; data were not corrected for probe recovery. Probes were implanted into the striatum (A, +0.7; L, 0.3; V, 6.5; with respect to bregma [Paxinos and Watson, 1986]) of rats that had been anesthetized with ketamine/xylazine (87/13%; 1 ml/kg i.p.). Animals were used 2 days after surgery. The probes were perfused at a flow rate of 1.5 µl/min, using a CMA microperfusion pump (Carnegie Medicin, Acton, MA), with aCSF (in mM): Na⁺ 145, K⁺ 2.7, Mg⁺⁺ 1.0, Ca⁺⁺ 1.2, adjusted to pH 7.4 ± 0.2 with phosphate buffer, 2.0. Samples collected every 20 min were analyzed immediately by HPLC-electrochemical detection. At the end of each experiment, animals were decapitated and probe locations were confirmed by visual examination of probe tracks.

HPLC with electrochemical detection. Dialysate samples were analyzed by reversed-phase HPLC coupled with electrochemical detection. The mobile phase was composed of 75 mM NaH₂PO₄, 1 mM SDS, 0.1 mM EDTA, 15% acetonitrile and 20% methanol; this was adjusted to pH 5.7 with sodium hydroxide. This mobile phase was delivered at a flow rate of 1 ml/min (LC-10AD pump; Shimadzu, Columbia, MD) through an HR-80 column (C18, 4.6 × 80 mm, 3 µm; ESA, Bedford, MA). DA and 5-HT were detected using a coulometric detector (Coulochem II; ESA, Bedford, MA) coupled to a dual electrode analytical cell (model 5014). The potential of the first electrode was set at 175 mV, and that of the second at +175 mV. Under these conditions, the sensitivities for DA and 5-HT were 2 fmol/20 µl.

Statistical analysis. DA and 5-HT contents of each dialysate sample were calculated as fmol in 20-µl sample volume. Data represent mean ± S.E.M. For the analysis of within-group drug effects, the average value for base line (three 20-min samples before drug administration) and values of nine samples collected immediately after drug administration were compared using one-way analysis of variance with repeated measures over time (P < .05) followed by Dunnett's pos- hoc test. A value of P < .05 defined statistical significance.

Reagents. d-fen was from Servier Amerique (Neuilly-sur-Seine Cedex, France). DA, 5-HT, tetrodotoxin and (+)-amphetamine hydrochloride were from Sigma Chemical (St. Louis, MO). NaH₂PO₄, SDS and EDTA were from Fluka (Buchs, Switzerland). Methanol and acetonitrile were from EM Science (Cherry Hill, NJ). Methiothepin mesylate was from Research Biochemical (Natick, MA). Other reagents used were of the highest grade commercially available.

	Results

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Effect of a single dose of d-fen on DA and 5-HT concentrations in dialysates. Base-line DA and 5-HT levels in striatal dialysates collected before drug administration were 74.5 ± 17.6 and 3.8 ± 1.3 fmol/20 µl (n = 50), respectively. Treatment with anorectic doses of d-fen (0.5-1.0 mg/kg i.p.) significantly increased dialysate 5-HT concentrations (fig. 1a); the maximum increases were by 200 ± 28% (P < .05) and 359 ± 66% (P < .01), respectively. In contrast, DA concentrations in the dialysates were not significantly increased 3 hr after the administration of the drug (fig. 1b). A higher d-fen dose, 2.5 mg/kg, increased both DA (152 ± 16%, P < .01) and 5-HT (472 ± 62%, P < .01) concentrations (fig. 1, a and b). This increase in DA release was maximal 160 min after administration of the drug, whereas the increase in 5-HT release was statistically significant 20 min after administration of d-fen and remained so for the next 3 hr (fig. 1, a and b).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Effect of d-fen on the release of 5-HT (top) and DA (bottom) in the rat striatum. After the collection of three base-line samples (20 min each), d-fen was administered intraperitoneally (the first fraction), and sample collection continued for the next 3 hr. Data points are mean ± S.E.M over time (for some points, S.E.M. were too small to be plotted), n = 4-7. * P < .05; ** P < .01 vs. base line.

Effect of methiothepin on the increases in DA concentrations induced by d-fen. The mixed-acting serotonin antagonist methiothepin (20 µM) was administered locally through the microdialysis probe at the same time that d-fen (2.5 mg/kg) was given intraperitoneally. Methiothepin by itself significantly increased 5-HT release without affecting that of DA (data not shown). It also blocked the ability of the high dose of d-fen (2.5 mg/kg) to release DA (fig. 2) but did not affect 5-HT release by d-fen.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Effect of local application of methiothepin on the release of DA induced by d-fen. After collection of three base-line samples (20 min each), methiothepin (20 µM) was applied through the dialysis probe, starting at the same time (the first fraction) that d-fen (2.5 mg/kg) was administered intraperitoneally, and was present throughout the experiment (3 hr). Data points are mean ± S.E.M., n = 4-7. *P < .05; **P < .01 vs. base line.

