Dopamine Transporter Loss in 6-OHDA Parkinson’s Model Is Unmet by Parallel Reduction in Dopamine Uptake

The dopamine transporter (DAT) regulates synaptic dopamine (DA) in striatum and modulation of DAT can affect locomotor activity. Thus, in Parkinson’s disease (PD), DAT loss could affect DA clearance and locomotor activity. The locomotor benefits of L-DOPA may be mediated by transport through monoamine transporters and conversion to DA. However, its impact upon DA reuptake is unknown and may modulate synaptic DA. Using the unilateral 6-OHDA rat PD model, we examined [³H]DA uptake dynamics in relation to striatal DAT and tyrosine hydroxylase (TH) protein loss compared with contralateral intact striatum. Despite >70% striatal DAT loss, DA uptake decreased only ∼25% and increased as DAT loss approached 99%. As other monoamine transporters can transport DA, we determined if norepinephrine (NE) and serotonin (5-HT) differentially modulated DA uptake in lesioned striatum. Unlabeled DA, NE, and 5-HT were used, at a concentration that differentially inhibited DA uptake in intact striatum, to compete against [³H]DA uptake. In 6-OHDA lesioned striatum, DA was less effective, whereas NE was more effective, at inhibiting [³H]DA uptake. Furthermore, norepinephrine transporter (NET) protein levels increased and desipramine was ∼two-fold more effective at inhibiting NE uptake. Serotonin inhibited [³H]DA uptake, but without significant difference between lesioned and contralateral striatum. L-DOPA inhibited [³H]DA uptake two-fold more in lesioned striatum and inhibited NE uptake ∼five-fold more than DA uptake in naïve striatum. Consequently, DA uptake may be mediated by NET when DAT loss is at PD levels. Increased inhibition of DA uptake by L-DOPA and its preferential inhibition of NE over DA uptake, indicates that NET-mediated DA uptake may be modulated by L-DOPA when DAT loss exceeds 70%. These results indicate a novel mechanism for DA uptake during PD progression and provide new insight into how L-DOPA affects DA uptake, revealing possible mechanisms of its therapeutic and side effect potential.     

Disruption of the PDZ domain–binding motif of the dopamine transporter uniquely alters nanoscale distribution,dopamine homeostasis,and reward motivation

The dopamine (DA) transporter (DAT) is part of a presynaptic multiprotein network involving interactions with scaffold proteins via its C-terminal PDZ domain–binding sequence. Using a mouse model expressing DAT with mutated PDZ-binding sequence (DAT-AAA), we previously demonstrated the importance of this binding sequence for striatal expression of DAT. Here, we show by application of direct stochastic reconstruction microscopy not only that the striatal level of transporter is reduced in DAT-AAA mice but also that the nanoscale distribution of this transporter is altered with a higher propensity of DAT-AAA to localize to irregular nanodomains in dopaminergic terminals. In parallel, we observe mesostriatal DA adaptations and changes in DA-related behaviors distinct from those seen in other genetic DAT mouse models. DA levels in the striatum are reduced to ∼45% of that of WT, accompanied by elevated DA turnover. Nonetheless, fast-scan cyclic voltammetry recordings on striatal slices reveal a larger amplitude and prolonged clearance rate of evoked DA release in DAT-AAA mice compared with WT mice. Autoradiography and radioligand binding show reduced DA D2 receptor levels, whereas immunohistochemistry and autoradiography show unchanged DA D1 receptor levels. In behavioral experiments, we observe enhanced self-administration of liquid food under both a fixed ratio of one and progressive ratio schedule of reinforcement but a reduction compared with WT when using cocaine as reinforcer. In summary, our data demonstrate how disruption of PDZ domain interactions causes changes in DAT expression and its nanoscopic distribution that in turn alter DA clearance dynamics and related behaviors.     

