Differential effects of dopamine and psychoactive drugs on dopamine transporter phosphorylation and regulation

The dopamine transporter (DAT) is a phosphoprotein whose activity and phosphorylation state are acutely regulated by both protein kinase C (PKC) and substrate transport. DAT is a major site of action for psychostimulant and therapeutic drugs that either block transport or are transported substrates, but the effects of such drugs on DAT phosphorylation and regulation are not well understood. To examine these issues we subjected rDAT LLC-PK(1) cells to acute in vitro pretreatments with the endogenous, psychostimulant, and therapeutic compounds dopamine (DA), (-)-cocaine, 2 beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (beta-CFT), GBR 12909, mazindol, and methylphenidate (MPH), in the presence or absence of the PKC activator phorbol 12 myristate 13 acetate (PMA), followed by analysis of DAT metabolic phosphorylation and transport activity. Basal phosphorylation of DAT was not affected by any of the uptake blockers tested, and PMA-stimulated phosphorylation was not affected by cocaine, beta-CFT, mazindol or MPH, but was strongly suppressed by GBR 12909. Pretreatment of cells with cocaine or MPH had no effect on subsequent DA transport activity or the extent of PMA-induced transport down-regulation, whereas GBR 12909 inhibited PMA-induced DAT internalization. These findings indicate that these DAT phosphorylation and down-regulation properties are unaffected by some classes of uptake blocking drugs, but that differential regulatory effects may be exerted by GBR compounds. Pretreatment of cells with DA had no obvious effect on basal or PMA-stimulated DAT phosphorylation but led to cocaine-blockable transport down-regulation. DA-induced down-regulation was blocked by the PKC inhibitor bisindoylmaleimide I and was not additive with down-regulation induced by PMA, consistent with PKC serving as a common step and point of integration for these DA and PMA induced processes. The results of this study provide information on the potential for endogenous and psychoactive compounds to modulate or be modulated by DAT phosphorylation-mediated regulatory mechanisms that may contribute to drug behavioral or therapeutic properties.     

Positron emission tomographic studies of brain dopamine and serotonin transporters in abstinent (±)3,4-methylenedioxymethamphetamine (“ecstasy”) users: relationship to cognitive performance

BACKGROUND: (+/-)3,4-Methylenedioxymethamphetamine (MDMA, "ecstasy") is a recreational drug and brain serotonin (5-HT) neurotoxin. Under certain conditions, MDMA can also damage brain dopamine (DA) neurons, at least in rodents. Human MDMA users have been found to have reduced brain 5-HT transporter (SERT) density and cognitive deficits, although it is not known whether these are related. This study sought to determine whether MDMA users who take closely spaced sequential doses, which engender high plasma MDMA concentrations, develop DA transporter (DAT) deficits, in addition to SERT deficits, and whether there is a relationship between transporter binding and cognitive performance. MATERIALS AND METHODS: Sixteen abstinent MDMA users with a history of using sequential MDMA doses (two or more doses over a 3- to 12-h period) and 16 age-, gender-, and education-matched controls participated. Subjects underwent positron emission tomography with the DAT and SERT radioligands, [(11)C]WIN 35,428 and [(11)C]DASB, respectively. Subjects also underwent formal neuropsychiatric testing. RESULTS: MDMA users had reductions in SERT binding in multiple brain regions but no reductions in striatal DAT binding. Memory performance in the aggregate subject population was correlated with SERT binding in the dorsolateral prefrontal cortex, orbitofrontal cortex, and parietal cortex, brain regions implicated in memory function. Prior exposure to MDMA significantly diminished the strength of this relationship. CONCLUSIONS: Use of sequential MDMA doses is associated with lasting decreases in brain SERT, but not DAT. Memory performance is associated with SERT binding in brain regions involved in memory function. Prior MDMA exposure appears to disrupt this relationship. These data are the first to directly relate memory performance to brain SERT density.     

