Methylphenidate (trade name: Ritalin) resulted in a conditioned place preference, but in contrast to work in juveniles, there were no sex differences. In addition, methylphenidate produced a significant decrease in the dopamine transporter compared to controls that may have implications towards development and plasticity of the dopamine system.

The dopamine transporter (DAT) is a plasma membrane-bound protein, localized on peri-synaptic terminals of dopaminergic (DA) neurons. DAT is responsible for terminating DA signaling by rapid removal of the transmitter from the synaptic cleft region. DA signaling relies on a critical balance between release and removal of the neurotransmitter within synaptic clefts. Recycling of DAT between intracellular endosomal compartments and the plasma membrane regulates DAT function. This dynamic trafficking occurs in both a constitutive and regulated manner to increase or decrease the number of transporters on the cell surface available for transmitter reuptake. Therapeutic drugs and/or drugs of abuse, including psychostimulants and ethanol, cause maladaptive changes in DA signaling in mesolimbic areas of the brain, leading to addictive behaviors. DAT is the primary site of action for psychostimulants such as, cocaine, methylphenidate, and amphetamine. These drugs can alter the function and/or regulation of the transporter. Ethanol, one of the most widely abused drugs in society, is known to activate DA pathways in reward and reinforcement areas of the brain. However, the effect of ethanol on DAT function and regulation is less clear. The studies presented here explore the action of ethanol on DAT function in mammalian cell systems, and the subcellular trafficking mechanisms that regulate the transporter. To delineate mechanisms of ethanol action on DAT, several lines of HEK-293 cells stably expressing DAT or ethanol-insensitive DAT mutants were generated. Short-term ethanol exposure was found to potentiate DAT function, and ethanol sensitivity is mediated by specific amino acids in the first intracellular loop. This increase in function was accompanied by an enhancement of DAT expressed on the cell surface. The changes in DAT localization and the absence of consensus phosphorylation sites in the ethanol sensitive regions of the transporter, led to the hypothesis that ethanol modulates DAT uptake by altering the dynamic trafficking of the transporter. In the present studies, we found ethanol directly regulates DAT function by altering specific step of the endosomal recycling pathway. Further analysis of the ethanol-sensitive first intracellular loop revealed this region might also play a role in conformational changes required for substrate binding. The findings presented in these studies describe a novel molecular mechanism of ethanol action on DAT, and provide a framework to further understand the action of ethanol on synaptic dopamine regulation. / text

Στο κεντρικό νευρικό σύστημα (ΚΝΣ), η ντοπαμίνη ελέγχει σημαντικές φυσιολογικές λειτουργίες όπως η έκκριση ορμονών, η ρύθμιση της κίνησης, γνωστικές πορείες και περίπλοκες συμπεριφορές που έχουν σχέση με το συναίσθημα και την ανταμοιβή. Η επαναπρόσληψη της ντοπαμίνης διαμέσου του μεταφορέα της νευρωνικής πλασματικής μεμβράνης (DAT) είναι σημαντική για τη διατήρηση της ομοιόστασης της στο ΚΝΣ. Πιστεύεται ότι ο DAT ελέγχει την ένταση και τη διάρκεια της ντοπαμινεργικής νευροδιαβίβασης. Ο DAT αποτελεί μοριακό στόχο θεραπευτικών ουσιών για την θεραπεία νοητικών δυσλειτουργιών όπως η σχιζοφρένεια και η κατάθλιψη. Επιπροσθέτως είναι στόχος εθιστικών ουσιών όπως η κοκαΐνη και η αμφεταμίνη. Οι ψυχοδιεγερτικές και οι θεραπευτικές ουσίες δεσμεύονται στον DAT, αλλάζουν την λειτουργία του και ως εκ τούτου επιτείνουν την ένταση και διάρκεια της ντοπαμινεργικής νευροδιαβίβασης. Η παρεγκεφαλίδα δέχεται μια κατεχολαμινεργική νεύρωση η οποία θεωρείται ότι είναι νοραδρενεργική. Εν τούτοις βιοχημικές, φαρμακολογικές και ανατομικές μελέτες υποδεικνύουν ότι επίσης δέχεται μια μικρή ντοπαμινεργική νεύρωση από την κοιλιακή περιοχή της καλύπτρας και την συμπαγή μοίρα της μέλαινας ουσίας. Πρόσφατες ανοσοϊστοχημικές μελέτες μας για τον DAT υπέδειξαν ότι νευρώνες της παρεγκεφαλίδας μπορεί να εκφράζουν τον μεταφορέα. Στην παρούσα μελέτη ο εντοπισμός του μεταφορέα της ντοπαμίνης (DAT) στην παρεγκεφαλίδα φυσιολογικών μυών, τόσο σε επίπεδο πρωτεΐνης όσο και σε επίπεδο mRNA έγινε με την μέθοδο Western Blot, IP και RT-PCR αντίστοιχα. Η πρωτεΐνη ανιχνεύθηκε στο κλάσμα των συναπτοσωμάτων, ενώ το mRNA σε ολικό εκχύλισμα παρεγκεφαλίδας. Τα αποτελέσματα έρχονται να επιβεβαιώσουν την ύπαρξη ντοπαμινεργικών κυττάρων στην παρεγκεφαλίδα. / In the Central Nervous System (CNS) the neurotransmitter dopamine (DA) controls important functions including hormone secretion, locomotion, cognitive processes and complex behaviours that are associated with emotion and reward. Dopamine uptake through the neuronal plasma membrane DA transporter is essential for the maintenance of normal DA homeostasis in the brain. It is believed that DAT controls the intensity and the duration of dopamine neurotransmittion. DAT is the molecular target for therapeutic agents used in the treatment of mental disorders, such as schizophrenia and depression. In addition, DAT is the target for cocaine and amphetamine. Psychostimulants and therapeutic substances alter its transporter function and therefore prolong the intensity and duration of dopaminergic neurotransmittion.The cerebellum receives a catecholaminergic input that is generally accepted to be noradrenergic. However, biochemical, pharmacological and anatomical evidence indicate that the cerebellum also receives a small dopaminergic input from ventral tegmental area and substantia nigra pars compacta. Recent immunohistochemical studies have indicated that cerebellar neurons express the transporter. In the current study, cerebellar dopamine transporter protein and mRNA were studied using the methods of western blotting and RT-PCR. Our results confirm the existence of dopaminergic neurons in cerebellum.