Prenatal Amphetamine Exposure Effects on Dopaminergic Receptors and Transporter in Postnatal Rats

We investigated the influence of prenatal amphetamine exposure (PAE) on dopamine (DA) receptors, and dopamine transporter (DAT) in various striatal and limbic subregions and locomotor activity induced by novel environmental conditions and amphetamine at two postnatal ages, 35 days old (prepubertal) and 60 days old (postpubertal). Experiments were carried out on pregnant female Sprague–Dawley rats, which were daily injected with either d-amphetamine sulfate (1 mg/kg) or saline solution (0.9%) for 11 days, from gestation day 11–21. In PAE rats compared to control we found the following: at pre-pubertal age, an enhancement of DA D1 in the dorsolateral area of the caudate-putamen (CPu), CPu-ventral and shell of the nucleus accumbens (NAcc) with a decrement of the DA D3 receptors in NAcc, olfactory tubercle (OT), and the islands of Calleja (IoC); whereas at postpubertal age, an increase in the levels of DAT in the NAcc and fundus of the CPu, and OT along with a decrease in the expression of DA D2 receptors only in the NAcc shell were found in PAE rats compared to control. In addition, amphetamine induces a marked decrease in locomotor activity at postpubertal age in rats with PAE. These results suggest a differential effect of amphetamines on the DAT mechanism of the nervous system during embryonic development of animals with implications in behavior and drug addictions at adulthood age.     

Dopamine down-regulates activity of alkaline phosphatase in Drosophila: The role of D2-like receptors

The effect of a rise in dopamine (DA) level as a result of a mutation, stress or pharmacological treatment on the activity of the enzyme of its synthesis, alkaline phosphatase (ALP) in females of Drosophila virilis and Drosophila melanogaster has been studied. It has been found that regardless of its nature, a rise in DA level has a negative effect on ALP activity, which indicates that DA down-regulates activity of the enzyme. The effects of bromocriptine (an agonist of Drosophila dopamine 2-like receptor (DD2R)) on ALP activity have been studied. ALP activity was found to drop in response to bromocriptine in flies. Conversely ALP activity was increased in flies with reduced DD2R expression (i.e. Actin5C-Gal4 > UAS-ds-DD2R RNA-interference flies) vs. corresponding controls (i.e. Actin5C-Gal4 > w1118 flies). Bromocriptine treatment of RNAi flies rescues ALP activity to the level typical of Actin5C-Gal4 > w1118 flies. A change in DD2R number or availability was found not to prevent the response of ALP to heat stress, but to change the intensity of its response to the stress exposure. The role of D2-like receptors in down-regulation of ALP activity by DA and in ALP response to stressor in Drosophila is discussed.     

Microglial calcium signal acts as a rapid sensor of single neuron damage in vivo

In the healthy adult brain microglia, the main immune-competent cells of the CNS, have a distinct (so-called resting or surveying) phenotype. Resting microglia can only be studied in vivo since any isolation of brain tissue inevitably triggers microglial activation. Here we used in vivo two-photon imaging to obtain a first insight into Ca²⁺ signaling in resting cortical microglia. The majority (80%) of microglial cells showed no spontaneous Ca²⁺ transients at rest and in conditions of strong neuronal activity. However, they reliably responded with large, generalized Ca²⁺ transients to damage of an individual neuron. These damage-induced responses had a short latency (0.4-4 s) and were localized to the immediate vicinity of the damaged neuron (< 50 μm cell body-to-cell body distance). They were occluded by the application of ATPγS as well as UDP and 2-MeSADP, the agonists of metabotropic P2Y receptors, and they required Ca²⁺ release from the intracellular Ca²⁺ stores. Thus, our in vivo data suggest that microglial Ca²⁺ signals occur mostly under pathological conditions and identify a Ca²⁺ store-operated signal, which represents a very sensitive, rapid, and highly localized response of microglial cells to brain damage. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

β-Phenylethylamine requires the dopamine transporter to increase extracellular dopamine in Caenorhabditis elegans dopaminergic neurons

