Vaccine-driven pharmacodynamic dissection and mitigation of fenethylline psychoactivity.

Fenethylline, also known by the trade name Captagon, is a synthetic psychoactive stimulant that has recently been linked to a substance-use disorder and 'pharmacoterrorism' in the Middle East. Although fenethylline shares a common phenethylamine core with other amphetamine-type stimulants, it also incorporates a covalently linked xanthine moiety into its parent structure. These independently active pharmacophores are liberated during metabolism, resulting in the release of a structurally diverse chemical mixture into the central nervous system. Although the psychoactive properties of fenethylline have been reported to differ from those of other synthetic stimulants, the in vivo chemical complexity it manifests upon ingestion has impeded efforts to unambiguously identify the specific species responsible for these effects. Here we develop a 'dissection through vaccination' approach, called DISSECTIV, to mitigate the psychoactive effects of fenethylline and show that its rapid-onset and distinct psychoactive properties are facilitated by functional synergy between theophylline and amphetamine. Our results demonstrate that incremental vaccination against a single chemical species within a multi-component mixture can be used to uncover emergent properties arising from polypharmacological activity. We anticipate that DISSECTIV will be used to expose unidentified active chemical species and resolve pharmacodynamic interactions within other chemically complex systems, such as those found in counterfeit or illegal drug preparations, post-metabolic tissue samples and natural product extracts.

Repeated administration of Yokukansan inhibits DOI-induced head-twitch response and decreases expression of 5-hydroxytryptamine (5-HT)2A receptors in the prefrontal cortex.

Behavioral and psychological symptoms of dementia (BPSD) are commonly seen in patients with Alzheimer's disease (AD) and other forms of senile dementia. BPSD have a serious impact on the quality of life of dementia patients, as well as their caregivers. However, an effective drug therapy for BPSD has not been established. Recently, the traditional Japanese medicine Yokukansan (YKS, Yi-gan san in Chinese) has been reported to improve BPSD in a randomized, single-blind, placebo-controlled study. Moreover, abnormalities of the serotonin (5-HT) system such as 5-HT2A receptors have been reported to be associated with BPSD of AD patients. In the present study, we investigated the effect of YKS on head-twitch response induced by 2,5-dimethoxy-4-iodoamphetamine (DOI, 5 mg/kg, i.p.) in mice, a behavioral response that is mediated, in part, by 5-HT2A receptors. Acute treatment with YKS (100 and 300 mg/kg, p.o.) had no effect on the DOI-induced head-twitch response, whilst 14 days repeated treatment with YKS (300 mg/kg, p.o.) significantly inhibited this response. Moreover, repeated treatment with YKS (300 mg/kg, p.o.) decreased expression of 5-HT2A receptors in the prefrontal cortex, which is part of the circuitry mediating the head-twitch response. These findings suggest that the inhibition of DOI-induced head-twitch response by YKS may be mediated, in part, by altered expression of 5-HT2A receptors in the prefrontal cortex, which suggests the involvement of the 5-HT system in psychopharmacological effects of YKS.

Serotonergic agents that activate 5HT2A receptors prevent NMDA antagonist neurotoxicity.

Phencyclidine, ketamine, and other agents that block NMDA glutamate receptors trigger a schizophrenia-like psychosis in humans and induce pathomorphological changes in cerebrocortical neurons in rat brain. Accumulating evidence suggests that a complex network disturbance involving multiple transmitter receptor systems is responsible for the neuronal injury, and it is proposed that a similar network disturbance is responsible for the psychotomimetic effects of NMDA antagonists, and might also be involved in the pathophysiology of schizophrenia. In the present study we present evidence that serotonergic agents possessing 5HT2A agonist activity prevent NMDA antagonist neurotoxicity in rat brain. It is proposed that 5HT2A agonists may also prevent the psychotomimetic effects of NMDA antagonists. Among the 5HT2A agonists examined and found to be neuroprotective are LSD and related hallucinogens. The apparent contradiction in proposing that these agents might have antipsychotic properties is resolved by evidence linking their hallucinogenic activity to agonist action at 5HT2C receptors, whereas antipsychotic activity would be attributable to agonist action at 5HT2A receptors.

