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The design and construction of an electrochemical detector with two working electrodes located on the opposite walls of a thin-layer cell and its use as a detector for High Pressure Liquid Chromatography (HPLC) in the analysis of catecholamines in human cerebrospinal fluid are described. The location of the electrodes in this manner permits an electrochemically reversible or quasireversible couple to be electrolized more than once as it passes through the detector. If one electrode is held at a potential where oxidation takes place and the second electrode at a potential where reduction of this oxidized form back to the starting material occurs, then the current produced increases proportionately to the number of conversions that take place. A comparison of this cell in the dual-working-electrode and single-working-electrode mode shows an improvement in the signal-to-noise ratio by a factor of six. This HPLC system with electrochemical detection has been used for the first time to detect norephinephrine (141 pg/mL) and dopamine (262 pg/mL) in human cerebrospinal fluid.

Narcolepsy is a disabling sleep disorder that is characterized by persistent sleepiness, and cataplexy – an involuntary loss of waking muscle tone. Cataplexy and narcolepsy are caused by the loss of hypocretin containing neurons in the hypothalamus. However, it is hypothesized that dopamine is also involved in sleep and motor control and plays a role in cataplexy. This study investigated how manipulating dopamine affected sleep and cataplexy in narcoleptic mice devoid of hypocretin. We used d-amphetamine to increase endogenous dopamine levels and quinpirole (D2 agonist) to agonize D2 receptor sites. Amphetamine promoted wakefulness while decreasing sleep in wild-type mice, but was less effective in narcoleptic mice. Amphetamine also reduced cataplexy as well as sleep attacks (an indicator of sleepiness) in narcoleptic mice. Quinpirole had no effect on sleep or wakefulness; however, it potently increased cataplexy without affecting sleep attacks in narcoleptic mice.

Repeated administration of psychostimulant drugs induces a long-term state of sensitization in the mesolimbic dopamine system. This hyperdopaminergic state is associated with enhanced reward-seeking behaviors. Such aberration of incentive motivational processing is suggested to facilitate the initiation and maintenance of compulsive drug-taking behaviors. A defining characteristic of addiction is the persistence to pursue drug reinforcement despite negative consequences associated with administration. Thus, it is likely that addicts frequently experience states of motivational conflict to both seek and avoid the drug. The present study investigated the effects of repeated cocaine exposure on goal-seeking behaviors in rats, utilizing conflict paradigms wherein positive and negative incentive motivations were simultaneously evoked. Here it was shown that cocaine-experienced rats displayed both enhanced approach and avoidance behaviors, depending upon the conditions put forth in each paradigm. The results contribute to elucidating the consequences of drug administration upon basic motivational processes that may influence compulsive drug-taking behaviors.

In this thesis I investigated the neural circuits that trigger cataplexy in mice. Specifically, I first addressed the theory that cataplexy is a REM sleep disorder. I then investigated a role for the noradrenergic and dopaminergic systems in murine cataplexy. Finally, I addressed the role of the amygdala in triggering cataplexy. From this work several specific conclusions can be drawn: 1. Cataplexy does not share a common executive mechanism with REM sleep, although the two may share a common mechanism that generates muscle atonia. Muscle tone during REM sleep and cataplexy is similar, however increasing REM sleep pressure does not increase cataplexy and positive affective stimuli that can increase cataplexy tend to decrease REM sleep. 2. Systemic manipulation of dopamine receptors can modulate cataplexy without affecting behavioral state. Specifically, manipulation of D2-like dopamine receptors at specific doses can modulate cataplexy while having no affect on sleep-wake state or sleep attacks, and manipulation of D1-like receptors potently affects sleep-wake state and sleep attacks without affecting cataplexy. 3. Systemic modulation of noradrenergic activity in orexin KO mice is sufficient to modulate cataplexy. Specifically, activation of excitatory α1 receptors reduces the occurrence of cataplexy while blockade of these receptors exacerbates it. 4. Withdrawal of an endogenous α1-mediated noradrenergic drive from motor neurons during wakefulness contributed to the loss of muscle tone during cataplexy. Re-establishing this excitatory drive exogenously alleviated cataplexy-dependant muscle atonia. 5. The amygdala is a critical part of the neural mechanism that triggers cataplexy in orexin KO mice. Ablation of the amygdala resulted in significant decreases in both baseline cataplexy and emotionally-induced cataplexy. The amygdala may trigger cataplexy through direct projections to brainstem areas that regulate muscle atonia.

The dopamine transporter, the principle binding site for such drugs of abuse as cocaine and amphetamines, has a critical role in limiting dopamine availability. Several lines of evidence, including variation of DAT density in human alcoholics and in vervet monkeys with a preference for alcohol, have implicated this locus as a candidate gene, which might increase vulnerability to alcoholism. The objective of this study was to identify polymorphisms in the regulatory region of the dopamine transporter and determine whether there was an association between any of the alleles and alcoholism. Five polymorphisms were identified: three in humans and two in vervet monkey subjects. Mutation analysis of this locus may be a critical step in identifying alleles which increase susceptibility to alcohol abuse in humans and vervet monkeys.