β-Phenylethylamine (βPEA) is an endogenous amine that has been shown to increase the synaptic levels of dopamine (DA). A number of in vitro and behavioral studies suggest the dopamine transporter (DAT) plays a role in the effects generated by βPEA, however the mechanism through which βPEA affects DAT has not yet been elucidated. Here, we used Caenorhabditis (C.) elegans DAT (DAT-1) expressing LLC-pk1 cells and neuronal cultures to investigate whether the βPEA-induced increase of extracellular DA required DAT-1. Our data show that βPEA increases extracellular dopamine both in DAT-1 transfected cells and cultures of differentiated neurons. RTI-55, a cocaine homologue and DAT inhibitor, completely blocked the βPEA-induced effect in transfected cells. However in neuronal cultures, RTI-55 only partly inhibited the increase of extracellular DA generated by βPEA. These results suggest that βPEA requires DAT-1 and other, not yet identified proteins, to increase extracellular DA when tested in a native system. Furthermore, our results suggest that βPEA-induced increase of extracellular DA does not require functional monoamine vesicles as genetic ablation of the C. elegans homologue vesicular monoamine transporter, cat-1, did not compromise the ability of βPEA to increase extracellular DA. Finally, our electrophysiology data show that βPEA caused fast-rising and self-inactivating amperometric currents in a subset of wild-type DA neurons but not in neurons isolated from dat-1 knockout animals. Taken together, these data demonstrate that in both DA neurons and heterogeneous cultures of differentiated C. elegans neurons, βPEA releases cytoplasmic DA through DAT-1 to ultimately increase the extracellular concentration of DA.     

Altered dopamine transporter function and phosphorylation following chronic cocaine self-administration and extinction in rats

Cocaine binds with the dopamine transporter (DAT), an effect that has been extensively implicated in its reinforcing effects. However, persisting adaptations in DAT regulation after cocaine self-administration have not been extensively investigated. Here, we determined the changes in molecular mechanisms of DAT regulation in the caudate-putamen (CPu) and nucleus accumbens (NAcc) of rats with a history of cocaine self-administration, followed by 3 weeks of withdrawal under extinction conditions (i.e., no cocaine available). DA uptake was significantly higher in the CPu of cocaine-experienced animals as compared to saline-yoked controls. DAT V_max was elevated in the CPu without changes in apparent affinity for DA. In spite of elevated CPu DAT activity, total and surface DAT density and DAT-PP2Ac (protein phosphatase 2A catalytic subunit) interaction remained unaltered, although p-Ser- DAT phosphorylation was elevated. In contrast to the CPu, there were no differences between cocaine and saline rats in the levels of DA uptake, DAT V_max and K_m values, total and surface DAT, p-Ser-DAT phosphorylation, or DAT-PP2Ac interactions in the NAcc. These results show that chronic cocaine self-administration leads to lasting, regionally specific alterations in striatal DA uptake and DAT-Ser phosphorylation. Such changes may be related to habitual patterns of cocaine-seeking observed during relapse.     

Comparative pharmacology of two D1-like dopamine receptors cloned from the silkworm Bombyx mori

Dopamine (DA) is a physiologically important biogenic amine in insect peripheral and nervous tissues. We recently cloned two DA receptors (BmDopR1 and BmDopR2) from the silkworm Bombyx mori and identified them as D1-like receptors, which activate adenylate cyclase to increase intracellular cAMP levels. In this study, these two receptors were stably expressed in HEK-293 cells, and the dose-responsiveness to DA and their pharmacological properties were examined using cAMP assays. BmDopR1 showed a dose-dependent increase in cAMP levels at DA concentrations up to 10^?7 M with EC₅₀ of 3.30 nM, while BmDopR2 required 10^?6 M DA for activation. In BmDopR1-expressing cells, DA at 10^?6–10^?4 M induced 30–50% lower cAMP production than 10^?7 M DA. BmDopR2-expressing cells showed a standard sigmoidal dose–response, with maximum cAMP levels attained with 10^?5–10^?4 M DA and EC₅₀ of 1.30 μM. Both receptors had similar agonist profiles, and the typical vertebrate D1-like receptor agonist SKF-38393 was ineffective. Experiments with antagonists revealed that BmDopR1 exhibits D1-like features. However, the pharmacology of BmDopR2 was distinct from D1-like receptors; the typical vertebrate D1-like receptor antagonist SCH-23390 was less potent than the nonselective antagonist flupenthixol and the D2-like receptor antagonist chlorpromazine. The rank order of activities of several antagonists for BmDopR1 and BmDopR2 was more similar to that of Drosophila melanogaster DA receptors than Apis mellifera DA receptors. These data suggest that DA receptors could be potential targets for specific insecticides or insectistatics.     

