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Serotonin (5-HT) transporter (SERT) substrates like fenfluramine and 3,4-methylenedioxymethamphetamine cause long-term depletion of brain 5-HT, while certain other substrates do not. The 5-HT deficits produced by SERT substrates are dependent upon transporter proteins, but the exact mechanisms responsible are unclear. Here, we compared the pharmacology of several SERT substrates: fenfluramine, d-fenfluramine, 1-(m-chlorophenyl)piperazine (mCPP) and 1-(m-trifluoromethylphenyl)piperainze (TFMPP), to establish relationships between acute drug mechanisms and the propensity for long-term 5-HT depletions. In vivo microdialysis was carried out in rat nucleus accumbens to examine acute 5-HT release and long-term depletion in the same subjects. In vitro assays were performed to measure efflux of [(3)H]5-HT in rat brain synaptosomes and transporter-mediated ionic currents in SERT-expressing Xenopus oocytes. When administered repeatedly to rats (6 mg/kg, i.p., four doses), all drugs produce large sustained elevations in extracellular 5-HT (>5-fold) with minimal effects on dopamine. Importantly, 2 weeks after dosing, only rats exposed to fenfluramine and d-fenfluramine display depletion of brain 5-HT. All test drugs evoke fluoxetine-sensitive efflux of [(3)H]5-HT from synaptosomes, but d-fenfluramine and its bioactive metabolite d-norfenfluramine induce significantly greater SERT-mediated currents than phenylpiperazines. Our data confirm that drug-induced 5-HT release probably does not mediate 5-HT depletion. However, the magnitude of transporter-mediated inward current may be a critical factor in the cascade of events leading to 5-HT deficits. This hypothesis warrants further study, especially given the growing popularity of designer drugs that target SERT.
Figure 1. Effects of repeated i.p. administrations of saline, fenfluramine (fen), d-fenfluramine (d-fen), mCPP, and TFMPP on extracellular 5-HT and dopamine (DA) in rats undergoing microdialysis in nucleus accumbens. Panel a depicts the effects of fen, d-fen, and saline on dialysate 5-HT, whereas panel b shows dialysate dopamine. Panel c depicts effects of mCPP, TFMPP, and saline on dialysate 5-HT, whereas panel d shows dialysate dopamine. Arrows indicate time of injections. Data are mean±SEM expressed as % baseline for N=5 rats per group.
Figure 2. Effects of repeated doses of saline, fenfluramine (fen), d-fenfluramine (d-fen), mCPP, or TFMPP on post-mortem tissue levels of 5-HT and dopamine (DA) in rat nucleus accumbens, measured 2 weeks after injections. Panel a shows the effects of fenfluramines while panel c shows the effects of phenylpiperazines. Post-mortem tissue data (a, c) are mean±SEM expressed as % control amine N=5 rats per group. The relationship between dialysate 5-HT and post-mortem tissue 5-HT is illustrated for fenfluramines and phenylpiperazine in panels b and d, respectively. Pearson's correlation coefficient (r) was calculated for effects of fenfluramines (b) or piperazines (d), based on mean dialysate 5-HT during the sampling period (pg/5 μl) and post-mortem tissue levels of 5-HT (ng/100 mg) for individual subjects. N=15 rats per plot.
Figure 3. Release of preloaded [3H]5-HT from rat brain synaptosomes evoked by d-fenfluramine (d-fen), d-norfenfluramine (d-norfen), mCPP, and TFMPP. Drug-induced [3H]-5-HT release was measured in the presence or absence of 10 nM fluoxetine for d-fen (a), d-norfen (b), mCPP (c), and TFMPP (d). Data are mean±SD for N=3 separate experiments.
Figure 4. SERT-generated currents in Xenopus oocytes induced by increasing concentrations of d-fenfluramine, d-norfenfluramine, mCPP, TFMPP, or MTA in comparison with the physiological substrate 5-HT (10 μM). Sample traces are shown in panels a–e, and drug concentrations are given in μM.
Figure 5. (a) Concentration-response curves, pooled from different oocytes and (b) comparison of the maximal current (Cmax) for d-fenfluramine, d-norfenfluramine, mCPP, TFMPP, and MTA compared in the same oocyte. Maximal current for d-fenfluramine was measured at 10 μM and for d-norfenfluramine at 100 μM, whereas maximal current for mCPP, TFMPP, and MTA was measured at 3 μM. Statistical analysis revealed significant differences at a level of p<0.01 (***).
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