MDMA: further evidence that its action in the medial prefrontal cortex is mediated by the serotonergic system.

Systemically administered (+/-)-MDMA (3,4-methylenedioxymethamphetamine, 'Ecstasy') suppressed the firing rates of the majority of neurons in the medial prefrontal cortex (mPFc). The responses of mPFc cells to (+/-)-MDMA is mimicked by (+)-MDMA but not (-)-MDMA. Furthermore, pretreatment with fluoxetine (a specific 5-HT uptake blocker) but not GBR 12909 (a specific dopamine uptake blocker) prevented the suppressant action of MDMA. These data support the notion that the 5-HT system mediates (+/-)-MDMA's action.     

MDMA ('ecstasy') enhances basal acetylcholine release in brain slices of the rat striatum

The pharmacological basis of acute (+/-)-MDMA (3, 4-methylenedioxymethamphetamine) intoxication still awaits full characterization. According to present knowledge, MDMA enhances the release of serotonin and dopamine in striatal slices and interacts with different types of receptors such as 5-HT2 (5-hydroxytryptamine or serotonin), M1 and M2 muscarinic acetylcholine (ACh), and histamine H1 receptors. Currently, no information is available about the influence of (+/-)-MDMA on striatal cholinergic neurotransmission. In the present study, we used the in vitro perfusion technique to investigate the effect of (+/-)-MDMA on ACh release in rat striatal slices. Perfusions with (+/-)-MDMA (10-300 microM) resulted in a dose-dependent increase of spontaneous ACh release (EC50 approximately 30 microM). The effect was reversible and Ca++- and tetrodotoxin-sensitive. To determine the neurochemical pathways underlying this response, we perfused with (+/-)-MDMA in the presence of various inhibitors of neurotransmitter receptors. Blockade of glutamate or muscarinic ACh receptors as well as 5-HT1, 5-HT2, 5-HT3C or dopamine D2 receptors did not modulate (+/-)-MDMA-induced ACh release. However, the presence of histamine H1 receptor antagonists in the perfusion medium abolished (+/-)-MDMA-induced ACh release. The present data clearly demonstrate that (+/-)-MDMA enhances the activity of striatal cholinergic neurons and suggest an involvement of histamine H1 receptors. The effect is not mediated by glutamate and does not involve the activation of receptors of dopamine D2, 5-HT1, 5-HT2, 5-HT3C or muscarinic ACh. Considering the relatively high affinity of (+/-)-MDMA for the H1 histamine receptor (Ki 6 microM), a direct activation of this type of receptor might represent a plausible mechanism for (+/-)-MDMA-induced ACh release.     

Release of serotonin induced by 3,4-methylenedioxymethamphetamine (MDMA) and other substituted amphetamines in cultured fetal raphe neurons: further evidence for calcium-independent mechanisms of release

The substituted amphetamines 3,4-methylenedioxyamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), p-chloroamphetamine (PCA) and fenfluramine (FEN) all exert their effects by releasing serotonin (5-HT) from presynaptic nerve terminals. In the current study, we examined the ability of these agents to induce the release of 5-HT in cultured fetal raphe neurons. The data indicate that the rank order of release potencies for these agents was (±)PCA > (+)MDMA = (+)MDA = (±)FEN. Studies examining the role of calcium in 5-HT release demonstrate that preventing calcium influx with L- and N-type calcium channel blockers inhibits potassium-stimulated release of [³H]5-HT but has no effect on release induced by the substituted amphetamines. Furthermore, omitting calcium from the extracellular media or depleting the vesicular pool of neurotransmitter with continual potassium stimulation did not affect the release of [³H]5-HT induced by these compounds. Administration of fluoxetine prior to the substituted amphetamines significantly attenuated the releasing effects of these agents, while producing no effect on potassium-stimulated release. These results are consistent with the notion that the amphetamines induce release of cytoplasmic 5-HT via the plasma membrane transporter.     