Amphetamine and Methamphetamine Differentially Affect Dopamine Transporters in Vitro and in Vivo

The psychostimulants d-amphetamine (AMPH) and methamphetamine (METH) release excess dopamine (DA) into the synaptic clefts of dopaminergic neurons. Abnormal DA release is thought to occur by reverse transport through the DA transporter (DAT), and it is believed to underlie the severe behavioral effects of these drugs. Here we compare structurally similar AMPH and METH on DAT function in a heterologous expression system and in an animal model. In the in vitro expression system, DAT-mediated whole-cell currents were greater for METH stimulation than for AMPH. At the same voltage and concentration, METH released five times more DA than AMPH and did so at physiological membrane potentials. At maximally effective concentrations, METH released twice as much [Ca²⁺]_i from internal stores compared with AMPH. [Ca²⁺]_i responses to both drugs were independent of membrane voltage but inhibited by DAT antagonists. Intact phosphorylation sites in the N-terminal domain of DAT were required for the AMPH- and METH-induced increase in [Ca²⁺]_i and for the enhanced effects of METH on [Ca²⁺]_i elevation. Calmodulin-dependent protein kinase II and protein kinase C inhibitors alone or in combination also blocked AMPH- or METH-induced Ca²⁺ responses. Finally, in the rat nucleus accumbens, in vivo voltammetry showed that systemic application of METH inhibited DAT-mediated DA clearance more efficiently than AMPH, resulting in excess external DA. Together these data demonstrate that METH has a stronger effect on DAT-mediated cell physiology than AMPH, which may contribute to the euphoric and addictive properties of METH compared with AMPH.The dopamine transporter (DAT)³ is a main target for psychostimulants, such as d-amphetamine (AMPH), methamphetamine (METH), cocaine (COC), and methylphenidate (Ritalin®). DAT is the major clearance mechanism for synaptic dopamine (DA) (1) and thereby regulates the strength and duration of dopaminergic signaling. AMPH and METH are substrates for DAT and competitively inhibit DA uptake (2, 3) and release DA through reverse transport (4–9). AMPH- and METH-induced elevations in extracellular DA result in complex neurochemical changes and profound psychiatric effects (2, 10–16). Despite their structural and pharmacokinetic similarities, a recent National Institute on Drug Abuse report describes METH as a more potent stimulant than AMPH with longer lasting effects at comparable doses (17). Although the route of METH administration and its availability must contribute to the almost four times higher lifetime nonmedical use of METH compared with AMPH (18), there may also be differences in the mechanisms that underlie the actions of these two drugs on the dopamine transporter.Recent studies by Joyce et al. (19) have shown that compared with d-AMPH alone, the combination of d- and l-AMPH in Adderall® significantly prolonged the time course of extracellular DA in vivo. These experiments demonstrate that subtle structural features of AMPH, such as chirality, can affect its action on dopamine transporters. Here we investigate whether METH, a more lipophilic analog of AMPH, affects DAT differently than AMPH, particularly in regard to stimulated DA efflux.METH and AMPH have been reported as equally effective in increasing extracellular DA levels in rodent dorsal striatum (dSTR), nucleus accumbens (NAc) (10, 14, 20), striatal synaptosomes, and DAT-expressing cells in vitro (3, 6). John and Jones (21), however, have recently shown in mouse striatal and substantia nigra slices, that AMPH is a more potent inhibitor of DA uptake than METH. On the other hand, in synaptosomes METH inhibits DA uptake three times more effectively than AMPH (14), and in DAT-expressing COS-7 cells, METH releases DA more potently than AMPH (EC₅₀ = 0.2 μm for METH versus EC₅₀ = 1.7 μm for AMPH) (5). However, these differences do not hold up under all conditions. For example, in a study utilizing C6 cells, the disparity between AMPH and METH was not found (12).The variations in AMPH and METH data extend to animal models. AMPH- and METH-mediated behavior has been reported as similar (22), lower (20), or higher (23) for AMPH compared with METH. Furthermore, although the maximal locomotor activation response was less for METH than for AMPH at a lower dose (2 mg/kg, intraperitoneal), both drugs decreased locomotor activity at a higher dose (4 mg/kg) (20). In contrast, in the presence of a salient stimuli, METH is more potent in increasing the overall magnitude of locomotor activity in rats yet is equipotent with AMPH in the absence of these stimuli (23).The simultaneous regulation of DA uptake and efflux by DAT substrates such as AMPH and METH, as well as the voltage dependence of DAT (24), may confound the interpretation of existing data describing the action of these drugs. Our biophysical approaches allowed us to significantly decrease the contribution of DA uptake and more accurately determine DAT-mediated DA efflux with millisecond time resolution. We have thus exploited time-resolved, whole-cell voltage clamp in combination with in vitro and in vivo microamperometry and Ca²⁺ imaging to compare the impact of METH and AMPH on DAT function and determine the consequence of these interactions on cell physiology.We find that near the resting potential, METH is more effective than AMPH in stimulating DAT to release DA. In addition, at efficacious concentrations METH generates more current, greater DA efflux, and higher Ca²⁺ release from internal stores than AMPH. Both METH-induced or the lesser AMPH-induced increase in intracellular Ca²⁺ are independent of membrane potential. The additional Ca²⁺ response induced by METH requires intact phosphorylation sites in the N-terminal domain of DAT. Finally, our in vivo voltammetry data indicate that METH inhibits clearance of locally applied DA more effectively than AMPH in the rat nucleus accumbens, which plays an important role in reward and addiction, but not in the dorsal striatum, which is involved in a variety of cognitive functions. Taken together these data imply that AMPH and METH have distinguishable effects on DAT that can be shown both at the molecular level and in vivo, and are likely to be implicated in the relative euphoric and addictive properties of these two psychostimulants.     