Effects of hallucinogens on locomotor and investigatory activity and patterns: influence of 5-HT2A and 5-HT2C receptors.

The 5-HT2A and 5-HT2C antagonists MDL 100,907 and SER-082 were tested with the 5-HT2A/C agonist DOI and the 5-HT1A/2A/2C agonist LSD in the Behavioral Pattern Monitor, which provides multiple measures of locomotor and investigatory activity. Previous investigations have shown that these measures load onto three independent behavioral factors: amount of activity, exploratory behavior, and behavioral organization. Rats pretreated with saline, MDL 100,907 (0.25-2.0 mg/kg), or SER-082 (0.5-1.0 mg/kg) were treated with saline, 0.25 mg/kg DOI, or 60 micrograms/kg LSD. All effects of DOI were blocked by all doses of MDL 100,907, but only by the highest dose of SER-082. While the effects of LSD on activity and exploratory behavior were largely unaffected, either pretreatment antagonized the effects of LSD on behavioral organization. Thus, all of these effects of DOI were attributable to 5-HT2A receptors, whereas the effect of LSD on behavioral organization was influenced by both 5-HT2A and 5-HT2C receptors.

Multiple serotonin receptor subtypes modulate prepulse inhibition of the startle response in rats.

The phenomenon of prepulse inhibition (PPI) of the acoustic startle reflex is widely used as an operational measure of sensorimotor gating mechanisms. Because sensorimotor gating abnormalities have been identified in schizophrenic patients, the exploration of the neural substrates involved in PPI may provide insight into the neural dysfunctions underlying this disorder. Both dopaminergic and glutamatergic systems are involved in the modulation of PPI in rats. In addition, the present studies demonstrate complex serotonergic influences in this phenomenon. Specifically, both the 5-HT2 agonist, DOI, (2,5-dimethoxy-4-iodoamphetamine), and the 5-HT1B agonist, RU 24969, [5-methoxy-3(1,2,3,6)tetrahydropyridin-4- yl]-1H-indole, potently and reversibly disrupted PPI. The 5-HT2C agonist mCPP, [1-(m)-chlorophenyl-piperazine], was ineffective. Furthermore, ketanserin (2.0 mg/kg) and haloperidol (0.1 mg/kg) but not (+/-)propranolol (20.0 mg/kg) blocked the effect of DOI. In addition, the same doses of haloperidol, and, to a lesser extent, (+/-)propranolol, prevented the disruption of PPI induced by RU 24969. Together with previous reports of 5-HT1A involvement in PPI, these results argue for multiple serotonergic mechanisms in the modulation of PPI.

Different role of 5-HT1A and 5-HT2 receptors in spinal cord in the control of nociceptive responsiveness.

The effects of the 5-hydroxytryptamine type-2 (5-HT2) receptor agonist (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and the 5-HT1A agonist (+)-8-hydroxy-2-(di-n-propylamino)-tetralin [(+)-8-OH-DPAT] on nociceptive responsiveness were compared in mice. Intrathecal administration of DOI (5-20 micrograms) produced a dose-dependent behavioural syndrome, consisting of biting or licking, directed towards the caudal part of the body and reciprocal hindlimb scratching. However, (+)-8-OH-DPAT (5-20 micrograms) did not produce the biting and scratching behaviour. The response to DOI (20 micrograms) was reversed by treatment with the substance P receptor antagonist, [D-Arg1, D-Trp7,9, Leu11]-SP (Spantide) (5 micrograms). The tail-flick reflex was markedly depressed 5-20 min after administration of (+)-8-OH-DPAT; DOI did not change the tail-flick reflex after 5 min but significantly inhibited the reflex response 10-20 min after injection. The data show that stimulation of 5-HT2 receptors, but not 5-HT1A receptors, induced a behavioural syndrome, which may reflect activation of nociceptive pathways. The tail-flick reflex was more markedly inhibited by stimulation of 5-HT1A than 5-HT2 receptors. Accordingly, 5-HT2 and 5-HT1A receptors seem to have a different function in the modulation of nociceptive responsiveness in the mouse.

