Dopamine Sequestration Research Articles

Effect of altered production and storage of dopamine on development and behavior in C. elegans.

The nematode, Caenorhabditis elegans (C. elegans), is an advantageous model for studying developmental toxicology due to its well-defined developmental stages and homology to humans. It has been established that across species, dopaminergic neurons are highly vulnerable to neurotoxicant exposure, resulting in developmental neuronal dysfunction and age-induced degeneration. C. elegans, with genetic perturbations in dopamine system proteins, can provide insight into the mechanisms of dopaminergic neurotoxicants. In this study, we present a comprehensive analysis on the effect of gene mutations in dopamine-related proteins on body size, development, and behavior in C. elegans. We studied C. elegans that lack the ability to sequester dopamine (OK411) and that overproduce dopamine (UA57) and a novel strain (MBIA) generated by the genetic crossing of OK411 and UA57, which both lack the ability to sequester dopamine into vesicles and, additionally, endogenously overproduce dopamine. The MBIA strain was generated to address the hypothesis that an endogenous increase in the production of dopamine can rescue deficits caused by a lack of vesicular dopamine sequestration. These strains were analyzed for body size, developmental stage, reproduction, egg laying, motor behaviors, and neuronal health utilizing multiple methods. Our results further implicate proper dopamine synthesis and sequestration in the regulation of C. elegans body size, development through larval stages into gravid adulthood, and motor functioning. Furthermore, our analyses demonstrate that body size in terms of length is distinct from the developmental stage as fully developed gravid adult C. elegans with disruptions in the dopamine system have decreased body lengths. Thus, body size should not be used as a proxy for the developmental stage when designing experiments. Our results provide additional evidence that the dopamine system impacts the development, growth, and reproduction in C. elegans. Furthermore, our data suggest that endogenously increasing the production of dopamine mitigates deficits in C. elegans lacking the ability to package dopamine into synaptic vesicles. The novel strain, MBIA, and novel analyses of development and reproduction presented here can be utilized in developmental neurotoxicity experiments.

Dopamine transporter and synaptic vesicle sorting defects underlie auxilin-associated Parkinson’s disease

Auxilin participates in the uncoating of clathrin-coated vesicles (CCVs), thereby facilitating synaptic vesicle (SV) regeneration at presynaptic sites. Auxilin (DNAJC6/PARK19) loss-of-function mutations cause early-onset Parkinson's disease (PD). Here, we utilized auxilin knockout (KO) mice to elucidate the mechanisms through which auxilin deficiency and clathrin-uncoating deficits lead to PD. Auxilin KO mice display cardinal features of PD, including progressive motor deficits, α-synuclein pathology, nigral dopaminergic loss, and neuroinflammation. Significantly, treatment with L-DOPA ameliorated motor deficits. Unbiased proteomic and neurochemical analyses of auxilin KO brains indicated dopamine dyshomeostasis. We validated these findings by demonstrating slower dopamine reuptake kinetics invivo, an effect associated with dopamine transporter misrouting into axonal membrane deformities in the dorsal striatum. Defective SV protein sorting and elevated synaptic autophagy also contribute to ineffective dopamine sequestration and compartmentalization, ultimately leading to neurodegeneration. This study provides insights into how presynaptic endocytosis deficits lead to dopaminergic vulnerability and pathogenesis of PD.

Proteome-Wide Profiling of Cellular Targets Modified by Dopamine Metabolites Using a Bio-Orthogonally Functionalized Catecholamine.