In an effort to identify novel binding sites for cocaine and its analogs, we carried out binding studies with the high-affinity and selective ligand [125I]RTI-121 in rat frontal cortical tissue. Very low densities of binding sites were found. Saturation analysis revealed that the binding was to both high- and low-affinity sites. Pharmacological competition studies were carried out with inhibitors of the dopamine, norepinephrine, and serotonin transporters. The various transporter inhibitors inhibited the. binding of 15 pM [125I]RTI-121 in a biphasic fashion following a two-site binding model. The resultant data were complex and did not suggest a simple association with any single transporter. Correlational analysis supported the following hypothesis: [125I] RTI-121 binds to known transporters and not to novel sites; these include dopamine, norepinephrine, and serotonin transporters. Immunoprecipitation of transporters photoaffinity labeled with [125]RTI-82 and subsequent analysis of SDS-page gels revealed the presence of authentic dopamine transporters in these samples; displacement of the photoaffinity label occurred with a typical dopamine transporter pharmacology. These data are compatible with the binding properties of RTI- 121 and the presence of several known transporters in the tissue studied.

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This dissertation work investigated the effect of acute and repeated in vivo administration of lobeline on dopamine transporter (DAT) and vesicular monoamine transporter (VMAT2) function. The effects of lobeline were then compared to the effects of acute and repeated in vivo administration of methylphenidate and amphetamine to determine if lobeline produced similar effects compared to these Attention Deficit Hyperactivity Disorder (ADHD) medications. These medications are considered the first line of pharmacotherapy for ADHD, although there is a growing concern associated with their potential for abuse and other side effects. This merits the need for novel ADHD treatments that have a safer side effect profile. If lobeline alters DAT and VMAT2 function in the same way as methylphenidate or amphetamine, further investigation may be necessary to evaluate lobeline as a potential treatment for ADHD. Kinetic analysis of [3H]dopamine (DA) was utilized to determine the effect on DAT and VMAT2 function in rat striatum. Results from the DAT experiments, revealed that lobeline as well as amphetamine had no effect on DAT function. However, methylphenidate increased DAT function after acute and 7-day treatment. None of the drug treatment regimens altered Km. To determine if the methylphenidateinduced increase in DAT function was due to DAT trafficking, biotinylation and Western blot analyses were performed. Acute administration of methylphenidate did not alter surface DAT, however repeated administration of methylphenidate for 7 days decreased intracellular DAT, suggesting that methylphenidate redistributes DAT in a time-dependent manner. Similar results were found in the VMAT2 experiments. Lobeline and amphetamine had no effect on VMAT2 function after acute or repeated administration. Amphetamine decreased the Km after repeated administration for 7 days. Methylphenidate increased VMAT2 function after acute and repeated administration for 7 days. The overall results of these experiments suggest that methylphenidate interacts with DAT and VMAT2 in a different manner than amphetamine and lobeline. In addition, since lobeline and amphetamine had no effect on DAT and VMAT2 function, further investigation is warranted to elucidate the underlying mechanisms of the therapeutic actions of these agents. This additional information will aid in the development of novel treatments for ADHD.