The effects of alpha 2-adrenoceptor antagonists on the inhibition of 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI)-induced head shakes by 5-HT1A receptor agonists in the mouse.

1. 8-Hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), gepirone, buspirone and ipsapirone dose-dependently antagonized the head-shakes induced by 1-(2,5-dimethoxy 4-iodophenyl)-2-amino propane hydrochloride (DOI) (1.0 mg kg-1) in mice, when these agents were given i.p. 10 min beforehand. 2. para-Chlorophenylalanine (pCPA) abolished the effect of 8-OH-DPAT (0.1 mg kg-1) and of buspirone (1.0 mg kg-1). (+/-)-Pindolol (5.0 mg kg-1) also antagonized the effect of 8-OH-DPAT (0.1 mg kg-1). 3. The alpha 2-adrenoceptor antagonists, RX811059 (1.0 mg kg-1), idazoxan (0.5 mg kg-1), yohimbine (1.0 mg kg-1) and 1-(2-pyrimidinyl)-piperazine (1-PP) (2.0 mg kg-1) i.p. prevented the antagonistic effect of 8-OH-DPAT (0.1 mg kg-1) on DOI-head-shakes. 4. Orally-administered buspirone, given 60 min beforehand, only reduced DOI-head-shakes at doses of 60 mg kg-1 and above. However, when buspirone (1.0 mg kg-1) was administered orally twice daily for 21 days, DOI-head-shakes were significantly reduced when tested 60 min after the first daily dose on days 5, 12 and 21 and 48 h after withdrawal. 5. A single oral dose of buspirone (1.0 mg kg-1) strongly antagonized DOI-head-shakes when given 24 h after the last of 4 daily doses of 1-PP (2.0 mg kg-1, p.o.) but had no effect on DOI-head-shakes 24 h after the last of 4 daily doses of water (p.o.). 6. A single oral dose of 1-PP (2.0 mg kg-1) abolished the inhibitory effect of i.p. buspirone(1.0 mg kg-1) on DOI-head-shakes in mice which had received water (p.o.) daily on the 4 previous days but not in mice which had received 1-PP (2.0mg kg-1, p.o.) on these days.7. The ability of 5-HT1A receptor agonists to antagonize DOI-head-shakes may be due to an effect at presynaptic 5-HT receptors. It is suggested that 1-PP, formed from buspirone, may act at a2-adrenoceptors to prevent acutely administered oral buspirone from antagonizing DOI-head shakes, but that tolerance occurs to this effect of I-PP, thus revealing the inhibitory effect of buspirone when the latter is given repeatedly.

Spinal 5-HT2 receptor-mediated facilitation of pudendal nerve reflexes in the anaesthetized cat.

1. 5-Hydroxytryptamine (5-HT) is intimately associated with central sympathetic and somatic control of the lower urinary tract. The sympathetic and somatic innervation of the lower urinary tract is conveyed through efferent axons of the hypogastric and pudendal nerves, respectively. 2. The present study examined the effects of 2,5-dimethoxy-4-iodophenylisopropylamine (DOI), a 5-HT2 receptor subtype-selective agonist, on evoked potentials recorded from the central ends of the hypogastric and pudendal nerves in response to electrical stimulation of afferent fibres in the pelvic and pudendal nerves, respectively. Various spinalization paradigms were employed to localize the site of action. All cats were pretreated with xylamidine (1 mg kg-1), a peripherally-restricted 5-HT2 receptor antagonist. 3. In acute spinal cats, DOI (0.01-3 mg kg-1, i.v.) reliably produced dose-dependent increases in the pudendal nerve reflex (to 228 +/- 31% of control). These increases were reversed by the 5-HT2 receptor-selective antagonist, LY53857 (0.3-3 mg kg-1, i.v.). On the other hand, in spinally-intact cats, DOI produced no significant changes in the pudendal reflex. However, within minutes of spinalization of DOI-pretreated cats, a marked increase (to 221 +/- 16% of control) in the pudendal reflex was observed which could be reversed by LY53857. No significant effects were observed on hypogastric reflexes in either acute spinal or spinally-intact cats following DOI administration. No effects were seen in either spinally-intact or acute spinal animals when LY53857 was administered as the initial drug. 4. These results indicate that activation of spinal 5-HT2 receptors facilitates pudendal reflexes. In spinally-intact cats, it is hypothesized that DOI activates supraspinal pathways that mediate inhibition of the pudendal reflexes and counteracts the facilitatory effects of spinal 5-HT2 receptor activation.