MDMA induced dopamine release in vivo: role of endogenous serotonin

Summary Acting as a substrate at the serotonin (5-HT) transporter, (+)-MDMA (3,4-methylenedioxymethamphetamine), is a potent releaser of 5-HT and causes toxicity to 5-HT neurons after repeated exposure. (+)-MDMA also releases dopamine (DA), although with less potency. Since we have shown previously that the intrastriatal application of 5-HT facilitates DA release, it was hypothesized that increased release of striatal 5-HT after MDMA may influence extracellular levels of DA. Using microdiaiysis in vivo, we found that (+)-MDMA (4.7 mol/kg, i.v.) administration increased extracellular striatal DA levels to 501% of control (p < 0.01, n=12). However, in the presence of fluoxetine (14.4 mol/kg, s.c.), which prevents (+)-MDMA effects on 5-HT release, the (+)-MDMA-induced increase in DA was significantly less (to 375% of control, p < 0.05, vs. no fluoxetine, n=8). In vitro studies with striatal slices, to test drug selectivity, showed that (+)-MDMA (0.3-3 M) increased extracellular levels of both DA and 5-HT in a dose-dependent manner. Fluoxetine (3 M) completely blocked the effects of (+)-MDMA on 5-HT release, but did not alter (+)-MDMA-induced DA release in vitro. The selective DA transport inhibitor GBR-12909 (1 M), blocked (+)-MDMA's effect on DA release. It is concluded that 5-HT release after (+)-MDMA treatment partially contributes to (+)-MDMA's effect on DA release in vivo.     

Feedback Stimulation of Somatodendritic Serotonin Release: A 5-HT3 Receptor-Mediated Effect in the Raphe Nuclei of the Rat

Slices from rat midbrain containing the raphe nuclei and from hippocampus were prepared, loaded with [³H]5-HT and superfused and the resting and the electrically stimulated [³H]5-HT release was measured. The 5-HT₃ receptor agonist 2-methyl-5-HT (1 to 10 μmol/l) increased the resting tritium outflow in superfused raphe nuclei slices, EC₅₀ 5.3 μmol/l. The 2-methyl-5-HT-induced increase of tritium outflow was an external Ca²⁺-independent process and was not altered by reserpine pretreatment but it was reversed by addition of the 5-HT uptake inhibitor fluoxetine (1 μmol/l). The 5-HT₃ receptor antagonists ondansetron and GYKI-46 903 (1 μmol/l) did not antagonize the stimulatory effect of 2-methyl-5-HT on resting tritium outflow. 2-Methyl-5-HT in lower concentration increased the electrically induced tritium overflow from raphe nuclei slices (EC₅₀ 0.56 μmol/l) and also from hippocampal slices preloaded with [³H]5-HT. These effects were reversed by 1 μmol/l of ondansetron and GYKI-46903. The 5-HT₃ receptor antagonists (1 μmol/l) were without effects on depolarization-evoked [³H]5-HT release at 2 Hz stimulation, when 10 Hz stimulation was used, ondansetron and GYKI-46 903 reduced the tritium overflow from raphe nuclei slices. These data indicate that 5-HT₃ receptors positively alter depolarization-induced somatodendritic 5-HT release in the raphe nuclei. They also show that 2-methyl-5-HT is able to evoke 5-HT release not only from vesicles but also from cytoplasmic stores via a transporter-dependent exchange process.     

Influence of inducers and inhibitors on the metabolism in vitro and neurochemical effects in vivo of MDMA