Membrane-permeable C-terminal Dopamine Transporter Peptides Attenuate Amphetamine-evoked Dopamine Release

The dopamine transporter (DAT) is responsible for sequestration of extracellular dopamine (DA). The psychostimulant amphetamine (AMPH) is a DAT substrate, which is actively transported into the nerve terminal, eliciting vesicular depletion and reversal of DA transport via DAT. Here, we investigate the role of the DAT C terminus in AMPH-evoked DA efflux using cell-permeant dominant-negative peptides. A peptide, which corresponded to the last 24 C-terminal residues of DAT (TAT-C24 DAT) and thereby contained the Ca²⁺-calmodulin-dependent protein kinase IIα (CaMKIIα) binding domain and the PSD-95/Discs-large/ZO-1 (PDZ)-binding sequence of DAT, was made membrane-permeable by fusing it to the cell membrane transduction domain of the HIV-1 Tat protein (TAT-C24WT). The ability of TAT-C24WT but not a scrambled peptide (TAT-C24Scr) to block the CaMKIIα-DAT interaction was supported by co-immunoprecipitation experiments in heterologous cells. In heterologous cells, we also found that TAT-C24WT, but not TAT-C24Scr, decreased AMPH-evoked 1-methyl-4-phenylpyridinium efflux. Moreover, chronoamperometric recordings in striatum revealed diminished AMPH-evoked DA efflux in mice preinjected with TAT-C24WT. Both in heterologous cells and in striatum, the peptide did not further inhibit efflux upon KN-93-mediated inhibition of CaMKIIα activity, consistent with a dominant-negative action preventing binding of CaMKIIα to the DAT C terminus. This was further supported by the ability of a peptide with perturbed PDZ-binding sequence, but preserved CaMKIIα binding (TAT-C24AAA), to diminish AMPH-evoked DA efflux in vivo to the same extent as TAT-C24WT. Finally, AMPH-induced locomotor hyperactivity was attenuated following systemic administration of TAT-C24WT but not TAT-C24Scr. Summarized, our findings substantiate that DAT C-terminal protein-protein interactions are critical for AMPH-evoked DA efflux and suggest that it may be possible to target protein-protein interactions to modulate transporter function and interfere with psychostimulant effects.     