Selective death of midbrain dopaminergic neurons is a hallmark pathology of Parkinson's disease (PD), but the molecular mechanisms that initiate the cascade of events resulting in neurodegeneration in PD remain unclear. Compelling evidence suggests that dysregulation of dopamine (DA) induces neuronal stress and damage responses that are operative processes in striatal degeneration preceding PD-like symptoms. Improper DA sequestration to vesicles raises cytosolic DA levels, which is rapidly converted into electrophilic dopaquinone species (DQs) that react readily with protein nucleophiles forming covalent modifications that alter the native structure and function of proteins. These so-called DA-protein adducts (DPAs) have been reported to play a role in neurotoxicity, and their abundance with respect to neurodegeneration has been linked to clinical and pathological features of PD that suggest that they play a causal role in PD pathogenesis. Therefore, characterizing DPAs is a critical first step in understanding the susceptibility of midbrain dopaminergic neurons during PD. To help achieve this goal, we report here a novel DA-mimetic (DAyne) containing a biorthogonal alkyne handle that exhibits a reactivity profile similar to DA in aqueous buffers. By linking DPAs formed with DAyne to a fluorescent reporter molecule, DPAs were visualized in fixed cells and within lysates. DAyne enabled global mapping of cellular proteins affected by DQ modification and their bioactive pathways through enrichment. Our proteomic profiling of DPAs in neuronal SH-SY5Y cells indicates that proteins susceptible to DPA formation are extant throughout the proteome, potentially influencing several diverse biological pathways involved in PD such as endoplasmic reticulum (ER) stress, cytoskeletal instability, proteotoxicity, and clathrin function. We validated that a protein involved in the ER stress pathway, protein disulfide isomerase 3 (PDIA3), which was enriched in our chemoproteomic analysis, is functionally inhibited by DA, providing evidence that dysregulated cellular DA may induce or exacerbate ER stress. Thus, DAyne provided new mechanistic insights into DA toxicity that may be observed during PD by enabling characterization of DPAs generated reproducibly at physiologically relevant quinone exposures. We anticipate our design and application of this reactivity-based probe will be generally applicable for clarifying mechanisms of metabolic quinone toxicity.

Acquired dysregulation of dopamine homeostasis reproduces features of Parkinson\u2019s disease

The catecholamine neurotransmitter dopamine has the potential to act as an endogenous neurotoxin when its vesicular sequestration is dysregulated. Despite postmortem analyses from patients with Parkinson’s disease that demonstrate decreased vesicular sequestration of dopamine with a corresponding increase in dopamine metabolism, dopamine’s contribution to nigrostriatal dopaminergic degeneration in Parkinson’s disease has been debated. Here, we present a new in vivo model demonstrating the induction of Parkinson’s disease-associated pathogenic mechanisms of degeneration resulting from acquired dysregulation of dopamine sequestration in nigrostriatal dopaminergic neurons in adult rats. Utilizing adeno-associated virus (serotype 2), viral-mediated small-hairpin RNA interference of endogenous vesicular monoamine transporter 2 (VMAT2) expression resulted in a loss of VMAT2 protein expression in transduced dopaminergic cell bodies in the substantia nigra with a corresponding loss of VMAT2 protein within the striatal terminals. The loss of VMAT2 resulted in an accumulation of cytosolic dopamine and subsequent increased dopamine metabolism, deficits in dopamine-mediated behaviors, and degeneration of nigrostriatal dopaminergic neurons that was rescued through reintroduction of exogenous VMAT2, demonstrating that the toxicity was specific to the loss of VMAT2. Analysis of parkinsonian pathogenic mechanisms of degeneration identified oxidative damage, activation of Parkinson’s disease-associated kinase LRRK2, and the formation of aberrant α-synuclein. This model demonstrates that a progressive acquired loss of VMAT2 expression in adulthood is sufficient to induce Parkinson’s disease-associated pathogenic mechanisms of degeneration and provides a new model to further investigate the consequences of cytosolic dopamine.

DJ-1 Protects Against Dopamine Toxicity: Implications for Parkinson's Disease and Aging

Parkinson's disease (PD) is a common age-related neurodegenerative disorder. Dopamine neurotoxicity, mediated through oxidative stress, is implicated in disease pathogenesis. The vesicular monoamine transporter-2 (VMAT2) transfers dopamine into synaptic vesicles preparing it for exocytotic release and preventing its cytoplasmic oxidation. DJ-1 mutations cause early-onset familial PD. Here, we show that DJ-1 protects dopaminergic neurons and controls the vesicular sequestration of dopamine by upregulating VMAT2. Overexpression of DJ-1 protected cells against dopamine toxicity, reduced oxidative stress, and increased VMAT2 expression and function. Reduced DJ-1 levels resulted in opposite effects. Dopamine vesicular sequestration and its release upon depolarization were dependent on DJ-1 levels. Transcriptional regulation of VMAT2 expression by DJ-1 was confirmed by chromatin immunoprecipitation assay. The results were corroborated in vivo using 6-hydroxydopamine hemiparkinsonian mouse model and transgenic DJ-1 knockout mice. Our experimental data point to a novel potential protective function of DJ-1, which could be used as a therapeutic tool.