(S)-(+)- and (R)-(-)-3,4-methylenedioxymethamphetamine (MDMA) were metabolized in vitro by rat liver microsomes via N-demethylation to 3,4-methylenedioxyamphetamine (MDA). Whereas no difference was found in the biotransformation of the two enantiomers in the male rat or in the phenobarbital (PB) treated animals of either sex, more than twice as much MDA was formed from (S)-(+)- than from (R)-(-)-MDMA in the untreated female rat. Although 3-methylcholanthrene (3MC) pretreated rat liver microsomes were less active than those from the untreated rats of the same sex, they formed more MDA from (+)- than from (-)-MDMA. The enantioselective metabolism thus appears to be associated with the relative abundance of individual cytochrome P-450 isozymes. (S)-(+)- and (R)-(-)-MDMA.HCl (20 mg/kg) were about equipotent in depleting serotonin (5-HT) levels in the frontal cortex at 3 hrs and 1 wk following oral administration to female rats. Pretreatment of rats with SKF-525A attenuated and that with PB enhanced the 5-HT depleting potential of either isomer at 3 hrs. The 5-HT depleting potency of (+)-MDMA was significantly greater than that of its (-)-antipode at 3 hr in PB pretreated, but not in SKF-525A pretreated animals. The results suggest that the neurochemical effects of MDMA are caused by the formation of an active metabolite in vivo, and since both enantiomers were N-demethylated in vitro to approximately the same extent by PB pretreated rat liver microsomes, the active metabolite may be other than MDA.     

Effect of nerve activity on the in vivo release of [3H]serotonin continuously formed from L-[3H]tryptophan in the caudate nucleus of the cat.

A new isotopic approach has been developed to study the in vivo release of serotonin (5-HT). 'Encéphale isolé' cats were implanted with a push-pull cannula in the ventrocaudal part of the head of the caudate nucleus to estimate the release of [3H]5-HT continuously synthesized from L-[3H]tryptophan. Both [3H]5-HT and [3H]tryptamine were found in superfusates. Resting steady state in the release of [3H]indoleamines was observed as soon as 20 min after the beginning of the superfusion with L-[3H]tryptophan; the levels of [3H]5-HT in superfusates were 2.5 times those of [3H]tryptamine and about 6 times the blank value. They were markedly enhanced in the presence of fluoxetine (5 x 10(-6)M), a blocker of the 5-HT uptake process. A marked increase in the release of [3H]5-HT was seen during the local depolarization of 5-HT terminals with potassium chloride (60 mM) or batrachotoxin (10(-6)M) or during the stimulation of 5-HT cell bodies in the nucleus raphe dorsalis with L-glutamic acid (5 x 10(-5)M). These treatments did not enhance the efflux of [3H]tryptamine. The potassium-evoked release of [3H]5-HT was reduced by LSD (10(-5)M). LSD added alone in the superfusing fluid was without effect. The batrachotoxin-evoked release of [3H]5-HT was inhibited in the presence of tetrodotoxin (9 x 10(-6)M). The spontaneous release of [3H]5-HT and [3H]tryptamine was markedly reduced in the presence of a calcium-free medium containing cobalt (10 mM). A transient slight reduction in the spontaneous release of [3H]5-HT was observed in the presence of tetrodotoxin (9 x 10(-6)M). The local cooling of 5-HT cell bodies with a cryoelectrode induced a slight reversible decrease in [3H]5-HT release. These last two treatments were without significant effect on [3H]tryptamine efflux in superfusates. These results indicate that the release of [3H]5-HT endogenously formed from [3H]tryptophan is dependent on nerve activity and that this is not the case for [3H]tryptamine. The advantages of the isotopic approach for in vivo studies on the release of 5-HT are discussed.     

Changes in 5-HT1A receptor binding and G-protein activation in the rat brain after estrogen treatment: comparison with tamoxifen and raloxifene