Effects of MDMA on Extracellular Dopamine and Serotonin Levels in Mice Lacking Dopamine and/or Serotonin Transporters

3,4-Methylendioxymethamphetamine (MDMA) has both stimulatory and hallucinogenic properties which make its psychoactive effects unique and different from those of typical psychostimulant and hallucinogenic agents. The present study investigated the effects of MDMA on extracellular dopamine (DA(ex)) and serotonin (5-HT(ex)) levels in the striatum and prefrontal cortex (PFC) using in vivo microdialysis techniques in mice lacking DA transporters (DAT) and/or 5-HT transporters (SERT). subcutaneous injection of MDMA (3, 10 mg/kg) significantly increased striatal DA(ex) in wild-type mice, SERT knockout mice, and DAT knockout mice, but not in DAT/SERT double-knockout mice. The MDMA-induced increase in striatal DA(ex) in SERT knockout mice was significantly less than in wildtype mice. In the PFC, MDMA dose-dependently increased DA(ex) levels in wildtype, DAT knockout, SERT knockout and DAT/SERT double-knockout mice to a similar extent. In contrast, MDMA markedly increased 5-HT(ex) in wildtype and DAT knockout mice and slightly increased 5-HT(ex) in SERT-KO and DAT/SERT double-knockout mice. The results confirm that MDMA acts at both DAT and SERT and increases DA(ex) and 5-HT(ex).     

Effects of annexins II and V on survival of neurons and astrocytes in vitro

To study the effects of annexins II and V on the survival and neurite outgrowth of primary cultured neurons and the survival of astrocytes after peroxide and hypoxia insults in vitro. METHODS: Annexins II and V proteins and/or corresponding antibodies were added to the medium of primary neocortical cultures. H202 and NaN3 were used to induce neuron injury, respectively. Lactate dehydrogenase (LDH) release was measured. RESULTS:Addition of annexin II or V into the culture medium did not affect the normal survival and neurite outgrowth of cortical neurons. However, when an antibody against annexin II or V was added to the culture, the survival and neurite outgrowth of these neurons markedly declined. Further, addition of the two annexins into cortical cultures after peroxide and hypoxia insults markedly reduced the LDH release and cell death. CONCLUSION: Annexins II and V are essential for the survival and neurite outgrowth of developing cortical neurons, the survival of glial cells,and protect neurons and glial cells against peroxide and hypoxia injuries.     

Effects of annexins II and V on survival of neurons and astrocytes in υitro

INTRODUCTIONAnnexins are a family of structurally and func-tionally related proteins that exhibit Ca2 -dependent bind-ing to phospholipids[1-4]. Previous studies suggest thatthese proteins play a role in the development of the cen-tral nervous system (CNS)[5]. In some pathologicalconditions such as traumatic hemorrhage, embolism,and thrombotic infarction[6] or some diseases of the CNSsuch as encephalomyelitis and Alzheimers disease[7],annexin expressions are upregulated. During deve…     

High concentrations of MDMA ('ecstasy') and its metabolite MDA inhibit calcium influx and depolarization-evoked vesicular dopamine release in PC12 cells

The popular synthetic drug of abuse 3,4-methylenedioxymethampetamine (MDMA) and its metabolite 3,4-methylenedioxyamphetamine (MDA) act mainly on the serotonergic system, though they also increase the amount of extracellular dopamine (DA) in the brain, presumably via reversal of the membrane dopamine transporter (DAT). As the involvement of exocytotic DA release is debated, we investigated if these drugs alter the intracellular calcium concentration ([Ca(2+)](i)) and subsequent DA exocytosis in single PC12 cells using respectively Fura-2 imaging and amperometry. MDMA and MDA did not change basal [Ca(2+)](i) or exocytosis, but inhibited depolarization-evoked increases in [Ca(2+)](i) and exocytosis following 15?min exposure to high concentrations of drugs (1?mM). Surprisingly, MDA was more potent in inhibiting exocytosis than MDMA and already inhibited exocytosis at concentrations that did not inhibit depolarization-evoked Ca(2+) influx (10-100?μM). Without 15?min pre-exposure, both drugs failed to inhibit depolarization-evoked Ca(2+) influx. These results indicate that at high concentrations both MDMA and MDA inhibit exocytosis via indirect inhibition of Ca(2+) influx, whereas at lower concentrations MDA may also reduce vesicle cycling. Our data suggest that the DAT-independent increase in extracellular DA in vivo is not due to direct stimulation of exocytosis, but rather to effects of these drugs on other neurotransmitter systems that innervate the dopaminergic system.     