HIV‐1 protein Tat inhibits vesicular monoamine transporter‐2 activity in rat striatum

HIV‐1 protein Tat inhibits vesicular monoamine transporter‐2 activity in rat striatum

Neuropharmacological Mechanisms Underlying the Neuroprotective Effects of Methylphenidate

Methylphenidate is a psychostimulant that inhibits the neuronal dopamine transporter. In addition, methylphenidate has the intriguing ability to provide neuroprotection from the neurotoxic effects of methamphetamine and perhaps also Parkinson’s disease; both of which may likely involve the abnormal accumulation of cytoplasmic dopamine inside dopaminergic neurons and the resulting formation of dopamine-associated reactive oxygen species. As delineated in this review, the neuroprotective effects of methylphenidate are due, at least in part, to its ability to attenuate or prevent this abnormal cytoplasmic dopamine accumulation through several possible neuropharmacological mechanisms. These may include 1) direct interactions between methylphenidate and the neuronal dopamine transporter which may attenuate or prevent the entry of methamphetamine into dopaminergic neurons and may also decrease the synthesis of cytoplasmic dopamine through a D2 receptor-mediated signal cascade process, and 2) indirect effects upon the functioning of the vesicular monoamine transporter-2 which may increase vesicular dopamine sequestration through both vesicle trafficking and the kinetic upregulation of the vesicular monoamine transporter-2 protein. Understanding these neuropharmacological mechanisms of methylphenidate neuroprotection may provide important insights into the physiologic regulation of dopaminergic systems as well as the pathophysiology of a variety of disorders involving abnormal dopamine disposition ranging from substance abuse to neurodegenerative diseases such as Parkinson’s disease.

Age‐dependent differences in dopamine transporter and vesicular monoamine transporter‐2 function and their implications for methamphetamine neurotoxicity

The abuse of methamphetamine (METH) is a serious public health problem because METH can cause persistent dopaminergic deficits in the brains of both animal models and humans. Surprisingly, adolescent postnatal day (PND)40 rats are resistant to these METH-induced deficits, whereas young adult PND90 rats are not. Studies described in this report used rotating disk electrode voltammetry and western blotting techniques to investigate whether there are age-dependent differences in monoamine transporter function in PND38-42 and PND88-92 rats that could contribute to this phenomenon. The initial velocities of dopamine (DA) transport into, METH-induced DA efflux from, and DA transporter (DAT) immunoreactivity in striatal suspensions are greater in PND38-42 rats than in PND88-92 rats. DA transport velocities into vesicles that cofractionate with synaptosomal membranes after osmotic lysis are also greater in PND38-42 rats. However, there is no difference in vesicular monoamine transporter-2 (VMAT-2) immunoreactivity between the two age groups in this fraction. This suggests that younger rats have a greater capacity to sequester cytoplasmic DA into membrane-associated vesicles due to kinetically upregulated VMAT-2 and also have increased levels of functionally active DAT. In the presence of METH, these may provide additional routes of cellular efflux for DA that is released from vesicles into the cytoplasm and thereby prevent cytoplasmic DA concentrations in younger rats from rising to neurotoxic levels after drug administration. These findings provide novel insight into the age-dependent physiological regulation of neuronal DA sequestration and may advance the treatment of disorders involving abnormal DA disposition including substance abuse and Parkinson's disease.

Method development and validation of an in vitro model of the effects of methylphenidate on membrane-associated synaptic vesicles

In vivo methylphenidate (MPD) administration decreases vesicular monoamine transporter-2 (VMAT-2) immunoreactivity in membrane-associated vesicles isolated from the striata of treated rats while concurrently kinetically upregulating VMAT-2-mediated vesicular dopamine (DA) sequestration. The functional consequences of these MPD-induced effects include an increase in both vesicular DA content and exocytotic DA release. This report describes experiments designed to develop and validate an in vitro MPD model to further elucidate the molecular mechanism(s) underlying the effects of MPD on the VMAT-2 in membrane-associated vesicles. Method development experiments revealed that in vitro MPD incubation of striatal homogenates, but not striatal synaptosomes, increased DA transport velocities and decreased VMAT-2 immunoreactivity in membrane-associated vesicles. An incubation time of 30 min with a MPD concentration of 10 mM was optimal. Method validation experiments indicated that in vitro MPD incubation kinetically upregulated VMAT-2 in membrane-associated vesicles, increased vesicular DA content, and increased exocytotic DA release. These results reveal that the in vitro MPD incubation model successfully reproduced the salient features of in vivo MPD administration. This in vitro MPD incubation model may provide novel insights into the receptor-mediated mechanism(s) of action of in vivo MPD in the striatum as well as the physiological regulation of vesicular DA sequestration and synaptic transmission. Accordingly, this in vitro model may help to advance the treatment of disorders involving abnormal DA disposition including Parkinson's disease, attention-deficit hyperactivity disorder, and substance abuse.