OBJECTIVE: It is thought that an imbalance in serotonergic neurotransmission may underlie many affective disorders. Thus, the serotonin-1A (5-HT1A) receptor is a target for antidepressant and neuroleptic drugs. It has been reported that estrogens modulate serotonergic neurotransmission. Therefore, we investigated the effect of long-term ovariectomy on 5-HT1A receptor-specific binding and G-protein activation in the brain. Correction therapy with estradiol was compared with treatments using the selective estrogen receptor modulators tamoxifen and raloxifene. METHODS: Four months after ovariectomy, Sprague-Dawley rats were treated with vehicle, 17beta-estradiol (80 microg/kg), tamoxifen (1 mg/kg) or raloxifene (1 mg/kg) subcutaneously for 2 weeks. Specific binding to 5-HT1A receptors was assessed by autoradiography of brain sections using the 5-HT1A agonist [3H]8-OH-DPAT. 5-HT1A receptor stimulation was measured using R-(+)-8-OH-DPAT-stimulated [35S]GTPgammaS-binding autoradiography. RESULTS: Ovariectomy decreased uterine weight, which was corrected by estradiol; tamoxifen and raloxifene partially corrected this decrease. Hormonal withdrawal and replacement left [3H]8-OH-DPAT-specific binding unchanged in the cortex. In contrast, ovariectomy induced a decrease in R-(+)-8-OH-DPAT-stimulated [35S]GTPgammaS-specific binding in the cortex; this was corrected by estradiol but was not corrected significantly by tamoxifen or raloxifene. In the hippocampus, ovariectomy had no effect on [3H]8-OH-DPAT-specific binding, whereas only 17beta-estradiol treatment decreased this binding in a subregion of the CA3. Ovariectomy increased R-(+)-8-OH-DPAT-stimulated [35S]GTPgammaS-specific binding in the dentate gyrus (but not in the CA1 or CA3); this was corrected by estradiol and raloxifene, but not by tamoxifen. In the dorsal raphe nucleus, ovariectomy increased [3H]8-OH-DPAT-specific binding and R-(+)-8-OH-DPAT-stimulated [35S]GTPgammaS-specific binding; estradiol corrected this increase, but this was not corrected significantly by tamoxifen or raloxifene. CONCLUSIONS: An overall stimulation by estradiol of 5-HT1A receptor-specific binding and coupling was observed, decreasing raphe somatodendritic receptors and increasing cortical postsynaptic receptors.     

Lack of serotonin neurotoxicity after intraraphe microinjection of (+)-3,4-methylenedioxymethamphetamine (MDMA).

Systemic administration of 3,4-methylenedioxymethamphetamine (MDMA) produces depletions of serotonin (5-HT) and its primary metabolite, 5-hydroxyindoleacetic acid (5-HIAA), decreases 5-HT reuptake sites and diminishes tryptophan hydroxylase activity in various forebrain regions. MDMA has been shown to be neurotoxic to the fine fibers originating from dorsal raphe (DR) 5-HT neurons but not the beaded fibers from the median raphe (MR) nucleus. In the present experiment, MDMA was microinjected directly into the DR or MR to determine whether differential neurotoxicity developed in the DR versus MR fiber systems as measured by 5-HT levels and immunocytochemistry. Two weeks following stereotaxic injection with either vehicle or (+)MDMA (50 micrograms base in 2 microliters) into the DR or MR, rat brains were assayed for 5-HT and catecholamine content or 5-HT immunocytochemistry. HPLC analysis revealed no significant changes in monoamine or metabolite concentrations in the hippocampus and striatum of rats administered intra-DR or -MR (+)MDMA. Raphe sections stained for 5-HT also did not reveal any apparent neurotoxicity. A single cerebral injection of (+)MDMA does not produce neurotoxicity to 5-HT neuronal systems originating in the raphe, although neurotoxicity of multiple MDMA injections into these raphe nuclei cannot be ruled out.     

Excitatory amino acid-evoked release of [3H]GABA from hippocampal neurons in primary culture

We investigated the release of gamma-[2,3-3H(N)]aminobutyric acid ([3H]GABA) from hippocampal neurons in primary cell culture. [3H]GABA release was stimulated by the excitatory amino acid neurotransmitter glutamate as well as by N-methyl-D-aspartate (NMDA) and kainate. Cell depolarization induced by raising [K+]o or by veratridine also stimulated [3H]GABA release. NMDA-induced release was completely blocked by 3-((+/-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP+), Mg2+ and Zn2+ whereas the release induced by glutamate and kainate was much less susceptible to inhibition by these substances. Furthermore, removal of external Ca2+ inhibited NMDA-induced release, but not that induced by glutamate, kainate, veratridine or 50 mM K+. Removal of external Na+ reduced [3H]GABA release evoked by all stimuli, but to different extents. All of the excitatory amino acids tested increased [Ca2+]i within hippocampal neurons as assessed by fura-2 based microspectrofluorimetry. This increase in [Ca2+]i was completely dependent on the presence of external Ca2+. These results suggest that Ca2+-dependent and -independent forms of GABA release from hippocampal interneurons may occur. [3H]GABA release evoked by glutamate, kainate, veratridine or 50 mM K+, appeared to be mediated by the reversal of electrogenic, Na+-coupled GABA uptake. Release was inhibited by nipecotic acid, an inhibitor of the Na+-coupled GABA uptake system. However, release induced by NMDA may also include a Ca2+-dependent component.     