Effect of tetrodotoxin on the increases in DA and 5-HT concentrations induced by d-fen. The sodium channel blocker tetrodotoxin, administered through the microdialysis probe 80 min before d-fen was given intraperitoneally, completely blocked the d-fen-induced increase in DA release (fig. 3) and reduced the response of 5-HT by 70% (fig. 4). Tetrodotoxin also reduced basal levels of DA and 5-HT in the dialysates.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Effect of TTX (a sodium channel blocker, 1 µM) on the release of DA induced by d-fen (2.5 mg/kg i.p). After collection of three base-line samples (20 min each), TTX was applied through the dialysis probe starting 80 min before (the first fraction) the administration of d-fen (the fourth fraction), and was present throughout the experiment. Data are mean ± S.E.M., n = 5-7. *P < .05; **P < .01 vs. base line.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Effect of TTX on the release of 5-HT induced by d-fen (2.5 mg/kg i.p.). Animals were treated as in figure 3. Data are mean ± S.E.M., n = 5-7. *P < .05; **P < .01 vs. base line.

Effects of amphetamine, phentermine or amphetamine plus tetrodotoxin on DA and 5-HT concentrations in dialysates. Administration of amphetamine (1 mg/kg i.p.) increased extracellular DA concentrations by 72 ± 13 fmol/20 µl (fig. 5) without affecting those of 5-HT (Data not shown). Pretreatment with tetrodotoxin (1 µM in perfusates; 80 min before amphetamine administration) failed to block completely this amphetamine-induced rise in DA (57 ± 14 fmol/20 µl; fig. 5). Phentermine (2 mg/kg i.p.) also increased striatal DA concentrations (148 ± 17%; P < .01; fig. 6) without affecting those of 5-HT (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Effect of TTX (a sodium channel blocker, 1 µM) on the release of DA induced by amphetamine (1.0 mg/kg s.c.). Animals were treated as in figure 3. Data are mean ± S.E.M., n = 4. **P < .01 vs. base line.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Effect of phentermine (2 mg/kg i.p.) on the release of DA in the striatum. After collection of three base-line samples (20 min each), phentermine was administered intraperitoneally (the first fraction), and sample collection continued for the next 3 hr. Data are mean ± S.E.M., n = 4. *P < .05; **P < .01 vs. base line.

Effect of local application of 5-HT on DA concentrations in dialysates. Local application of 5-HT (3 or 10 µM, through the microdialysis probe) increased DA release by 31 ± 6% or 129 ± 29%, respectively (fig. 7).

View larger version (11K): [in this window] [in a new window]   Fig. 7.   Effect of local application of 5-HT on the release of DA in the striatum. 5-HT was applied through the dialysis probe for 20 min as indicated on the graph. Data were graphed as mean ± S.E.M., n = 4. *P < .05; **P < .01 vs. base line.

	Discussion

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D-fen, although in substantially higher doses (2.5 mg/kg), which release much larger amounts of 5-HT, thereby also elevates DA release. That this effect is indirect, and mediated by 5-HT, is indicated by our findings that (1) tetrodotoxin application, which decreased d-fen-induced 5-HT release by 70% (fig. 4), completely blocked d-fen-induced release of DA (fig. 3). (2) Local application of 5-HT to the striatum also increased DA release (fig. 7). (3) Methiothepin, a mixed-acting 5-HT receptor antagonist, blocked the release of DA induced by d-fen (2.5 mg/kg; fig. 2). In contrast to d-fen, amphetamine and phentermine increased DA release without affecting that of 5-HT, and local application of tetrodotoxin did not completely block this response to amphetamine (figs. 5 and 6).

Our data apparently provide the first evidence, based on in vivo microdialysis, that lower doses of d-fen (0.5 or 1.0 mg/kg) enhance 5-HT release in the rat striatum. Similar findings were obtained by other investigators using relatively higher doses in frontocortex, hypothalamus and nucleus accumbens (Laferrere and Wurtman 1989; Gardier et al., 1994). d-Fen increased dialysate DA concentrations at the highest dose (2.5 mg/kg) tested in our experimental conditions (fig. 1b). Similar increases were obtained by De Deurwaerdere et al. (1995) in animals given 5 and 10 mg/kg but not at 2.5 mg/kg. This difference may have resulted from our use of unanesthetized animals (Hamilton et al., 1992; Dringerberg and Vanderwolf, 1995). In previous studies, amphetamine had been shown to increase the release of both DA and 5-HT, whereas under our experimental conditions, either it or phentermine increased DA but not 5-HT. This difference may reflect our use of a lower amphetamine dose (1 mg/kg) than that used by Hernandez et al. (2 mg/kg; 1987) as well as the use of this drug through microdialysis probe (Ferre et al., 1994). Kuczenski and Segal (1990) previously demonstrated that 1 mg/kg d-amphetamine increased concentrations of DA and but not 5-HT in the striatum and that higher doses would produce increases in both monoamines. Our data on the effects of phentermine on neurotransmitter release, as studied by in vivo microdialysis, are apparently the first to be described for this anorexic drug.