Methylphenidate‐Induced Alterations in Synaptic Vesicle Trafficking and Activity

The psychostimulant, methylphenidate (MPD), is commonly prescribed to treat attention-deficit hyperactivity disorder. MPD binds to the neuronal dopamine (DA) transporter, where it blocks the inward transport of DA. The present study expands upon these findings by examining the effects of in vivo MPD administration on the vesicular monoamine transporter-2 (VMAT-2) in membrane-associated vesicle and cytoplasmic vesicle subcellular fractions (i.e., those vesicles that do and do not co-fractionate with synaptosomal membranes after osmotic lysis, respectively) isolated from lysates of rat striatal synaptosomes. The results indicate that a single MPD administration redistributes VMAT-2 and associated vesicles within nerve terminals away from the synaptosomal membranes and into the cytoplasm, as assessed 1 hour after treatment. DA transport is also increased by MPD in both vesicle fractions (on account of vesicle trafficking in the cytoplasmic vesicles and to kinetic upregulation of the VMAT-2 in the membrane-associated vesicles). This, in turn, leads to an increase in the DA content of both vesicle fractions as well as an increase in the velocity and magnitude of K(+)-stimulated DA release from striatal suspensions. Taken together, these data show that the trafficking, DA sequestration function, DA content, and exocytotic DA release function of synaptic vesicles can all be pharmacologically manipulated by in vivo MPD treatment. These findings may provide important insights useful for understanding and treating disorders involving abnormal DA transmission including drug abuse, Parkinson's disease, and attention-deficit hyperactivity disorder.

Methylphenidate-Induced Increases in Vesicular Dopamine Sequestration and Dopamine Release in the Striatum: The Role of Muscarinic and Dopamine D2 Receptors

Methylphenidate (MPD) administration alters the subcellular distribution of vesicular monoamine transporter-2 (VMAT-2)-containing vesicles in rat striatum. This report reveals previously undescribed pharmacological features of MPD by elucidating its receptor-mediated effects on VMAT-2-containing vesicles that cofractionate with synaptosomal membranes after osmotic lysis (referred to herein as membrane-associated vesicles) and on striatal dopamine (DA) release. MPD administration increased DA transport into, and decreased the VMAT-2 immunoreactivity of, the membrane-associated vesicle subcellular fraction. These effects were mimicked by the D2 receptor agonist quinpirole and blocked by the D2 receptor antagonist eticlopride. Both MPD and quinpirole increased vesicular DA content. However, MPD increased, whereas quinpirole decreased, K(+)-stimulated DA release from striatal suspensions. Like MPD, the muscarinic receptor agonist, oxotremorine, increased K(+)-stimulated DA release. Both eticlopride and the muscarinic receptor antagonist scopolamine blocked MPD-induced increases in K(+)-stimulated DA release, whereas the N-methyl-d-aspartate receptor antagonist (-)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) was without effect. This suggests that D2 receptors mediate both the MPD-induced redistribution of vesicles away from synaptosomal membranes and the MPD-induced up-regulation of vesicles remaining at the membrane. This results in a redistribution of DA within the striatum from the cytoplasm into vesicles, leading to increased DA release. However, D2 receptor activation alone is not sufficient to mediate the MPD-induced increases in striatal DA release because muscarinic receptor activation is also required. These novel findings provide insight into the mechanism of action of MPD, regulation of DA sequestration/release, and treatment of disorders affecting DA disposition, including attention-deficit hyperactivity disorder, substance abuse, and Parkinson's disease.