Evidence that 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI)-induced hyperthermia in rats is mediated by stimulation of 5-HT2A receptors.

The effects of various 5-HT receptor subtype-selective antagonists were studied on phenylisopropylamine hallucinogen 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI)-induced hyperthermia in Wistar rats, in an attempt to characterize the 5-HT receptor subtype mediating DOI-induced hyperthermia. Intraperitoneal administration of DOI to rats produced hyperthermia with a peak effect at 60 min. Pretreatment with propranolol (beta-adrenoceptor antagonist that also has binding affinity for 5-HT1A, 5-HT1B and 5-HT2C sites), MDL-72222 or ondansetron (5-HT3 antagonists) did not attenuate DOI-induced hyperthermia. In contrast, pretreatment with metergoline (5-HT1/5-HT2 antagonist), ketanserin, LY53857, mesulergine, mianserin and ritanserin (5-HT2C/5-HT2A antagonists), as well as spiperone (5-HT1A/5-HT2A/D2 antagonist), significantly attenuated DOI-induced hyperthermia. Furthermore, daily administration of DOI (2.5 mg/kg per day) for 17 days did not produce either tolerance to its hyperthermic effect or modify m-CPP-induced hyperthermia in rats. These findings suggest that DOI-induced hyperthermia in rats is mediated by stimulation of 5-HT2A receptors.

Anandamide inhibits the DOI-induced head-twitch response in mice.

RATIONALE: Recently, Delta(9)-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, and synthetic cannabinoid receptor agonists reportedly reduced the head-twitches induced by the 5-HT(2A/2C) receptor agonist 1-(2,5-dimethoxy 4-iodophenyl)-2-amino propane (DOI) in mice, which is mediated via the activation of 5-HT(2A) receptor. However, the effect of endogenous cannabinoid anandamide on the head-twitch response has not been studied. OBJECTIVES: In this study, we investigated the effect of anandamide on the DOI-induced head-twitch response in mice. METHODS: Five minutes after the injection of DOI (5 mg/kg IP), the number of head-twitches was counted for a 5-min period. THC or anandamide was injected IP 60 min or 10 min before the number of head-twitches was counted, respectively. RESULTS: THC and anandamide each reduced the DOI-induced head-twitch response. The inhibition of the DOI-induced head-twitch response by THC was reversed by SR141716A (N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide), a CB(1) receptor antagonist, while the effect of anandamide was not blocked by SR141716A. Cyclooxygenase (COX) inhibitors such as aspirin and indomethacin reversed the inhibition of the DOI-induced head-twitch response by anandamide. On the other hand, COX inhibitors did not affect the inhibition of the DOI-induced head-twitch response by THC. CONCLUSIONS: Taken together, these findings suggest that the endocannabinoid anandamide may inhibit 5-HT(2A) receptor-mediated function via the arachidonic acid cascade, but not via a direct interaction with the CB(1) cannabinoid receptor, and that the mechanism of its action is clearly different from that of THC.

1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane (DOI) exerts an anorexic action that is blocked by 5-HT2 antagonists in rats.