Ca-dependent release of [H]GABA in cultured chick retina cells

Depolarization by K+ (50 mM) of cultured chick retina cells released 1.14 +/- 0.28% of the accumulated [3H] gamma-aminobutyric acid (GABA) in the absence of Ca2+, but when 1.0 mM Ca2+ was present, the internal free calcium ion concentration [Ca2+]i rose by about 750 nM and the [3H]GABA release about doubled to a value of 2.22 +/- 0.2% of the total [3H]GABA. Nitrendipine (0.1 microM), a blocker of the L-type Ca2+ channels, blocked the [Ca2+]i response to K+ depolarization by about 65%, and the omega-Conotoxin GVIA (omega-CgTx) (0.5 microM), a blocker of the N-type of Ca2+ channels, inhibited by 27% the [Ca2+]i rise due to K+ depolarization. Parallel experiments showed that nitrendipine inhibits [3H]GABA release to the level observed in the absence of Ca2+, whereas omega-CgTx did not inhibit significantly the release of [3H]GABA. The results also show that the release of [3H]GABA due to K(+)-depolarization in the absence of Ca2+ can be totally blocked by 1-(2-(((Diphenylmethylene) amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride (NNC-711), an inhibitor of the GABA carrier. However, in the presence of Ca2+, NNC-711 blocks the release only by about 66%, corresponding to the Ca(2+)-independent release. Thus, it is concluded that [3H]GABA is released in chick retina cells by the exocytotic mechanism, which is Ca(2+)-dependent, and by reversal of the carrier, which is Ca(2+)-independent, in much the same way as has been found for other GABAergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics and autoradiography of high affinity uptake of serotonin by primary astrocyte cultures

Primary astrocyte cultures prepared from the cerebral cortices of neonatal rats showed significant accumulation of serotonin (5-hydroxytryptamine; [3H]-5-HT). At concentrations in the range of 0.01 to 0.7 microM [3H]-5-HT, this uptake was 50 to 85% Na+ dependent and gave a Km of 0.40 +/- 0.11 microM [3H]-5-HT and a Vmax of 6.42 +/- 0.85 (+/- SEM) pmol of [3H]-5-HT/mg of protein/4 min for the Na+-dependent component. In the absence of Na+ the uptake was nonsaturable. Omission of the monoamine oxidase inhibitor pargyline markedly reduced the Na+-dependent component of [3H]-5-HT uptake but had a negligible effect on the Na+-independent component. This suggest significant oxidative deamination of serotonin after it has been taken up by the high affinity system, followed by release of its metabolite. We estimated that this system enabled the cells to concentrate [3H]-5-HT up to 44-fold at an external [3H]-5-HT concentration of 10(-7) M. Inhibition of [3H]-5-HT uptake by a number of clinically effective antidepressants was also consistent with a specific high affinity uptake mechanism for 5-HT, the order of effectiveness of inhibition being chlorimipramine greater than fluoxetine greater than imipramine = amitriptyline greater than desmethylimipramine greater than iprindole greater than mianserin. Uptake of [3H]-5-HT was dependent on the presence of Cl- as well as Na+ in the medium, and the effect of omission of both ions was nonadditive. Varying the concentration of K+ in the media from 1 to 50 mM had a limited effect on [3H]-5-HT uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduced uptake and release of 5-hydroxytryptamine and taurine in the cerebral cortex of epileptic El mice