VMAT2 and dopamine neuron loss in a primate model of Parkinson’s disease

We used positron emission tomography (PET) to measure the earliest change in dopaminergic synapses and glial cell markers in a chronic, low-dose MPTP non-human primate model of Parkinson's disease (PD). In vivo levels of dopamine transporters (DAT), vesicular monoamine transporter-type 2 (VMAT2), amphetamine-induced dopamine release (AMPH-DAR), D2-dopamine receptors (D2R) and translocator protein 18 kDa (TSPO) were measured longitudinally in the striatum of MPTP-treated animals. We report an early (2 months) decrease (46%) of striatal VMAT2 in asymptomatic MPTP animals that preceded changes in DAT, D2R, and AMPH-DAR and was associated with increased TSPO levels indicative of a glial response. Subsequent PET studies showed progressive loss of all pre-synaptic dopamine markers in the striatum with expression of parkinsonism. However, glial cell activation did not track disease progression. These findings indicate that decreased VMAT2 is a key pathogenic event that precedes nigrostriatal dopamine neuron degeneration. The loss of VMAT2 may result from an association with alpha-synuclein aggregation induced by oxidative stress. Disruption of dopamine sequestration by reducing VMAT2 is an early pathogenic event in the dopamine neuron degeneration that occurs in the MPTP non-human primate model of PD. Genetic or environmental factors that decrease VMAT2 function may be important determinants of PD.

Vesicular monoamine transporter 2 regulates the sensitivity of rat dopaminergic neurons to disturbed cytosolic dopamine levels

An abnormal accumulation of cytosolic dopamine resulting in reactive oxygen species and dopamine–quinone products may play an important role in the rather selective degeneration of substantia nigra pars compacta (SNc) dopaminergic neurons in Parkinson's disease. The neuronal-specific vesicular monoamine transporter (VMAT2), responsible for uptake of dopamine into vesicles, has been shown to play a central role both in intracellular dopamine homeostasis and sequestration of dopaminergic neurotoxins. Direct or indirect enhancement of VMAT2 activity could therefore have neuroprotective effects by decreasing cytosolic dopamine levels. Here, we demonstrate that transfection of VMAT2 in the dopaminergic cell line, PC12, increases intracellular dopamine content, augments potassium-induced dopamine release and attenuates cell death induced by the cytosolic dopamine enhancer, methamphetamine, suggesting an enhancement in vesicular dopamine storage. In rat ventral mesencephalic cultures highly enriched for dopaminergic neurons, lentiviral delivery of recombinant VMAT2 using a neuronal-specific promoter also resulted in elevated intracellular dopamine content and neurotransmitter release after depolarization. The opposite was seen after downregulation of VMAT2 using virally delivered shRNAs. Furthermore, using this VMAT2 knockdown model, we are the first to report a direct link between enhanced cytoplasmic dopamine levels, measured following mild permeabilization of the plasma membrane using digitonin, and neurite degeneration in primary dopaminergic neurons. In conclusion, our data support the hypothesis that an increase in vesicular sequestration of dopamine by modulation of VMAT2 activity could restore neuronal function and enhance dopaminergic cell survival in conditions of dysregulated dopamine homeostasis such as Parkinson's disease.

Methylphenidate Administration Alters Vesicular Monoamine Transporter-2 Function in Cytoplasmic and Membrane-Associated Vesicles

In vivo methylphenidate (MPD) administration increases vesicular monoamine transporter-2 (VMAT-2) immunoreactivity, VMAT-2-mediated dopamine (DA) transport, and DA content in a nonmembrane-associated (referred to herein as cytoplasmic) vesicular subcellular fraction purified from rat striatum: a phenomenon attributed to a redistribution of VMAT-2-associated vesicles within nerve terminals. In contrast, the present study elucidated the nature of, and the impact of MPD on, VMAT-2-associated vesicles that cofractionate with synaptosomal membranes after osmotic lysis (referred to herein as membrane-associated vesicles). Results revealed that, in striking contrast to the cytoplasmic vesicles, DA transport velocity versus substrate concentration curves in the membrane-associated vesicles were sigmoidal, suggesting positive cooperativity with respect to DA transport. Additionally, DA transport into membrane-associated vesicles was greater in total capacity in the presence of high DA concentrations than transport into cytoplasmic vesicles. Of potential therapeutic relevance, MPD increased DA transport into the membrane-associated vesicles despite rapidly decreasing (presumably by redistributing) VMAT-2 immunoreactivity in this fraction. Functional relevance was suggested by findings that MPD treatment increased both the DA content of the membrane-associated vesicle fraction and K(+)-stimulated DA release from striatal suspensions. In summary, the present data demonstrate the existence of a previously uncharacterized pool of membrane-associated VMAT-2-containing vesicles that displays novel transport kinetics, has a large sequestration capacity, and responds to in vivo pharmacological manipulation. These findings provide insight into both the regulation of vesicular DA sequestration and the mechanism of action of MPD, and they may have implications regarding treatment of disorders involving abnormal DA disposition, including Parkinson's disease and substance abuse.