（一）黄皮酰胺体外对胆碱能神经元生长的促进作用

AIM: To study the neurotrophic effects of (-) and (+)clausenamide on frontal cortex neurons in culture. METHODS: The activity of the choline acetyltransferase (ChAT) was determined by spectrophotometric method; protein content was assayed by Folin phenol method. RESULTS: (-)Clausenamide increased the activity of ChAT and protein content in cultured neurons, as well as stimulated proliferation of neuronal cells, support survival and neurite outgrowth of neurons. The neurotrophic action of (-)clausenamide (0.001-10 mumol.L-1) was similar to that of nerve growth factor. The (+)clausenamide had no neurotrophic action, even at high concentrations (0.1-10 mumol.L-1), but neurons were damaged. CONCLUSION: (-)Clausenamide stimulated central cholinergic neuron development.     

MDMA (Ecstasy) and human dopamine,norepinephrine, and serotonin transporters: implications for MDMA-induced neurotoxicity and treatment

Rationale 3,4-Methylenedioxymethamphetamine (MDMA, designated as “Ecstasy” if illicitly marketed in tablet form) induces significant decrements in neuronal serotonin (5-HT) markers in humans, nonhuman primates, and rats as a function of dosing and dosing regimen. In rats, MDMA-mediated effects are attributed, in part, to selective high-affinity transport of MDMA into 5-HT neurons by the 5-HT transporter (SERT), followed by extensive 5-HT release.Objectives To clarify whether SERT-selective effects of MDMA at human monoamine transporters can account for the reported MDMA-induced selective toxicity of serotonin neurons in primate brain.Methods We investigated the interaction of [³H](±, RS)- (+, S)- and (−, R)-MDMA with the human SERT, dopamine (DA) transporter (DAT), and norepinephrine (NE) transporter (NET) in stably transfected human embryo kidney (HEK)-293 cells.Results The human DAT, NET, and SERT actively transported [³H]RS(±)-MDMA saturably, stereoselectively, and in a temperature-, concentration-, and transporter-dependent manner. MDMA exhibited the highest affinity for the NET≫SERT≥DAT, the same rank order for MDMA inhibition of [³H]DA, [³H]NE, and [³H]5-HT transport and stimulated release of the [³H]monoamines, which differed from reports derived from rodent monoamine transporters. The extent of MDMA-induced release of 5-HT was higher compared with release of DA or NE.Conclusions The affinity of MDMA for the human SERT in transfected cells does not clarify the apparent selective toxicity of MDMA for serotonin neurons, although conceivably, its higher efficacy for stimulating 5-HT release may be a distinguishing factor. The findings highlight the need to investigate MDMA effects in DAT-, SERT-, and NET-expressing neurons in the primate brain and the therapeutic potential of NET or DAT inhibitors, in addition to SERT-selective inhibitors, for alleviating the pharmacological effects of MDMA.Klaus A. Miczek as Principal Editor—the special issue “A Contemporary View of MDMA.”This research was presented by C. Verrico et al. in abstract forms at the annual meeting of the Society for Neuroscience 2003 and 2004.     

Role of dopamine transporters in the behavioral effects of 3,4-methylenedioxymethamphetamine (MDMA) in nonhuman primates

Rationale  The interoceptive and reinforcing effects of 3,4-methylenedioxymethamphetamine (MDMA) are similar to those of psychostimulants, but the role of dopamine in the behavioral effects of MDMA is not well documented, especially in primates. Objective  The aim of this study was to assess the role of dopamine in the behavioral effects of MDMA in two nonhuman primate species. Methods  The behavioral effects of MDMA, with and without serotonergic or dopaminergic pretreatments, were studied in squirrel monkeys trained to respond under a fixed-interval schedule of stimulus termination; effects on caudate dopamine levels were studied in a separate group of squirrel monkeys using in vivo microdialysis. Positron emission tomography neuroimaging with the dopamine transporter (DAT) ligand [¹⁸F]FECNT was used to determine DAT occupancy by MDMA in rhesus monkeys. Results  MDMA (0.5–1.5 mg/kg) did not induce behavioral stimulant effects, but the highest dose of MDMA suppressed responding. Pretreatment with fluoxetine (3.0 mg/kg) or the selective 5HT_2A antagonist M100907 (0.03–0.3 mg/kg) attenuated the rate suppressing effects of MDMA. In contrast, pretreatment with the selective dopamine transporter inhibitor RTI-177 (0.1 mg/kg) did not alter the rate suppressing effects of MDMA. Administration of MDMA at a dose that suppressed operant behavior had negligible effects on extracellular dopamine. The percent DAT occupancy of MDMA at a dose that suppressed operant behavior also was marginal and reflected low in vivo potency for DAT binding. Conclusions  Collectively, these results indicate that behaviorally relevant doses of MDMA do not induce behavioral stimulant or dopamine transporter-mediated effects in nonhuman primates.     