Previous literature suggests that the anorexic action of peripherally administered serotonin (5-HT) is mediated by 5-HT2 receptors. This study, therefore, examined the effect of DOI, a non-indole 5-HT2 agonist, on deprivation-induced feeding. Rats were first adapted to a schedule in which a milk diet was presented for 6 h daily. Intraperitoneal (IP) administration of DOI (1.0-11.2 mumol/kg) inhibited feeding in a dose-related fashion (ID50 = 2.6 mumol/kg). One hour pretreatment with 6.0 mumol/kg of the 5-HT2 antagonists ketanserin and LY53857 completely reversed the anorexic action of an equimolar dose of DOI. Neither ketanserin nor LY53857, alone, altered baseline feeding. Further testing demonstrated the antagonistic effect of LY53857 (0.047-6.0 mumol/kg, IP) to be dose related, with an ID50 of 0.14 mumol/kg. Ten minute pretreatment with 1-(1-naphthyl)-piperazine (1-NP; 2.0 or 4.0 mumol/kg), a mixed-acting agent with 5HT2 blocking actions, also attenuated the anorexic effect of 6.0 mumol/kg DOI. Unlike ketanserin and LY53857, however, 1-NP did reduce food intake by itself. By contrast with ketanserin, LY53857 and 1-NP, the peripherally-acting 5-HT2 antagonist xylamidine failed to alter the anorexic effect of DOI. Taken together, these results suggest that central 5-HT2 receptors are important in the control of ingestive behavior.

Prevention of DOI-mediated wet dog shakes by inhibitors of nitric oxide synthase.

The purpose of this study was to determine if (+/-)-1-(2, 5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI)-induced wet dog shakes (WDS) required the involvement of nitric oxide synthases (NOS). Systemic administration of the general NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME), but not its d-isomer, and the neuronal NOS (nNOS) inhibitor 7-nitroindazole completely blocked DOI-mediated WDS in a dose dependent manner. The data provides evidence that serotonin 5HT2 receptors are coupled to nNOS activation in the rat brain.

Dextromethorphan antagonizes the acute depletion of brain serotonin by p-chloroamphetamine and H75/12 in rats.

A role for calcium in p-chloroamphetamine-induced neurotoxicity has been inferred previously from protective effects of dextromethorphan. We found that dextromethorphan reduces rat brain concentrations of 5-hydroxyindoleacetic acid and blocks the acute, non-neurotoxic depletion of brain serotonin by p-chloroamphetamine and by H75/12. Inhibition of the membrane transporter on brain serotonin neurons by dextromethorphan in vivo might explain its protective effect against p-chloroamphetamine neurotoxicity.

Effects of (+/-)-DOI on medial prefrontal cortical cells: a microiontophoretic study.

The relatively selective 5-HT2 receptor agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane [+/-)-DOI) ejected by microiontophoresis at low currents potentiates glutamate (GLU)-induced excitation and at higher currents (greater than 20 nA) invariably inhibits both spontaneous and GLU-induced activity of cells in the medial prefrontal cortex (mPFc). The inhibitory action of DOI is blocked by the 5-HT2 antagonists, ritanserin, metergoline and spiperone, but not other receptor antagonists. Microiontophoresis of 1 M Mg2+ for 5-20 min did not alter DOI's inhibitory effect suggesting that DOI's action is a direct one. These results show that DOI predominantly inhibits mPFc neuronal activity and this inhibitory action is mediated by 5-HT2 receptors.

Fluoxetine gradually increases [125I]DOI-labelled 5-HT2A/2C receptors in the hypothalamus without changing the levels of Gq- and G11-proteins.

The time course of fluoxetine-induced supersensitivity of hypothalamic 5-HT2A/2C receptors was examined. Daily injections of fluoxetine (7 or 14 days) significantly increased agonist ([125I]DOI)-labeled high-affinity-state 5-HT2A/2C receptors in the hypothalamus, but not frontal cortex. No change was observed in the density of [3H]ketanscrin-labeled 5-HT2A receptors in either brain region. The levels of Gq- and G11- proteins in the hypothalamus and cortex were not altered by fluoxetine. These results suggest that fluoxetine gradually increases the G-protein coupling of 5-HT2A/2C receptors without altering the levels of Gq- or G11-proteins.

Antagonism of the effects of the hallucinogen DOM and the purported 5-HT agonist quipazine by 5-HT2 antagonists.