Vesicular monoamine transporter 2: Role as a novel target for drug development

In the central nervous system, vesicular monoamine transporter 2 (VMAT2) is the only transporter that moves cytoplasmic dopamine (DA) into synaptic vesicles for storage and subsequent exocytotic release. Pharmacologically enhancing DA sequestration by VMAT2, and thus preventing the oxidation of DA in the cytoplasm, may be a strategy for treating diseases such as Parkinson's disease. VMAT2 may also be a novel target for the development of treatments for psychostimulant abuse. This review summarizes the possible role of VMAT2 as a therapeutic target, VMAT2 ligands reported in the literature, and the structure-activity relationship of these ligands, including tetrabenazine analogs, ketanserin analogs, lobeline analogs, and 3-amine-2-phenylpropene analogs. The molecular structure of VMAT2 and its relevance to ligand binding are briefly discussed.

Age‐Dependent Effects of Methamphetamine on VMAT‐2

Abuse of methamphetamine (METH) among adolescents and young adults is concerning since studies have demonstrated that multiple administrations of high-dose METH induce persistent dopaminergic deficits. METH has also been shown to reduce dopamine (DA) uptake by the vesicular monoamine transporter-2 (VMAT-2) and to reduce the amount of VMAT-2 protein in a purified vesicular fraction. VMAT-2 plays a critical role in the sequestration of DA in dopaminergic nerve terminals. This function is important since DA can oxidize rapidly to form highly reactive species. It is likely that disruption of this normal intracellular processing of DA contributes to oxidative sequences ultimately leading to persistent deficits. Interestingly, METH appears to be less toxic in adolescent rats compared to young adult rats. VMAT-2 is proposed to play an important role in the age-dependent difference. Since the effect of METH on the function and quantity of VMAT-2 has primarily been studied in young adult rats and since developmental changes in the dopaminergic system are reported to occur between adolescence and adulthood, it is important to determine if there is an age-dependent difference in response of VMAT-2 to METH.

Gain-of-function haplotypes in the vesicular monoamine transporter promoter are protective for Parkinson disease in women

The vesicular monoamine transporter can protect against toxins that induce an acute parkinsonian syndrome. It has been hypothesized that cytoplasmic dopamine has subacute toxic effects in Parkinson Disease (PD) leading to neuronal death and clinical symptoms. Regulatory polymorphisms in the brain form of the vesicular monoamine transporter (VMAT2) which affect its quantitative expression might therefore serve as genetic risk factors for PD. We have screened the promoter region of the gene for VMAT2 (SLC18A2) and identified several novel polymorphisms that form discrete haplotypes. We have tested the common halpotypes in SLC18A2 for functional effects in reporter gene assays and found that there are several gain-of-function haplotypes that display significantly increased transcriptional activity from the reference element. These gain-of-function haplotypes were tested for association with PD and found to confer a protective effect that was selective for females. This finding is consistent with the prediction that increased sequestration of dopamine in secretory vesicles by VMAT2 is protective for PD.

Reduced Extracellular Dopamine and Increased Responsiveness to Novelty: Neurochemical and Behavioral Sequelae of Intermittent Hypoxia