Inbred mutant El mice are highly susceptible to convulsive seizures upon 'throwing' stimulation, and the inhibition of 5-hydroxytryptamine (5-HT) and taurine activities appears to be involved in the El mouse seizures. Uptake and release of [3H]5-HT and [3H]taurine into and from cerebral neurocortical slices using a superfusion system were investigated in both non-stimulated and stimulated El mice [El(-), El(+)] and in ddY mice, which do not have a convulsive disposition. Release was defined as 40 mM K+-stimulated release. 5-HT and taurine uptake in El(+) was lower than El(-) but no difference in either uptake was found between ddY and El(-). Release of 5-HT and taurine in El(-) was higher than in ddY whereas their release in El(+) was lower than in El(-). The taurine level in the cerebral neocortex of El(-) and El(+) was higher than in ddY. These results suggest that the synaptic function of the 5-HT and taurine containing neurons is suppressed and that dysfunction of these inhibitory neurons is involved in the seizure susceptibility in the El mice.     

Veratridine-induced breakdown of cytosolic acetylcholine in rat hippocampal minces: An intraterminal form of acetylcholinesterase or choline O-acetyltransferase?

Rat hippocampal minces were loaded with N-methyl-[3H]acetylcholine ([3H]ACh) in the presence of the 'poorly penetrating' acetylcholinesterase (EC 3.1.1.7, AChE) inhibitor echothiophate and the effect of the depolarizing agent veratridine determined on the subcellular storage and release of [3H]ACh and [3H]choline. Results indicated that veratridine stimulated the release of [3H]ACh from a crude vesicular fraction (P3) by a Ca2+-dependent process, while simultaneously accelerating the breakdown of cytosolic (S3) [3H]ACh. A portion of the [3H]choline derived from the hydrolyzed S3 [3H]ACh was donated to the P3 fraction for [3H]ACh formation and release. When the identical experiment was done using hippocampal minces from septal lesioned rats, veratridine did not stimulate either the Ca2+-dependent release of [3H]ACh or the hydrolysis of cytosolic [3H]ACh. Incubation of control hippocampal minces with paraoxon, an AChE inhibitor which can penetrate cholinergic nerve terminals more rapidly than echothiophate, prevented veratridine from stimulating the Ca2+-dependent release of [3H]ACh from the P3 fraction. Instead, it then stimulated the Ca2+-independent release of [3H]ACh from the S3 fraction. When minces were incubated with the choline O-acetyltransferase (EC 2.3.1.6, ChAT) inhibitor 4-(1-naphthyl)vinyl pyridine (NVP), veratridine was no longer able to stimulate the Ca2+-dependent release of labelled ACh either. Instead, veratridine stimulated the Ca2+-independent release of labelled ACh from the S3 fraction. NVP also abolished the veratridine-induced, Ca2+-dependent release of total ACh. Both paraoxon and NVP inhibited the reversible reaction of ionically bound ChAT prepared from rat brain when tested in vitro, yet paraoxon was much less potent than NVP, and was unable to inhibit this reaction at the low concentration which prevented the veratridine induced breakdown of S3 [3H]ACh during mince incubation. Veratridine depolarization of hippocampal minces stimulated the activity of a membrane-bound fraction of ChAT associated with the P3 fraction, but this fraction of ChAT did not become more sensitive to inhibition by paraoxon during tissue incubation. Veratridine depolarization of minces also increased the activity of membrane-bound AChE, but this enzyme was not inhibited by the low NVP concentration which prevented the veratridine-induced breakdown of S3 [3H]ACh. The veratridine-induced increase in membrane-bound ChAT activity was dependent on the presence of extracellular Ca2+ in the incubation medium.(ABSTRACT TRUNCATED AT 400 WORDS)     

The neurotoxic effects of 3,4-methylenedioxymethamphetamine (MDMA) and methamphetamine on serotonin, dopamine, and GABA-ergic terminals: an in-vitro autoradiographic study in rats