Synthetic cathinones represent threatening and high abuse-potential designer drugs. These are analogs of cathinone (the b-keto analog of amphetamine (AMPH)) a naturally occurring stimulant found in the plant Catha Edulis. Methcathinone (MCAT) was the first synthetic analog of cathinone to be identified in 1987 by Glennon and co-workers and it exerted its action primarily through the dopamine transporter (DAT). Most central stimulants exert their action via monomaine transporters by causing either the release (e.g. cathinone analogs such as MCAT) or by preventing the reuptake (e.g. cocaine) of the neurotransmitter dopamine (DA) thus increasing the extracellular synaptosomal concentration of this neurotransmitter. In 2010, a new class of designer cathinone-like drugs called ‘bath salts’, initially a combination of methylenedioxypyrovalerone (MDPV), methylone (methylenedioxymethcathione, MDMC) and mephedrone (MEPH), soared to popularity. It caused extremely detrimental side effects; it was exceedingly popular for its recreational use and posed a threat to public health. At the time, their mechanisms of action were unknown. Our group identified that MDPV produced actions distinct from other cathinone analogs (i.e., it was identified as the first cathinone-like compound to act as a reuptake inhibitor at the dopamine transporter (DAT)). These findings suggested that not all cathinone-like compounds act uniformly and this insinuated that unique structural features on the cathinone scaffold might contribute to different effects at the transporter level. The overall goal of this project was to study the mechanisms of action of synthetic cathinones (including ‘bath salts’) at the monoamine transporters. We investigated the contribution of each of various structural features on the cathinone scaffold (i.e, the terminal amine, a and b positions, and the phenyl ring). We also constructed homology models of the human dopamine and serotonin transporters (hDAT and hSERT respectively) to help explain differences in selectivity between the neurochemical and behavioral aspects of DAT and SERT. Overall we found that structural features contributed to similar or distinctive mechanisms of action and also contributed to selectivity at monoamine transporters. Our studies provide information that can be useful to drug and health regulatory agencies to help prevent, treat, or curb the future abuse of such drugs.

In two experiments, we analyzed the effects of methylphenidate (MPH) on conditioned place preference (CPP) in adolescent male and female rats, and the effects of MPH on the dopamine transporter (DAT). In Experiment 1, male and female rats were conditioned for 5 consecutive days from postnatal day (P)44 to P48 with saline, 1, or 5mg/kg MPH. On the post conditioning preference test, the group administered the 1mg/kg dose of MPH resulted in no significant preference compared to controls, whereas the 5mg/kg dose of MPH produced a robust significant preference for the paired context, but there were no sex differences. Analysis of the DAT revealed that animals conditioned with the 5mg/kg dose of MPH demonstrated a significant decrease of the dopamine transporter (DAT) in the nucleus accumbens and striatum compared to controls. In Experiment 2, animals were conditioned using an every second day paradigm from P33–41 to model a previous MPH treatment regimen that had revealed sex differences in behavioral sensitization. MPH produced an increased preference for the paired context on a post-conditioning preference test in Experiment 2, but as in Experiment 1, no sex differences were observed. These data show that a relatively high dose of MPH has rewarding associative effects in both adolescent male and female rats reliably across two different conditioning paradigms and ages in adolescence, but no sex difference. In addition, MPH results in a significant decrease of the DAT in drug reward brain areas which has implications toward plasticity of the brain's reward system.

Parkinson’s disease (PD) is a common neurodegenerative disorder that is characterized by the degeneration of dopaminergic (DA) neurons in the substantia nigra and motor deficits. Although the majority of PD cases are sporadic, several genetic defects in rare familial cases have been identified. Animal models of these genetic defects have been created and have provided unique insights into the molecular mechanisms of the pathogenesis of PD. However, the etiology of PD is still not well understood. Here, taking advantage of the unique features offered by zebrafish, I characterized the functions of PINK1 (PTEN-induced kinase 1) gene, which is associated with recessive familial PD, in the development and survival of DA neurons. In zebrafish, antisense morpholino knockdown of pink1 did not cause a large loss of DA neurons in the ventral diencephalon (vDC), but the patterning of these neurons and their projections were perturbed. The pink1 morphants also showed impaired response to touch stimuli and reduced swimming behaviour. Moreover, the pink1 knockdown caused a significant reduction in the number of mitochondria, as well as mitochondrial morphological defects such as smaller size or loss of cristae, thus affecting mitochondrial function. These results suggest that zebrafish pink1 plays conserved important roles in the development of DA neurons and in the mitochondrial morphology and function. To better follow DA neurons after injury or administration of toxins, I generated a transgenic zebrafish line, Tg(dat:EGFP), in which the green fluorescent protein (GFP) is expressed under the control of cis-regulatory elements of dopamine transporter (dat). In Tg(dat:EGFP) fish, all major groups of DA neurons are correctly labeled with GFP, especially the ones in the vDC, which are analogous to the ascending midbrain DA neurons in mammals. In addition, we observed that the DA neurons in the vDC could partially be replaced after severe laser cell ablation. This suggests that zebrafish may have the unique capacity of regenerating DA neurons after injury. Taken together, my studies suggested that zebrafish could be a useful alternative animal model for the study of the molecular mechanisms underlying PD and for the screening of potential therapeutic compounds for PD.