Vesicular monoamine transporter and dopamine D1-receptor protein expression are upregulated within the striatum of adults rats exposed to intermittent hypoxic insults as neonates. These observations prompted us to test the hypothesis that intermittent hypoxic insults, occurring during this period of critical brain development, lead to persistent reductions in extracellular levels of dopamine within the striatum. We also tested the hypothesis that post-hypoxic rats exhibit increased novelty-induced behavioral activation and increased basal levels of locomotor activity, two indexes of impaired dopaminergic functioning. Extracellular levels of dopamine were measured over a 50-hour interval via in-vivo microdialysis. Responsivity to novelty and basal levels of locomotor activity were measured via cubicles equipped with infrared transmitters and photosensors. Between postnatal days 7 and 11, rat pups were exposed to intermittent hypoxia (20-second bursts of hypoxic gas consisting of 10% oxygen balance nitrogen; 60 events/hour) or compressed air for 6 hours during their major sleep period. On postnatal day 35, locomotor activity was continuously recorded over a 72-hour period. On postnatal day 42, extracellular levels of dopamine were determined in microdialysis samples collected at 30-minute intervals continuously for 50 hours. Post-hypoxic rats exhibited heightened behavioral activation when placed into the novel environment of locomotor-recording cubicles. Following 24 hours of adaptation to the cubicles, post-hypoxic rats exhibited locomotor hyperactivity during each dark phase of the circadian cycle, their typical waking period. These same rats also demonstrated reduced levels of extracellular dopamine during both the dark and light phases of the circadian cycle. We demonstrate increased responsivity to novelty, locomotor hyperactivity, and reduced levels of extracellular dopamine within the striata of juvenile rats exposed to intermittent hypoxic insults between postnatal days 7 and 11. These data, in conjunction with our previous observations, support our hypothesis that intermittent hypoxic insults occurring during a period of critical brain development lead to sequestration of dopamine presynaptically within nigrostriatal axons. We postulate that neonatally occurring hypoxic insults are one potential pathogenic mechanism underlying disorders of minimal brain dysfunction, such as attention-deficit/hyperactivity disorder, characterized by executive dysfunction and hyper responsiveness to novel stimuli, which is responsive to agents promoting enhanced extracellular levels of synaptic dopamine.

Psychostimulants and Vesicle Trafficking: A Novel Mechanism and Therapeutic Implications

The monoamine vesicular transporter 2 (VMAT-2) has been associated with dopamine (DA) sequestration and protection against neurodegeneration caused by the intracellular oxidation of this monoamine. The data presented herein suggest that methylphenidate treatment enhances the amount of VMAT-2 protein and possibly its activity in the presynaptic cytosol, where it is able to increase the sequestration of DA and likely protect against its instability. In contrast, methamphetamine (METH) has an opposite effect on cytosolic VMAT-2 resulting in degradation of DA terminals. The fact that posttreatment of methylphenidate after a neurotoxic regimen of METH protects against resulting loss of DA parameters suggests that treatment with methylphenidate, or other DA transporter blockers, may be protective against degenerative disorders of DA pathways, such as Parkinson's disease.

Methylphenidate alters vesicular monoamine transport and prevents methamphetamine-induced dopaminergic deficits.

It has been hypothesized that high-dose methamphetamine treatment rapidly redistributes cytoplasmic dopamine within nerve terminals, leading to intraneuronal reactive oxygen species formation and well characterized persistent dopamine deficits. We and others have reported that in addition to this persistent damage, methamphetamine treatment rapidly decreases vesicular dopamine uptake, as assessed in purified vesicles prepared from treated rats; a phenomenon that may contribute to aberrant intraneuronal dopamine redistribution proposedly caused by the stimulant. Interestingly, post-treatment with dopamine transporter inhibitors protect against the persistent dopamine deficits caused by methamphetamine; however, mechanisms underlying this phenomenon have not been elucidated. Also of interest are findings that dopamine transporter inhibitors, including methylphenidate, rapidly increase 1) vesicular dopamine uptake, 2) vesicular monoamine transporter-2 (VMAT-2) ligand binding, and 3) VMAT-2 immunoreactivity in a vesicular subcellular fraction prepared from treated rats. Therefore, we hypothesized that methylphenidate post-treatment might protect against the persistent striatal dopamine deficits caused by methamphetamine by rapidly affecting VMAT-2 and vesicular dopamine content. Results reveal that methylphenidate post-treatment both prevents the persistent dopamine deficits and reverses the acute decreases in vesicular dopamine uptake and VMAT-2 ligand binding caused by methamphetamine treatment. In addition, methylphenidate post-treatment reverses the acute decreases in vesicular dopamine content caused by methamphetamine treatment. Taken together, these findings suggest that methylphenidate prevents persistent methamphetamine-induced dopamine deficits by redistributing vesicles and the associated VMAT-2 protein and presumably affecting dopamine sequestration. These findings not only provide insight into the neurotoxic effects of methamphetamine but also mechanisms underlying dopamine neurodegenerative disorders, including Parkinson's disease.