重组人Neurturin对体外培养的胚胎大鼠多巴胺能神经元的保护作用

目的建立大鼠胚胎多巴胺能神经元原代培养方法 ,研究重组人Neurturin(rhNTN)体外对黑质多巴胺能神经元的营养和保护作用。方法孕 14dWister大鼠 ,取出胎鼠中脑腹侧区组织 ,经胰酶消化、分散 ,接种至涂有多聚左旋赖氨酸基质的 6孔板中 ,37℃培养 2 4h后将培养液更换为B2 7无血清培养基 ,实验组同时加入纯化的rhNTN。继续培养 ,于培养第 7、14、2 1天直接观察及TH免疫细胞化学染色观察结果。结果培养 14、2 1d ,对照组中多数神经元已经萎缩、死亡 ,少量存活的多巴胺能神经元胞体小 ,突起细短 ;实验组中存活的多巴胺能神经元数目较多 ,神经元胞体粗壮 ,突起长 ,生长状况良好。结论rhNTN能促进中脑黑质多巴胺能神经元的存活及突起生长 ,对多巴胺能神经元具营养和保护作用。     

PCB-induced inhibition of the vesicular monoamine transporter predicts reductions in synaptosomal dopamine content.

Both Aroclor mixtures and individual non-coplanar polychlorinated biphenyl (PCB) congeners reduce dopamine (DA) concentrations in cells in culture and in the brains of developing and adult laboratory animals. These reductions may involve inhibition of the dopamine transporter (DAT) and the vesicular monoamine transporter (VMAT) responsible, respectively, for the uptake of extracellular DA and the packaging of nerve terminal cytosolic DA into synaptic vesicles. However, the relative contribution of each monoamine transporter to the PCB-induced reductions in tissue DA has not been determined. Accordingly, we exposed striatal synaptosomes from adult rats to individual PCB congeners, a commercial mixture of PCBs or known monoamine transporter inhibitors; measured synaptosomal DA; and related these changes to media DA and concentrations of 3,4-dihydroxyphenylacetic (DOPAC). PCB-induced elevations in media DA concentrations are not sufficient to explain the reductions in tissue DA because known DAT inhibitors elevate media DA to a much greater extent than PCBs and yet induce similar decreases in tissue DA concentrations. On the other hand, PCB-induced elevations in DOPAC, reflective of increases in nerve terminal cytosolic DA, are sufficient to explain the reductions in tissue DA, because a known VMAT inhibitor elevates DOPAC and reduces tissue DA to an extent similar to that seen with PCBs. Taken together, these results suggest that elevations in DOPAC, reflective of increases in nerve terminal cytosolic DA due to VMAT inhibition, rather than elevations in media DA due to DAT inhibition, are largely responsible for the observed decreases in tissue DA content.     

Effects of subchronic methamphetamine exposure on basal dopamine and stress-induced dopamine release in the nucleus accumbens shell of rats