Rats trained to discriminate 1.0 mg/kg of 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM) from saline in a two-lever operant choice task were administered doses of mescaline, LSD, 5-methoxy-N,N-dimethyltryptamine (5-OMe DMT), quipazine, TFMPP and RU-24969. The DOM-stimulus generalized to the three hallucinogenic agents and to quipazine, but not to the purported serotonin agonists TFMPP or RU-24969. Pretreatment of the animals with the 5-HT2 antagonists ketanserin and pirenperone antagonized the effect produced by DOM. Pirenperone also blocked DOM-stimulus generalization to mescaline, LSD, 5-OMe DMT and quipazine. The results of this study suggest that the discriminative stimulus effects of DOM, the three hallucinogenic agents to which DOM-stimulus generalization occurred, and quipazine, may involve those sub-populations of serotonin receptors that are labeled by tritiated ketanserin (i.e. 5-HT2 sites).

Reversal of the acute effects of 3,4-methylenedioxymethamphetamine by 5-HT uptake inhibitors.

Recent evidence suggests that the acute 3,4-methylenedioxymethamphetamine (MDMA)-induced loss of tryptophan hydroxylase activity (TPH) may be due to the oxidation of critical sulf-hydryl groups on the molecule. To determine if TPH activity could be regenerated in vivo we administered a 5-HT uptake inhibitor at various times immediately after MDMA. Although enzyme activity began to decline immediately following MDMA administration, rats receiving the uptake inhibitor 1 h post MDMA showed a rapid recovery of TPH activity. Administration of an uptake inhibitor 3 h post MDMA was without effect on the time course of TPH inactivation. The results suggest that systems exist within the serotonergic neuron for the reductive regeneration of active TPH. Furthermore, these systems are acutely compromised following the administration of MDMA.

The selective 5-HT2 receptor antagonist amperozide attenuates 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane-induced inhibition of male rat sexual behavior.

This study was aimed at exploring the role of 5-HT2/5-HT1C neurotransmission in male rat sexual behavior. The administration of the 5-HT2/5-HT1C agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (1 mg/kg), suppressed sexual activity in most of the animals. The suppressive effect of DOI was antagonized by treatment with amperozide, a selective 5-HT2 receptor antagonist, in doses which did not by themselves affect sexual activity. In addition, several other serotonin antagonists were tested with varying affinity profiles for 5-HT2/5-HT1C receptors, including ketanserin, ritanserin, and mesulergine. All these compounds antagonized the suppressive action of DOI. In contrast, no antagonizing effect was obtained by treatment with (-)-alprenolol, a 5-HT1A antagonist. The present findings suggest that 5-HT2/5-HT1C receptors might be involved in the neural control of male rat sexual behavior, presumably by exerting an inhibitory influence on the behavior.

Effect of cysteine on the persistent depletion of brain monoamines by amphetamine, p-chloroamphetamine and MPTP.

The administration of L-cysteine (500 mg/kg i.p.) 30 min before and 5 h after the administration of (+)-amphetamine sulfate markedly attenuated the persistent decreases in striatal dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in rats one week after the administration of a single dose of amphetamine (9.2 mg/kg i.p.) to iprindole-treated animals and in mice one week after the last of four daily injections of amphetamine (30 mg/kg i.p.). Cysteine prevented the persistent decreases in striatal serotonin (5HT) and 5-hydroxyindoleacetic acid (5HIAA) one week after the administration of p-chloroamphetamine to rats, but failed to alter the persistent decreases in striatal DA, DOPAC and HVA in mice one week after the last of four daily doses of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 30 mg/kg s.c.). The results suggest that the mechanisms by which amphetamine and p-chloroamphetamine, but not MPTP, produce persistent depletions of striatal monoamines involve the generation of neurotoxic electrophilic intermediates which can be inactivated by the administration of cysteine.

5-hydroxytryptamine uptake blockers attenuate the 5-hydroxytryptamine-releasing effect of 3,4-methylenedioxymethamphetamine and related agents.

This study examined the [3H]5-HT-releasing properties of 3,4-methylenedioxymethamphetamine (MDMA) and related agents, all of which cause significant release of [3H]5-HT from rat brain synaptosomes. 5-HT uptake blockers dose dependently block MDMA-induced [3H]5-HT release. The EC50s of the uptake drugs in blocking MDMA-induced release correlate with their affinity for the 5-HT uptake site labeled by [3H]paroxetine (r = 0.98; P less than 0.01). These data demonstrate that the 5-HT uptake carrier plays a significant role in the release of 5-HT induced by MDMA and related agents.