Attention deficit hyperactivity disorder (ADHD) is a neurobehavioral disorder that affects 11% of children in the US alo­ne. Methylphenidate (MPH) is the most commonly prescribed drug for the treatment of ADHD. Given the fact that ADHD symptoms persist in up to 50% of patients, many children receive MPH from childhood to early adulthood. Unfortunately, most of the scientific literature focuses on the short-term consequences of MPH, even though individuals are taking MPH for many years. Previous research has shown that long-term exposure to MPH causes dopamine-releasing neurons within the nigrostriatal pathway to die when exposed to the Parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPH acts by blocking dopamine transporters and norepinephrine transporters, preventing the reuptake and removal of these neurotransmitters following release and increasing the time outside of the protective environment of the neuron’s vesicles. We hypothesize that spontaneous oxidation of excess dopamine to a quinone metabolite is rendering these neurons within this particular pathway to be more sensitive to MPTP. The dopamine quinone may be bound by the antioxidant glutathione (GSH) in an effort to protect the cell against oxidative stress. However, as the finite amount of GSH is depleted, the quinone may lead to the production of highly reactive molecules, leading to mitochondrial damage and cell death which may be accelerated by MPTP. In order to examine this hypothesis, we chose to study adolescent male Swiss-Webster mice, which have been shown to be resistant to MPTP’s toxic effects. They were divided into 3 cohorts and administered either saline (control), 1 mg/kg MPH (therapeutic dose) or 10 mg/kg (abusive dose) via intraperitoneal (IP) injections for 12 weeks. Mice were injected twice daily, Monday through Friday, mimicking a school-week dosing schedule. After 12 weeks, all animals received a drug washout period of 7 days. Then, half of each cohort was treated with MPTP (4 x 20 mg/kg, every 2 hours), while the other half was administered 4 injections of sterile saline. Either 3 or 7 days after MPTP or saline treatment, the mice were sacrificed, brains were removed, and the substantia nigra (SN) and striatum (STR) were collected. These areas of the brain make up the nigrostriatal pathway and are affected by Parkinson’s disease. Oxidative stress related to increased dopamine levels was determined using the glutathione assay to measure GSH content, near-infrared fluorescence dot blots to measure free and protein-bound ortho-quinones, and an ATP luciferase assay to measure mitochondrial function. Interestingly, there was a significant decrease in GSH as the dose of MPH was increased with both saline and MPTP samples. Furthermore, a significant increase in quinones was observed as the dose of MPH increased. We also expect to see a decrease in ATP inversely proportional to the dose of MPH indicating increased oxidative stress. In conclusion, it appears that long-term exposure to MPH sensitizes dopaminergic neurons within the nigrostriatal pathway to oxidative stress, rendering them vulnerable to further insults, such as MPTP exposure. As such, these studies provide insight into the risks of long-term psychostimulant exposure.

Epidemiological studies implicate pesticide exposure as a possible etiologic factor in idiopathic Parkinson's Disease, which results from degeneration of nigrostriatal neurons, along with reduced levels of the neurotransmitter, dopamine. Behavioral and neurochemical analyses in C57BL6 mice were performed following a subchronic dosing regime with the organochlorine insecticide heptachlor or the pyrethroid deltamethrin. Results were compared to those induced by the established parkinsonian neurotoxicant, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). At the end of the treatment period, mice were assessed for effects on behavior, as well as levels of striatal dopamine, nerve terminal respiration, and synaptosomal dopamine transport. The primary behavioral effect of deltamethrin was incoordination, while heptachlor caused hyperexcitability and increased locomotion. The major neurochemical effect observed for both compounds was upregulation of the presynaptic dopamine transporter (DAT) by 70% and 100% for deltamethrin and heptachlor, respectively. The insecticides exerted only modest effects on striatal levels of dopamine and its metabolite, dihydroxyphenylacetic acid. However, doses of heptachlor higher than those which caused induction of DAT (e.g. greater than or equal to 25 mg/kg), when administered subchronically, were found to cause convulsions in some animals and caused marked, dose-dependent depression of basal striatal tissue respiration rates. No synergism was observed between the effects of insecticides and MPTP. Enhanced transport was thought to be a compensatory effect from increased release of transmitters by the insecticides, <i>in vivo</i>. Striatal dopamine, GABA and glutamate nerve terminals were differentially sensitive to the releasing effects of heptachlor compared to cortical serotonin terminals, and responded in the following rank order of sensitivity: dopamine > GABA > glutamate > serotonin. Additional experiments to characterize the mechanism(s) by which cyclodienes facilitate release of neurotransmitters in synaptosomes demonstrated a lack of distinct Ca²⁺ component and no involvement of retrograde DAT activity, suggesting that released label was of vesicular origin, but did not require Ca²⁺. Insecticidal toxicants, such as organochlorines and pyrethroids, which augment dopamine release and increase the maximal rate of dopamine uptake, may inundate the cytosol of nigrostriatal neurons with high concentrations of free dopamine, which has been shown by other researchers to induce apoptosis and may thereby contribute to the development of Parkinson's disease. Funding for this work was provided under grant number HHHREP 94-01 by the Hawaii Heptachlor Foundation, a non-profit organization. The Hawaii Heptachlor Foundation may be contacted at the following address: Ocean View Center PH#3, 707 Richards St., Honolulu, HI 96813. / Ph. D.