Rationale Subchronic administration of stimulants reduces basal dopamine (DA) concentrations and blocks stress-induced DA release in the nucleus accumbens (NA) of rats during withdrawal. However, no studies have attempted to relate early withdrawal from chronic drug exposure to stress reactivity and changes in DA transmission. Objectives The effects of subchronic low-dose methamphetamine (METH) administration on regional changes in dopamine transporter (DAT) and norepinephrine transporter (NET) immunoreactivity and function during early withdrawal were examined. The effects of subchronic METH on stress responsivity measured by DA release in the nucleus accumbens shell (NA SHELL) and core (NA CORE) during acute restraint stress were also examined. Methods Male rats received single injections of METH (2.0 mg/kg i.p.) or saline (SAL) for 10 days and then were killed 24 h after the last injection. DAT and NET protein in NA, striatum (STR), medial prefrontal cortex (mPFC), and hippocampus were assayed by Western blot analysis. Experiment 2 measured basal extracellular DA concentrations and restraint-stress-induced DA release in vivo in the NA SHELL and CORE of SAL- and METH-pretreated rats after 24-h withdrawal. Experiment 3 examined the in vivo regulation of extracellular DA in the NA SHELL and/or CORE after local administration of GBR12909 (50 μM) or nisoxetine (100 μM; NA SHELL). Results Subchronic METH increased DAT but not NET immunoreactivity in the NA compared to the STR and mPFC. METH reduced basal extracellular DA and blocked restraint-stress-induced DA release in the NA SHELL. DA uptake blockade increased extracellular DA more in the NA SHELL of METH rats, whereas NE uptake blockade increased basal DA concentrations to a similar extent in METH and SAL rats. Conclusions These results suggest that subchronic METH exposure selectively increases NA DAT and consequently reduces basal and stress-induced DA release in the NA SHELL during early withdrawal.     

Long-term central 5-HT depletions resulting from repeated administration of MDMA enhances the effects of single administration of MDMA on schedule-controlled behavior of rats

The behavioral effect of single administration of +/- 3,4-methylene-dioxymethamphetamine (MDMA) on rats performing on the differential-reinforcement-of-low-rate 72-second schedule (DRL 72-sec) was compared before and after a period of repeated administration of MDMA known to deplete 5-hydroxytryptamine (5-HT) levels in the brain. Single administration of MDMA decreased reinforcement rate (1, 2, 4, 6 mg/kg) and increased response rate (4,6 mg/kg) of rats performing on the DRL 72-sec schedule. This effect is typical of amphetamines and other psychomotor stimulants. Four weeks after repeated administration of MDMA (6 mg/kg twice daily for 4 days) there was an increase in sensitivity to the effect of single administration of MDMA. Doses of 2, 4 and 6 mg/kg of MDMA resulted in increases in response rate that were significantly greater after repeated MDMA administration than before. Doses of 0.5, 2, and 6 mg/kg of MDMA resulted in decreases of reinforcement rate that were significantly greater after repeated MDMA administration than before. Repeated administration of MDMA resulted in long-term depletion of serotonin levels by 30-50% in the amygdala, neostriatum, hippocampus and the frontal cortex. Norepinephrine and dopamine (DA) levels were not significantly different from control in any of the brain regions analyzed. The behavioral and neurochemical results suggest that serotonergic neurons normally exert an inhibitory action upon the psychomotor stimulant effects of MDMA. Since the psychomotor stimulant effects of amphetamines appear to be mediated primarily by the dopamine system, these results provide evidence that 5-HT and DA may represent opposing systems in the DRL schedule-controlled behavior.     

The abused inhalant toluene increases dopamine release in the nucleus accumbens by directly stimulating ventral tegmental area neurons.

Recreational abuse of toluene-containing volatile inhalants by adolescents is a significant public health problem. The mechanisms underlying the abuse potential of such substances remain unclear, but could involve increased activity in mesoaccumbal dopamine (DA) afferents innervating the nucleus accumbens (ACB). Here, using in vitro electrophysiology, we show that application of behaviorally relevant concentrations of toluene directly stimulates DA neurons in the ventral tegmental area (VTA), but not surrounding midbrain regions. Toluene stimulation of VTA neurons persists when synaptic transmission is reduced. Moreover, unlike non-DA neurons, the magnitude of VTA DA neuron firing does not decline during longer exposures designed to emulate 'huffing'. Using dual-probe in vivo microdialysis, we show that perfusion of toluene directly into the VTA increases DA concentrations in the VTA (somatodendritic release) and its terminal projection site, the ACB. These results provide the first demonstration that even brief exposure to toluene increases action potential drive onto mesoaccumbal VTA DA neurons, thereby enhancing DA release in the ACB. The finding that toluene stimulates mesoaccumbal neurotransmission by activating VTA DA neurons directly (independently of transynaptic inputs) provide insights into the neural substrates that may contribute to the initiation and pathophysiology of toluene abuse.     