d-fen increases dialysate 5-HT levels by blocking its reuptake (Garattini et al., 1978), and its active metabolite, dexnorfenfluramine, also releases 5-HT directly and activates postsynaptic 5-HT₂ receptors (Garattini et al., 1987; Spedding et al., 1996). Pretreatment with tetrodotoxin, a sodium channel blocker, reduced 5-HT release by 70% (fig. 4), suggesting that a sodium channel-dependent mechanism participates in this response. This finding is in agreement with previous data showing that the 5-HT-releasing effect of a much higher dose of d-fen (10 mg/kg) in vivo depends on nerve terminal depolarization (Gardier et al., 1994). Inasmuch as tetrodotoxin did not fully block the rise in dialysate 5-HT after d-fen, the mechanism of action of d-fen probably involves both tetrodotoxin-sensitive and -insensitive mechanisms. Thus, the tetrodotoxin-induced decrease in 5-HT release is expected to result in a decrease in DA release.

Our finding that 5-HT increased DA release (fig. 7) is consonant with the findings of previous studies. A treatment that increased intrasynaptic concentrations of endogenous 5-HT, the 5-HT reuptake blocker alaproclate administered through a microdialysis, probe also increased striatal DA release in conscious rats (Yadid et al., 1994). Striatal infusion of fenfluramine increased extracellular DA in a fluoxetine-sensitive fashion, thus demonstrating the ability of endogenous 5-HT to elicit DA release (Benloucif and Galloway, 1991). Similarly, 5-HT administered into the striatum of anesthetized rats through a microdialysis probe increased extracellular DA in a concentration-dependent manner (Benloucif and Galloway, 1991; Benloucif et al., 1993; Bonhomme et al., 1995; de Deurwaerdere et al., 1996). The known effects of local applications of 5-HT agonists and antagonists on DA release in vivo imply a stimulatory role for 5-HT in striatal DA release (Galloway et al., 1993; West and Galloway, 1996) as well as DA release in other brain tissues (Chen et al., 1992; Parsons and Justice, 1993; Iyer and Bradberry, 1996). In some studies using different experimental systems (e.g., electrophysiological and behavioral approaches), 5-HT produced effects consistent with inhibition of DA release (Osborne et al., 1978; Costall et al., 1979; Yamamoto and Morayama, 1980).

The amount of increase in DA release induced by 10 µM 5-HT was in parallel with literature values. Iyer and Bradberry (1996) demonstrated that the response to 10 µM 5-HT perfusion (for 20 min) was an increase in DA release by 149%. We also observed a similar increase (129%) in our system with the same concentration of 5-HT perfusion. However, this effect was quite different than observed by West and Galloway (1996) and Yadid et al. (1994). The discrepancy might be explained by the use of chlorate hydrate anesthesia in the former study and high calcium/high potassium-containing aCSF in the later study. The concentration of 5-HT in the striatal dialysates was increased 400%, from 4 to 20 fmol/20 µl, by the 2.5 mg/kg dose of d-fen in our experimental system. This translates into 1 nM 5-HT concentrations in dialysates or 10 nM 5-HT around the probe. This increment in 5-HT resulted in an 152% increase in DA release. When 5-HT (10 µM) was locally applied for 20 min, the concentration around the probe can be calculated as 20 pmol/20 µl (or 1 µM; with probe recovery of 10%). This application of 5-HT resulted in an 129% increase in striatal DA release. The calculated concentration of 5-HT around the probe (1 µM) is probably considerably higher than that observed after intraperitoneal d-fen administration (10 nM). However, the neurotransmitter concentration around the probe does not necessarily tell us the concentrations in nerve synapses. The difference in 5-HT concentrations around the probe obtained after intraperitoneal d-fen (2.5 mg/kg) administration and after local perfusion of 5-HT (10 µM) could reflect differences in the direction of flow of the 5-HT (i.e., from nerve terminals to extracellular space after d-fen and from perfusion site (extracellular space) to the nerve terminals after 5-HT perfusion). Perhaps intrasynaptic 5-HT concentrations after d-fen are considerably higher than those measured in dialysates.