No description available.

The ability to objectively index dopamine (DA) levels in the brain has the potential to revolutionize the field of neuropsychopharmacology, as having a peripheral biomarker of brain DA would enable the objective monitoring of the progression of Parkinson's disease (PD) and other DA-dependent psychiatric states. Of particular relevance to commercialization, it would provide an objective measure of treatment efficacy. We used a DA-depletion approach to determine if peripheral D2Rs are a biomarker for brain DA; mainly, the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin model and PD subjects, which are well-known models of DA depletion in the midbrain of rodents and humans, respectively. Dopamine depletion in the substantia nigra resulted a significant change to DA and norepinephrine (NE) levels in the plasma. Interestingly, these changes could be tracked as a time course from baseline to 15 days after injection with MPTP. Also, the proportion of D2R expressing leukocytes steadily increased (specifically B cells and T cells) during this same time of DA depletion. These results suggest that changes to the dopamine neuron population in the substantia nigra can be tracked with DA and NE level changes and D2R expression on B and T cells, providing a possible biomarker for nigral DA neuron loss to further investigate. In two cohorts of studies comparing subjects with PD vs controls, we found that Parkinson's subjects displayed significantly decreased D2R expression in all populations except for (natural killer) NK cells, CD16+ monocytes, and cytotoxic T cells. We also found that subjects with PD show increased levels in epinephrine (EPI) and DA as compared to control subjects. We did not, however, find any statistically significant correlations between the recorded leukocytic D2R downregulation in PD patients and their elevated DA and EPI plasma levels. Therefore, the results of this study did not provide a clear indication how brain DA levels are being represented in the periphery. Regardless, the modulation of peripheral D2Rs in PD and MPTP seen in this study do show that substantia nigra DA depletion in humans and rodents do manifest in the periphery. Although our study didn't result in a clear narrative of how nigral and peripheral DA system mirror each other, our result provide more evidence D2Rs may be both biomarkers and important substrates for treatment of DA-dependent disorders. Our results give a foundation from which future studies can investigate this connection further.

No description available.

Methylphenidate (MPH) is the most commonly prescribed drug to treat Attention Deficit Hyperactivity Disorder (ADHD). ADHD is now considered a life-long disorder; therefore, patients take MPH from adolescence into adulthood, highlighting the need for research studying chronic MPH use. MPH increases dopamine and norepinephrine within the synaptic cleft; therefore, chronic use of MPH may lead to changes within important dopaminergic pathways. One pathway, the mesolimbic pathway, includes the hippocampus, an area where adult neurogenesis occurs. We investigated the effects of chronic low and high doses of MPH on neurogenesis and examined levels of a few key proteins linked to cell proliferation in the hippocampus. Low dose MPH appears to increase cell proliferation and cell survival in the hippocampus, and these effects are accompanied by increases in vascular endothelial growth factor (VEGF), the receptor for brain-derived neurotrophic factor (TrkB), and beta-catenin. While high dose MPH may initially increase neuronal proliferation, newly-generated neurons are unable to survive long-term, and decreases in VEGF, TrkB, and beta-catenin are observed with chronic high dose MPH. Another major dopaminergic pathway is the nigrostriatal pathway, which is involved in motor control and degenerates with Parkinson’s disease. Chronic use of MPH appears to sensitize dopaminergic neurons within this pathway to the Parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), but the cause of this sensitization is unknown. The autooxidation of excess dopamine forms dopamine-quinones that lead to free radical production, but the antioxidant, glutathione, can protect neurons. However, we showed that chronic MPH increases dopamine-quinone formation and causes a subsequent glutathione depletion within the striatum. Therefore, oxidative stress may sensitize dopamine neurons to MPTP. We also assessed the vulnerability of dopaminergic neurons in the nigrostriatal pathway to MPTP after chronic MPH in females. Interestingly, proestrus (high estrogen) females were more sensitive to MPTP than anestrus (low estrogen) females. Similar to males, chronic MPH caused a depletion in glutathione that was further decreased following MPTP exposure. However, chronic MPH did not significantly alter dopaminergic neuronal numbers or quinone formation in females. These studies highlight some of the potential effects of chronic MPH use.