Damage to serotonin (5-HT) terminals following doses of 3,4-methylenedioxymethamphetamine (MDMA) is well documented, and this toxicity is thought to be related to dopamine release that is potentiated by the 5-HT(2A/2C) agonist effects of the drug. Although MDMA and methamphetamine (METH) have some similar dopaminergic activities, they differ in their 5-HT agonistic properties. It is reasoned that the study of the resultant toxicity following equimolar doses of MDMA and METH on both dopamine and 5-HT terminals should offer a comparison of the ability of these drugs to induce neurotoxicity. In order to measure the toxic effects to the brain, rats were given equimolar doses of MDMA (40 mg/kg/day) and METH (32 mg/kg/day) in subcutaneously implanted osmotic minipumps for a period of 5 days, and in-vitro autoradiography using [3H]-paroxetine, [3H]-mazindol, [3H]-methylspiperone, and [3H]-flunitrazepam, was performed on brain sections. The results showed that METH was more toxic to 5-HT terminals than MDMA in forebrain regions, including the anterior cingulate, caudate nucleus, nucleus accumbens, and septum. METH was also more toxic than MDMA to dopamine terminals in the habenula, and posterior retrosplenial cortex. Therefore, we find that METH was more toxic to 5-HT and dopamine terminals in specific brain regions in both pre and post-synaptic sites following continuous equimolar dosing.     

Neurochemical neutralization of amphetamine-type stimulants in rat brain by the indatraline analog (-)-HY038

Amphetamine-type stimulants are substrates for the proteins that serve as transporters for the biogenic amines dopamine (DA), serotonin (5HT), and norepinephrine (NE) and release these neurotransmitters from neurons located in the peripheral and central nervous system. Using indatraline as a lead compound, we sought to develop a long-acting depot medication that would neutralize the deleterious effects of amphetamine-type stimulants. Our first efforts produced (+/-)-HY038, and its two stereoisomers, which are hydroxy-substituted analog of indatraline. The K(i) values for [(3)H]DA reuptake inhibition by (-)-HY038 and (+)-HY038 were 3.2 +/- 0.1 and 32 +/- 1 nM. Similar results were obtained for [(3)H]5HT reuptake inhibition. (-)-HY038 and (+)-HY038 were slightly less potent at inhibiting [(3)H]NE reuptake (K(i) values of 20 +/- 2 and 159 +/- 12 nM). Low doses of (-)-HY038 blunted the ability of AMPH to release [(3)H]DA by shifting the AMPH dose-response curve to the right in a dose-dependent manner. (-)-HY038 also inhibited the ability of (+)-methamphetamine and (+/-)-3,4-methylenedioxymethamphetamine ((+/-)-MDMA) to release [(3)H]DA. Low doses of (-)-HY038 blunted the ability of these stimulants to release [(3)H]NE and [(3)H]5HT by shifting their dose-response curves to the right in a manner similar to that seen for inhibition of [(3)H]DA release. These data indicate that (-)-HY038 inhibits the ability of AMPH, (+)-methamphetamine and (+/-)-MDMA to release DA, NE, and 5HT and therefore might have the potential to neutralize the neurotoxic and cardiovascular side-effects of substrate-type stimulants.     

Effect of nerve activity on the in vivo release of [H]serotonin continuously formed from l-[H]tryptophan in the caudate nucleus of the cat

A new isotopic approach has been developed to study the in vivo release of serotonin (5-HT). ‘Ence´phale isole´cats were implanted with a push-pull cannula in the ventrocaudal part of the head of the caudate nucleus to estimate the release of [³H]5-HT continuously synthesized froml-[³H]tryptophan. Both [³H]5-HT and [³H]tryptamine were found in superfusates. Resting steady state in the release of [³H]indoleamines was observed as soon as 20 min after the beginning of the superfusion withl-[³H]tryptophan; the levels of [³H]5-HT in superfusates were 2.5 times those of [³H]tryptamine and about 6 times the blank value. They were markedly enhanced in the presence of fluoxetine (5 × 10^-6M), a blocker of the 5-HT uptake process.A marked increase in the release of [³H]5-HT was seen during the local depolarization of 5-HT terminals with potassium chloride (60 mM) or batrachotoxin (10^-6M) or during the stimulation of 5-HT cell bodies in the nucleus raphe dorsalis withl-glutamic acid (5 × 10^-5M). These treatments did not enhance the efflux of [³H]tryptamine. The potassium-evoked release of [³H]5-HT was reduced by LSD (10^-5M). LSD added alone in the superfusing fluid was without effect. The batrachotoxin-evoked release of [³H]5-HT was inhibited in the presence of tetrodotoxin (9 × 10^-6M).The spontaneous release of [³H]5-HT and [³H]tryptamine was markedly reduced in the presence of a calcium-free medium containing cobalt (10 mM). A transient slight reduction in the spontaneous release of [³H]5-HT was observed in the presence of tetrodotoxin (9 × 10^-6M). The local cooling of 5-HT cell bodies with a cryoelectrode induced a slight reversible decrease in [³H]5-HT release. These last two treatments were without significant effect on [³H]tryptamine efflux in superfusates.     