Dopamine Dynamics and Signaling in Drosophila: An Overview of Genes,Drugs and Behavioral Paradigms

Changes in dopamine (DA) signaling have been implicated in a number of human neurologic and psychiatric disorders. Similarly, defects in DA signaling in the fruit fly, Drosophila melanogaster, have also been associated with several behavioral defects. As most genes involved in DA synthesis, transport, secretion, and signaling are conserved between species, Drosophila is a powerful genetic model organism to study the regulation of DA signaling in vivo. In this review, we will provide an overview of the genes and drugs that regulate DA biology in Drosophila. Furthermore, we will discuss the behavioral paradigms that are regulated by DA signaling in flies. By analyzing the genes and neuronal circuits that govern such behaviors using sophisticated genetic, pharmacologic, electrophysiologic, and imaging approaches in Drosophila, we will likely gain a better understanding about how this neuromodulator regulates motor tasks and cognition in humans.     

Biotic and abiotic regulation of resting spore formation in vivo of obligate aphid pathogen Pandora nouryi: Modeling analysis and biological implication

Entomophthoralean fungus Pandora nouryi is an obligate aphid pathogen that enables to produce resting spores (azygospores) for surviving host absence. To explore possible mechanisms involved in the regulation of resting spore formation in vivo, host cohorts consisting of 40-60 nymphs of green peach aphid Myzus persicae produced within 24 h on cabbage leaf discs in petri dishes were exposed to spore showers of P. nouryi at the concentrations (C) from a very few to nearly 2000 conidia/mm² and then reared for 7-11 days at the regimes of 10-25 °C (T) and 8-16 h daylight (H_L) or ambient (17.5 ± 3.1 °C, 13:11 L:D). Aphid mortalities observed from 35-83 cohorts (showered separately) at each regime showed typical sigmoid trend and fit well a general logistic equation (0.79 ? r² ? 0.88), yielding similar LC₅₀ estimates of 1.7-6.1 conidia/mm². The proportions (P) of cadavers forming resting spores in the cohorts also fit the same equation (0.73 ? r² ? 0.85) at all tested regimes except at 10 °C, a low temperature for the host-pathogen interaction. This indicates the dependence of resting spore formation on the spore concentration. The effects of T and H_L on P over C were well elucidated by the fitted modified logistic equations P = 0.578/{1 + exp[1.710 − (0.136 − 0.0053T)C]} and P = 0.534/{1 + exp[1.639 + (0.034 − 0.0053H_L)C]} (both r² = 0.79). Our results highlight that the resting spore formation in vivo of P. nouryi is regulated primarily by the concentration of host-infecting conidia discharged from cadavers and facilitated by lower temperature and longer daylight.     

Computational and Biochemical Docking of the Irreversible Cocaine Analog RTI 82 Directly Demonstrates Ligand Positioning in the Dopamine Transporter Central Substrate-binding Site

The dopamine transporter (DAT) functions as a key regulator of dopaminergic neurotransmission via re-uptake of synaptic dopamine (DA). Cocaine binding to DAT blocks this activity and elevates extracellular DA, leading to psychomotor stimulation and addiction, but the mechanisms by which cocaine interacts with DAT and inhibits transport remain incompletely understood. Here, we addressed these questions using computational and biochemical methodologies to localize the binding and adduction sites of the photoactivatable irreversible cocaine analog 3β-(p-chlorophenyl)tropane-2β-carboxylic acid, 4′-azido-3′-iodophenylethyl ester ([¹²⁵I]RTI 82). Comparative modeling and small molecule docking indicated that the tropane pharmacophore of RTI 82 was positioned in the central DA active site with an orientation that juxtaposed the aryliodoazide group for cross-linking to rat DAT Phe-319. This prediction was verified by focused methionine substitution of residues flanking this site followed by cyanogen bromide mapping of the [¹²⁵I]RTI 82-labeled mutants and by the substituted cysteine accessibility method protection analyses. These findings provide positive functional evidence linking tropane pharmacophore interaction with the core substrate-binding site and support a competitive mechanism for transport inhibition. This synergistic application of computational and biochemical methodologies overcomes many uncertainties inherent in other approaches and furnishes a schematic framework for elucidating the ligand-protein interactions of other classes of DA transport inhibitors.     