Methiothepin blocked d-fen-induced DA release (fig. 2), possibly by blocking postsynaptic 5-HT receptors on the dopaminergic nerve terminals. Methiothepin is a nonselective 5-HT antagonist and might also block DA receptors at higher doses (Christensen, 1985; Meltzer et al., 1989). Thus, it could have stimulated DA release if it also blocked inhibitory D₂ DA receptors. Inasmuch as methiothepin had no effect on DA release when given alone, and because, moreover, the drug blocked d-fen-induced DA release in our animals, the drug apparently was not acting via D₂ receptors.

Methiothepin also increased striatal 5-HT levels when given alone, possibly by blocking presynaptic inhibitory 5-HT_1B receptors. This would be compatible with the view that presynaptic 5-HT_1B autoreceptors in the striatum of the conscious rat receive tonic activation, as opposed to the reported lack of tonic activation observed in the hippocampus of anesthetized rats (Hjorth and Sharp, 1993). Methiothepin, on the other hand, did not affect d-fen-induced 5-HT release. This would indicate that 5-HT release by d-fen interferes with the ability of methiothepin to block inhibitory presynaptic serotonin autoreceptors.

Pretreatment with tetrodotoxin completely blocked the increase in extracellular DA concentrations induced by a high dose of d-fen (2.5 mg/kg i.p.; fig. 3), implying that a sodium channel-dependent mechanism is solely responsible for this increase. We hypothesized that d-fen would increase dialysate DA concentrations by enhancing serotoninergic transmission at axo-axonal synapses (e.g., of raphe terminals on nigrostriatal terminals; Soubrie et al., 1984). Hence, if tetrodotoxin suppressed d-fen-induced 5-HT release, as it was found to do (fig. 4), the blockade of DA release could be anticipated. Thus, the effect of 5-HT induced by d-fen on DA release is dependent on intact impulse flow in the nerve terminal. We eliminated the possibility that d-fen might increase DA concentrations via an amphetamine-like mechanism because tetrodotoxin completely blocked increases in extracellular DA concentrations induced by d-fen but not by amphetamine (figs. 3 and 5).

De Deurwaerdere et al. (1995) previously showed that the release of striatal DA by d-fen is not blocked in halothane-anesthetized rats after destruction of 5-HT terminals (by 5,7-dihydroxytryptamine injected into dorsal raphe nucleus). They thus concluded that striatal 5-HT is not required for this response. That study did not measure extracellular 5-HT concentrations after treatment with d-fen and also used higher doses of d-fen (5 and 10 mg/kg) than those used in the present study. Low concentrations of d-fen (0.5 µM) did not induce DA release from striatal synaptosomes, whereas higher concentrations (10 µM) did induce some overflow of prelabeled [³H]DA, although the overflow was much lower than that of [³H]-5-HT (Gobbi et al., 1993). Other investigators also showed that although high concentrations of d-fen can suppress DA reuptake, the IC₅₀ value for this effect (16.3 µM) is 40 times that for the inhibition of 5-HT re-uptake (0.4 µM; Garattini et al., 1992). It is thus possible that in vivo, sufficiently high concentrations of d-fen might have both direct and indirect effects on dopamine release. However, the pretreatment of animals with a DA uptake blocker, nomifensine (0.1-1 µM), failed to modify significantly the release of striatal DA induced by local application of d-fen (25 µM), whereas the 5-HT uptake blocker citalopram slightly but significantly reduced DA release induced by the same application of d-fen (De Deurwaerdere et al., 1995). Even though the metabolite d-norfenfluramine can activate 5-HT₂ receptor subtypes in rat brain (Spedding et al., 1996), these receptors apparently are not involved in the effect of serotonin on extracellular dopamine concentrations in the rat striatum (Bonhomme et al., 1995). A possible explanation for the discrepancy between the lack of involvement of 5-HT in the d-fen effect advanced by De Deurwaerdere et al. (1995) and the opposite conclusion of the present work is that some functionally intact (or hypersensitive) serotoninergic nerve endings remaining after the 5,7-dihydroxytryptamine lesion (that study did not measure 5-HT levels in striatal dialysates) responded to dexfenfluramine administration. If those authors had measured striatal 5-HT release and also found these levels increased in dialysates, their data would be compatible with our hypothesis that dexfenfluramine increases DA release via 5-HT.

Very high doses of d-fen can be shown to produce prolonged decreases in brain 5-HT levels and 5-HT uptake. Some investigators have suggested that the mechanism responsible for these decreases might involve DA release (Henderson et al., 1993; Gudelsky and Nash 1996). Our finding that d-fen can increase DA release at higher doses (2.5 mg/kg), and that most likely 5-HT probably mediates this response, is compatible with this hypothesis.

In conclusion, we suggest that d-fen can increase striatal DA release by a tetrodotoxin-sensitive mechanism and that this effect may be mediated by 5-HT.