Examination of the action of 3,4-methylenedioxymethamphetamine on rat A10 dopamine neurons

Extracellular single cell recording was used to examine the effect of intravenous administration of (−), (+), and (±)-3,4-methylenedioxymethamphetamine (MDMA) on A10 dopamine (DA) neurons in chloral hydrate anesthetized male rats. Both (±)-MDMA and (+)-MDMA inhibited the firing rate of most (79%) A10 DA cells. By contrast, (−)-MDMA induced either no effect or a slight increase in the firing rate of these cells. Analysis of the effects of (±)-MDMA on the firing pattern of the DA cells revealed an overall decrease in the percentage of spikes in bursts but both increases and decreases were seen in the coefficient of variation of interspike intervals. To determine the contribution of 5-HT and DA to the (±)-MDMA-induced inhibition of A10 DA cells rats were pretreated either with the 5-HT synthesis inhibitor p-chlorophenylalanine (PCPA) or the DA synthesis inhibitor α-methyl-p-tyrosine (AMPT). Pretreatment of rats with PCPA did not reduce the ability of (±)-MDMA to inhibit the DA cells. However, in rats pretreated with AMPT, the (±)-MDMA-induced inhibition was blocked and some cells (44%) showed instead an increase in firing rate following administration of (±)-MDMA. The administration of l-3,4-dihydroxyphenylalanine (L -DOPA) to AMPT-treated rats rapidly restored the inhibition of cell firing by (±)-MDMA. In conclusion, the results reported here demonstrate that MDMA has an overall inhibitory effect on A10 DA cells. Despite MDMA's greater potency in releasing 5-HT compared to DA, the inhibitory effect of this drug on A10 DA cells appears to be mediated by the latter transmitter. © 1996 Wiley-Liss, Inc.     

Direct effects of 3,4-methylenedioxymethamphetamine (MDMA) on serotonin or dopamine release and uptake in the caudate putamen, nucleus accumbens, substantia nigra pars reticulata, and the dorsal raphé nucleus slices

We examined the effects of pressure ejected 3, 4-methylenedioxymethamphetamine (MDMA) from a micropipette on direct chemically stimulated release, and on electrically stimulated serotonin (5-HT) or dopamine (DA) release in the caudate putamen (CPu), nucleus accumbens (NAc), substantia nigra pars reticulata (SNr), and the dorsal raphé nucleus (DRN) brain slices of rat, using fast cyclic voltammetry (FCV). MDMA is electroactive, oxidising at +1100 mV. When the anodic input waveform was reduced from +1.4 to +1.0 volt, MDMA was not electroactive. Using this waveform, pressure ejection of MDMA did not release 5-HT or DA in brain slices prepared from any of the nuclei studied. MDMA significantly potentiated electrically stimulated 5-HT release in the SNr and DA release in CPu. In the DRN or in the NAc, MDMA was without effect on peak electrically stimulated 5-HT or DA release. The rates of neurotransmitter uptake, expressed as t(1/2), were in all cases significantly decreased after MDMA. The results indicate that MDMA, unlike (+)amphetamine, is not as a releaser of DA or 5-HT, it is a potent inhibitor of both DA and 5-HT uptake.