Dopamine transporter inhibitory and antiparkinsonian effect of common flowering quince extract

Common flowering quince (FQ) is the fruit of Chaenomeles speciosa (Sweet) Nakai. FQ-containing cocktails have been applied to the treatment of neuralgia, migraine, and depression in traditional Chinese medicine. The present study assessed whether FQ is effective in dopamine transporter (DAT) regulation and antiparkinsonism by utilizing in vitro and in vivo assays, respectively. FQ at concentrations of 1-1000 microg/ml concentration-dependently inhibited dopamine uptake by Chinese hamster ovary (CHO) cells stably expressing DAT (D8 cells) and by synaptosomes. FQ had a slight inhibitory action on norepinephrine uptake by CHO cells expressing the norepinephrine transporter and no inhibitory effect on gamma-aminobutyric acid (GABA) uptake by CHO cells expressing GABA transporter-1 or serotonin uptake by the serotonin transporter. A viability assay showed that FQ mitigated 1-methyl-4-phenylpyridinium-induced toxicity in D8 cells. Furthermore, in behavioral studies, FQ alleviated rotational behavior in 6-hydroxydopamine-treated rats and improved deficits in endurance performance in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. Furthermore, immunohistochemistry revealed that FQ markedly reduced the loss of tyrosine hydroxylase-positive neurons in the substantia nigra in MPTP-treated mice. In summary, FQ is a selective, potent DAT inhibitor and has antiparkinsonian-like effects that are mediated possibly by DAT suppression. FQ has the potential to be further developed for Parkinson's disease treatment.     

The control of dopamine neuron development,function and survival: insights from transgenic mice and the relevance to human disease

Transgenic technology, especially the use of homologous recombination to disrupt specific genes to produce knockout mice, has added considerably to the understanding of dopamine (DA) neuron develop, survival and function. The current review summarizes results from knockout mice with the target disruption of genes involved in the development of DA neurons (engrailed 1 and 2, lmx1b, and Nurr1), in maintaining DA neurotransmission (tyrosine hydroxylase, vesicular monoamine transporter, DA transporter, DA D2 and D3 receptors) and important for DA neuron survival (alpha-synuclein, glia cell line-derived neurotrophic factor and superoxide dismutase). As alterations in DA neurotransmission have been implicated in a number of human neuropathologies including Parkinson's disease, schizophrenia and attention deficit/hyperactivity disorder, understanding how specific genes are involved in the function of DA neurons and the compensatory changes that result from loss or reduction in gene expression could provide important insight for the treatment of these diseases.     

The dopamine transporter: a vigilant border control for psychostimulant action

Neurotransmission within the mesocorticolimbic dopamine system has remained the central focus of investigation into the molecular, cellular and behavioral properties of psychostimulants for nearly three decades. The primary means by which dopamine transmission in the synapse is terminated is via the dopamine transporter (DAT), the presynaptic plasmalemmal protein that is responsible for the reuptake of released dopamine. Numerous abused as well as clinically important drugs have important pharmacological interactions with DAT. In general, these compounds fall into two categories: those that block dopamine transport (e.g., cocaine, methylphenidate) and those that serve as substrates for transport [e.g., dopamine, amphetamine and 3,4-methylenedioxymethamphetamine (MDMA or "ecstasy")]. Recent data from in vitro and in vivo studies have suggested that DAT, like other biogenic amine transporters, share several characteristics with classical ligand-gated ion channels. In addition, substrates for transport promote redistribution of DAT away from the plasma membrane, while transport inhibitors such as cocaine disrupt this process. In addition, presynaptic autoreceptors for dopamine have been implicated in the modulation of DAT surface expression and function. The present chapter summarizes some of the recent discoveries pertaining to the electrogenic properties of DAT and their potential relevance to the effects of amphetamine-like stimulants on DAT function. Although there are a number of intracellular and extracellular modulatory influences on dopamine clearance that may play particular roles in psychostimulant action, we specifically focus on the differential direct modulation of DAT function by transport substrates and inhibitors, and we also discusses the role of presynaptic D2 receptors in transport regulation.     