Changes in Neuronal Dopamine Homeostasis following 1-Methyl-4-phenylpyridinium (MPP+) Exposure

1-Methyl-4-phenylpyridinium (MPP⁺), the active metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, selectively kills dopaminergic neurons in vivo and in vitro via a variety of toxic mechanisms, including mitochondrial dysfunction, generation of peroxynitrite, induction of apoptosis, and oxidative stress due to disruption of vesicular dopamine (DA) storage. To investigate the effects of acute MPP⁺ exposure on neuronal DA homeostasis, we measured stimulation-dependent DA release and non-exocytotic DA efflux from mouse striatal slices and extracellular, intracellular, and cytosolic DA (DA_cyt) levels in cultured mouse ventral midbrain neurons. In acute striatal slices, MPP⁺ exposure gradually decreased stimulation-dependent DA release, followed by massive DA efflux that was dependent on MPP⁺ concentration, temperature, and DA uptake transporter activity. Similarly, in mouse midbrain neuronal cultures, MPP⁺ depleted vesicular DA storage accompanied by an elevation of cytosolic and extracellular DA levels. In neuronal cell bodies, increased DA_cyt was not due to transmitter leakage from synaptic vesicles but rather to competitive MPP⁺-dependent inhibition of monoamine oxidase activity. Accordingly, monoamine oxidase blockers pargyline and l-deprenyl had no effect on DA_cyt levels in MPP⁺-treated cells and produced only a moderate effect on the survival of dopaminergic neurons treated with the toxin. In contrast, depletion of intracellular DA by blocking neurotransmitter synthesis resulted in ∼30% reduction of MPP⁺-mediated toxicity, whereas overexpression of VMAT2 completely rescued dopaminergic neurons. These results demonstrate the utility of comprehensive analysis of DA metabolism using various electrochemical methods and reveal the complexity of the effects of MPP⁺ on neuronal DA homeostasis and neurotoxicity.     

Polymorphism of the COMT,MAO, DAT,NET and 5-HTT Genes,and Biogenic Amines in Parkinson’s Disease

Epinephrine (E) and sympathetic nerve stimulation were described by Thomas Renton Elliott in 1905 for the first time. Dopamine (DA), norepinephrine (NE), E, and serotonin (5-HT) belong to the classic biogenic amines (or monoamines). Parkinson’s disease (PD) is among the diseases in which it has been established that catecholamines may account for the neurodegeneration of central and peripheral catecholamine neural systems. PD is a chronic and progressive neurological disorder characterized by resting tremor, rigidity, and bradykinesia, affecting 2% of individuals above the age of 65 years. This disorder is a result of degeneration of DA-producing neurons of the substantia nigra and a significant loss of noradrenergic neurons in the locus coeruleus. In PD and other related neurodegerative diseases, catecholamines play the role of endogenous neurotoxins. Catechol-O-methyltransferase (COMT) and/or monoamine oxidase (MAO) catalyze the metabolism of monoamines. However, the monoamine transporters for DA, NE, and 5-HT namely DAT, NET, and SERT, respectively regulate the monoamine concentration. The metabolism of catecholamines and 5-HT involves common factors. Monoamine transporters represent targets for many pharmacological agents that affect brain function, including psychostimulators and antidepressants. In PD, polymorphisms of the COMT, MAO, DAT, NET, and 5- HTT genes may change the levels of biogenic amines and their metabolic products. The currently available therapies for PD improve the symptoms but do not halt the progression of the disease. The most effective treatment for PD patients is therapy with L-dopa. Combined therapy for PD involves a DA agonist and decarboxylase, MAOs and COMT inhibitors, and is the current optimal form of PD treatment maintaining monoamine balance.     