Studies into the quantitative structure act! Vlty relationships of rate s of I-methyl-4-phenyl-l,2,3,6-tetrahydropyridine oxidation catalyzed by monoamine oxidases A and B were performed to elucidate active site substrate conformation and oxidation mechanisms. Plotting experimental kinetic activity against molecular properties obtained by experiment and by computational chemistry methods demonstrated correlations with lipophilic, steric, and electronic factors. Compounds studied were 4-aryloxy analogs, 4- aromatic heterocycle analogs, and 4-phenyl analogs. The conformer with phenyl ring to tetrahydropyridine dihedral angles similar to a low energy conformer of I-methyl-4-(2'-methyl-phenyl)-1,2,3,6- tetrahydropyridine is the most active conformer. Results indicate that rate limiting single electron transfer mechanisms are more viable than hydrogen atom abstraction mechanisms. Results indicate that binding or dissociation is the rate limiting step for aryloxy-analog oxidation catalyzed by monoamine oxidase B whereas the catalytic event itself is the rate limiting step for the other analogs. Several equations were developed to describe quantitative structure activity relationships of oxidation rates. / Master of Science

The unexpected monoamine oxidase (MAO) substrate properties of 1-cyclopropyl-4-substituted-1 ,2,3,6-tetrahydropyridines have been interpreted in terms of partitioning of these tertiary cyclic allylamines between substrate turnover and ring opening inactivation processes. To evaluate further this proposal, we examined the bioactivities of the related saturated analogs. Several 1,4-disubstituted piperidine derivatives were synthesized and their interactions with MAO-A and MAO-B were characterized. These compounds displayed poor substrate properties toward MAO-A and MAO-B and led to the expected α-carbon oxidized metabolites which were fully characterized. Both the N-methyl and N-cyclopropyl derivatives were good inactivators of MAO-B, suggesting that some species other than the radical resulting from cyclopropyl ring opening is responsible for the inactivation. Both the N-methyl and N-cyclopropyl derivatives also inactivated MAO-A. In this instance, the N-cyclopropyl analogs were much more potent inactivators than the N-methyl analogs. These results suggest that the radical derived from cyclopropyl ring opening may be involved in this inactivation process. The MAO substrate/inactivator properties of these piperidine analogs are discussed in terms of current proposed mechanisms for the MAO catalyzed oxidation of amines. / Master of Science

Les troubles du sommeil, tels que la somnolence diurne excessive et les attaques narcoleptiques, sont fréquemment observés chez les patients Parkinsoniens. Malgré de nombreux efforts cliniques à l’échelle mondiale, il reste à déterminer si ces troubles sont causés par les lésions neuronales dopaminergiques (DAergiques) ou non DAergiques, par les troubles moteurs ou par les effets délétères des médicaments anti-parkinsoniens. Par une approche pluridisciplinaire (analyse EEG; mesure de l’activité locomotrice; tests pharmacologiques; immunohistochimie) chez le félin traité au MPTP, nous avons étudié la corrélation possible entre perte DAergique et troubles du cycle veille-sommeil (V/S). Le MPTP (5mg/kg/jour x5, i.p.) induit en période aiguë, une hypersomnie en sommeil lent (SL), une suppression du sommeil paradoxal (SP), ainsi qu’une diminution de la locomotion et une difficulté à l’initiation des mouvements. Les agonistes DAergiques (L-dopa; ropinirole) empêchent l’hypersomnie en SL de façon transitoire. Durant la période chronique, les taux d’éveil et de SL reviennent à la normale. Le SP augmente transitoirement et s’associe à des épisodes narcoleptiques. Les analyses ex-vivo révèlent une diminution du marquage TH (corps cellulaire de la substance noire ; fibres du striatum). Le marquage des neurones cholinergiques (cerveau antérieur basal et tegmentum mésopontique) semblent inchangée. Nos résultats montrent donc que le MPTP induit chez le félin des symptômes moteurs et des troubles du sommeil proches de ceux observés chez les patients parkinsoniens. Cette étude suggère également une corrélation possible entre les troubles du cycle V/S et la perte des cellules DAergiques / Motors disorders are not the only symptoms of Parkinson disease (PD), and sleep disorders such as somnolence and narcolepsy are frequently reported in PD patients. Despite much investigation worldwide, it remains unknown whether these disorders are caused by dopaminergic (DArgic), non-DArgic neural lesions, nocturnal motor disability or deleterious effect of anti-PD drugs. Using multiple experimental approaches (EEG and sleep-wake recordings/pharmacological dosing / immunohistochemistry) in cats treated with MPTP, which causes DArgic neuronal loss, we have studied the possible correlation between the induced effects on the sleep-wake (S/W) cycle and those on DArgic neurons. MPTP (5mg/kg/day x 5, i.p.) caused, during the acute period, a slow wave sleep hypersomnia (SWS, up to 80% of recorded time) and a suppression of paradoxical sleep (PS), accompanied with pronounced behavioural somnolence, marked decrease in locomotion and difficulty to initiate movements. DArgic agonists L-dopa and ropinirole transiently prevented hypersomnia in SWS. During the chronic period, whereas the amount of W and SWS returned to control, PS transiently increased, associated with narcolepsy-like episodes. Ex-vivo analyses revealed marked decrease in TH labelling (cell bodies in the substantia nigra and terminal-like dots in the striatum) whereas cholinergic neurons in the basal forebrain and mesopontine-tegmentum seemed unchanged. Thus, MPTP treated cats showed major signs of motor and S/W disorders similar to those seen in PD patients and so could serve as useful animal model. Our results also suggest a possible correlation/causality between the MPTP-induced S/W disorders and DArgic cell loss.

No description available.