MDMA-induced impairment in primates: antagonism by a selective norepinephrine or serotonin, but not by a dopamine/norepinephrine transport inhibitor

Human MDMA (R,S-3,4-methylenedioxymethamphetamine) users display selective cognitive deficits after acute MDMA exposure, frequently attributed to serotonin deficits. We postulated that MDMA will compromise executive function in primates and that an inhibitor of the serotonin transporter (SERT) and the norepinephrine transporter (NET) but not the dopamine (DAT) transporter, will prevent impairment. The potencies of DAT/NET, NET and SERT inhibitors to block transport of [(3)H]MDMA and [(3)H]monoamines were compared in vitro. Subsequently, cynomolgus monkeys (Macaca fasicularis) were trained to stable performance in a reversal learning task. Effects of once-weekly oral or i.m. dose of MDMA (1.5 mg/kg, n = 4) on performance were monitored, alone or after pretreatment with inhibitors of the SERT, DAT or NET (prior to i.m. MDMA). 1) Drug potencies for blocking [(3)H]MDMA or [(3)H]monoamine transport were not consistent; 2) Oral MDMA increased error rates in a cognitive task for up to three days following exposure, whereas intramuscular MDMA prevented subjects from performing the cognitive task on the day of administration, but not on subsequent days; 3) The SERT inhibitor citalopram and the NET inhibitor desipramine, but not the DAT/NET inhibitor methylphenidate, reversed the effects of MDMA on task performance and mandibular movements induced by i.m. MDMA and 4) MDMA altered sleep latency. Oral MDMA impairs executive function in monkeys for several days, a finding of potential relevance to MDMA consumption by humans. Reversal of impaired executive function by a NET inhibitor implicates the NET and norepinephrine in MDMA-induced cognitive impairment and may be relevant to therapeutic strategies.     

Effect of nerve growth factor (NGF) on survival of superior cervical ganglion (SCG) transplanted into the third ventricle in rats

Effect of short-term exposure to nerve growth factor (NGF) on neuron survival of superior cervical ganglion (SCG) transplanted into the third ventricle in rats and that on neurite outgrowth of SCG neurons in a tissue culture system were examined. Because SCG taken from 3 week-old rats exhibited a high survival rate in transplantation and they were highly sensitive to NGF in culture, they were used in the short-term NGF treatment experiment. SCG from 3 week-old rats were preincubated with NGF for 30 min at room temperature, and then they were cultured for 2 days or grafted for 14 days. Short-term exposure to NGF at the concentration of 10 micrograms/ml enhanced the survival of transplanted SCG neurons. The same concentration of NGF enhanced neurite outgrowth of SCG neurons in culture. These results suggest that short-term NGF pretreatment could increase the efficiency of neuronal transplantation.     

Dopamine D2 receptor regulation of the dopamine transporter expressed in Xenopus laevis oocytes is voltage-independent

Presynaptic dopamine D2 receptors (D2Rs) regulate dopamine transporter (DAT) activity in the brain. A potential mechanism was suggested by the observations that somatodendritic D2R activation produces hyperpolarization and the velocity of DAT expressed in Xenopus laevis oocytes varies with changes in membrane potential. To investigate whether D2R regulation of DAT function is voltage-dependent, we coexpressed the long isoform of the human (h) D2R and the hDAT in oocytes. Most DAT substrates fully activate D2Rs at concentrations used to measure uptake. Thus, DAT function was compared under conditions of maximal D2R activation (0.1-10 microM DA) or maximal D2R blockade (DA + 1 microM (-)-sulpiride). D2R activation significantly increased [3H]DA uptake into unclamped oocytes expressing relatively lower velocities. Uptake measured with a saturating concentration of DA suggested a D2R-induced increase in Vmax. The D2R-mediated enhancement of DA uptake was not associated with changes in resting membrane potential and was abolished by pertussis toxin pretreatment. Furthermore, in voltage-clamped oocytes, D2R activation enhanced both DA uptake and DAT-mediated steady-state currents by as much as 70%. Activation of D2Rs resulted in a 59% increase in cell surface binding of the cocaine analog [3H]WIN 35,428; this effect was also abolished by pertussis toxin pretreatment. Saturation experiments confirmed that D2R activation was associated with an increased Bmax and unchanged Ki for [3H]WIN 35,428. These results suggest that D2R-induced up-regulation of DAT activity occurs via a voltage-independent mechanism that depends on G(i/o) activation and a rapid increase in expression of functional DAT molecules at the cell surface.