Upregulation of tumor necrosis factor-alpha in nucleus accumbens attenuates morphine-induced rewarding in a neuropathic pain model

Treatment of neuropathic pain with opioid analgesics remains controversial and a major concern is the risk of addiction. Here, we investigated this issue with spared nerve injury (SNI) model of neuropathic pain in rats and mice. SNI prevented conditioned place preference (CPP) induced by low dose (3.5 mg/kg) of morphine (MOR), which was effective for anti-allodynia, but not by high dose (?5.0 mg/kg) of MOR. Tumor necrosis factor-alpha (TNF-α) was upregulated in nucleus accumbens (NAcc) following SNI. The inhibitory effect of SNI on MOR-induced CPP was blocked by either genetic deletion of TNF receptor 1 (TNFR1) or microinjection of anti-TNF-α into the NAcc and was mimicked by intra-NAcc injection of TNF-α in sham rats. Furthermore, SNI reduced dopamine (DA) level and upregulated dopamine transporter (DAT) in the NAcc, but did not affect total tyrosine hydroxylase (TH) or phospho-TH (p-TH), a rate-limiting enzyme of catecholamine biosynthesis, in ventral tegmental area (VTA). Accordingly, the increase in DA reuptake but not decrease in its synthesis may lead to the reduction of DA level. Finally, the upregulation of DAT in the NAcc of SNI animals was again blocked by either genetic deletion of TNFR1 or NAcc injection of anti-TNF-α, and was mimicked by NAcc injection of TNF-α in sham animals. Thus, our data provided novel evidence that upregulation of TNF-α in NAcc may attenuate MOR-induced rewarding by upregulation of DAT in NAcc under neuropathic pain condition.     

No differential regulation of dopamine transporter (DAT) and vesicular monoamine transporter 2 (VMAT2) binding in a primate model of Parkinson disease

Radioligands for DAT and VMAT2 are widely used presynaptic markers for assessing dopamine (DA) nerve terminals in Parkinson disease (PD). Previous in vivo imaging and postmortem studies suggest that these transporter sites may be regulated as the numbers of nigrostriatal neurons change in pathologic conditions. To investigate this issue, we used in vitro quantitative autoradioradiography to measure striatal DAT and VMAT2 specific binding in postmortem brain from 14 monkeys after unilateral internal carotid artery infusion of 1-Methyl-4-Phenyl-1,2,3,6-tetrahydropyridine (MPTP) with doses varying from 0 to 0.31 mg/kg. Quantitative estimates of the number of tyrosine hydroxylase (TH)-immunoreactive (ir) neurons in substantia nigra (SN) were determined with unbiased stereology, and quantitative autoradiography was used to measure DAT and VMAT2 striatal specific binding. Striatal VMAT2 and DAT binding correlated with striatal DA (r_s = 0.83, r_s = 0.80, respectively, both with n = 14, p<0.001) but only with nigra TH-ir cells when nigral cell loss was 50% or less (r = 0.93, n = 8, p = 0.001 and r = 0.91, n = 8, p = 0.002 respectively). Reduction of VMAT2 and DAT striatal specific binding sites strongly correlated with each other (r = 0.93, n = 14, p<0.0005). These similar changes in DAT and VMAT2 binding sites in the striatal terminal fields of the surviving nigrostriatal neurons demonstrate that there is no differential regulation of these two sites at 2 months after MPTP infusion.     

Paradoxical Abatement of Striatal Dopaminergic Transmission by Cocaine and Methylphenidate

We combined in vitro amperometric, optical analysis of fluorescent false neurotransmitters and microdialysis techniques to unveil that cocaine and methylphenidate induced a marked depression of the synaptic release of dopamine (DA) in mouse striatum. In contrast to the classical dopamine transporter (DAT)-dependent enhancement of the dopaminergic signal observed at concentrations of cocaine lower than 3 μm, the inhibitory effect of cocaine was found at concentrations higher than 3 μm. The paradoxical inhibitory effect of cocaine and methylphenidate was associated with a decrease in synapsin phosphorylation. Interestingly, a cocaine-induced depression of DA release was only present in cocaine-insensitive animals (DAT-CI). Similar effects of cocaine were produced by methylphenidate in both wild-type and DAT-CI mice. On the other hand, nomifensine only enhanced the dopaminergic signal either in wild-type or in DAT-CI mice. Overall, these results indicate that cocaine and methylphenidate can increase or decrease DA neurotransmission by blocking reuptake and reducing the exocytotic release, respectively. The biphasic reshaping of DA neurotransmission could contribute to different behavioral effects of psychostimulants, including the calming ones, in attention deficit hyperactivity disorder.