Controversy exists concerning the role that dopamine plays in analgesia. In the present studies, dopamine agonists produced analgesia, and D-amphetamine potentiated morphine analgesia, while treatment with 6-hydroxydopamine or mixed or selective D1 and D2 dopamine receptor antagonists attenuated or abolished morphine and D-amphetamine-induced analgesia, in the formalin test. Furthermore, microinjection of morphine into the ventral tegmental area (VTA) and ventral striatum produced analgesia, while intra-VTA microinjection of naloxone methylbromide antagonized the analgesia produced by systemic morphine, in the formalin test. In contrast, similar manipulations of dopamine had little or no effect in the tail flick test. Thus, dopamine appears to play a facilitatory role in formalin test analgesia, and there appear to be fundamental differences between the formalin and tail flick tests and parallels between the role of dopamine in the formalin test and in clinical pain, the vocalization after-discharge test and reward.

A theoretical post-translational processing model of the proprotein form of glial cell line-derived neurotrophic factor (GDNF) likely produces three biologically active peptides. The three prospective peptides formed are 5, 11, and 17 amino acid peptides, entitled dopamine neuron stimulating peptide -5 (DNSP-5), -11 (DNSP-11), and -17 (DNSP-17), respectively. The DNSPs were hypothesized to increase dopaminergic neuron function because of their relationship to GDNF: a molecule with established neurotrophic actions on dopaminergic neurons. The DNSPs have the potential to provide a therapeutic molecule similar to GDNF, but with increased ease of delivery and improved bioavailability. Neurochemical effects of DNSPs were examined in the nigrostriatal pathway of normal Fischer 344 rats, and DNSP-11 was found to be the most effective in increasing dopamine neurochemical function. Striatal microdialysis, four weeks after a single intranigral administration of DNSP-11, showed significant increases in the baseline concentrations of dopamine, DOPAC, and HVA. In addition, both, potassium and d-amphetamine-evoked dopamine overflow were significantly increased. DNSP-11 was delivered intranigrally to aged Fischer 344 rats to examine DNSP-11’s ability to improve dopaminergic function in aged dopamine neurons. DNSP-11 affected striatal dopaminergic function 28 days after treatment by decreasing baseline concentrations of dopamine and evoked dopamine release. Investigation of DNSP-11 continued, using two models of neurotoxin-induced dopamine neuron loss that model cell loss associated with Parkinson’s disease. The neuroprotective properties of DNSP-11 were evaluated by delivering DNSP-11 prior to the neurotoxic insult. DNSP-11 treatment was unable to protect dopaminergic neurons, but significantly increased dopamine metabolism. In a model of severe dopamine neuron loss, DNSP-11 treatment significantly improved apomorphine-induced rotation behavior, indicative of alterations in the function of nigrostriatal dopaminergic neurons. Subsequent examination of dopamine content within the SN revealed significant increases in dopamine and DOPAC levels by DNSP-11. Taken together, DNSP-11 treatments modified dopamine neurochemistry in all investigated rodent models. The observed long-term alterations of dopamine neurochemistry by DNSP-11 and subsequent behavioral changes support a potential use for DNSP-11 as a therapeutic for dopaminergic cell loss. Increased dopaminergic function by DNSP-11 is evidence for the novel concept that peptides contained within the prodomain of trophic factors can have neurotrophic actions.

Impulsive choice refers to the inability to delay gratification and is associated with increased drug abuse vulnerability. Understanding the underlying neural mechanisms linking impulsive choice and drug abuse can contribute to improved treatment options for individuals with substance use disorders. Evidence suggests a major role for nucleus accumbens core (NAcc) in impulsive choice and the reinforcing effects of drugs of abuse. The neurotransmitters glutamate (Glu) and dopamine (DA) are implicated in the neural adaptations observed in drug addiction; however, the role of intra-NAcc Glu and DA in impulsive choice is unclear. Rats were trained in a delay discounting task, in which animals chose between a small, immediate reinforcer and large, delayed reinforcer. Consistently choosing the small, immediate reinforcer was considered to reflect increased impulsivity. Following delay discounting, in vitro receptor autoradiography was performed to quantify the number of N-methyl-D-aspartate (NMDA) receptors and dopamine transporters (DAT) in NAcc and nucleus accumbens shell (NAcSh). In a separate experiment, rats were trained in delay discounting and were implanted with guide cannulae into NAcc. Following surgery, rats received microinfusions of either a) the Glu-selective ligands MK-801 (noncompetitive NMDA receptor channel blocker; 0, 0.3, and 1.0 μg), AP-5 (competitive NMDA receptor antagonist; 0, 0.3, and 1.0 μg), ifenprodil (NMDA NR2B subunit antagonist; 0, 0.3, and 1.0 μg), and CNQX (AMPA receptor antagonist; 0, 0.2, and 0.5 μg) or b) the DA-selective ligands SKF 38393 (D1-like receptor agonist; 0, 0.03, and 0.1 μg), SCH 23390 (D1-like receptor antagonist; 0, 0.3, and 1.0 μg), quinpirole (D2-like receptor agonist; 0, 0.3, and 1.0 μg), and eticlopride (D2-like receptor antagonist; 0, 0.3, and 1.0 μg). In NAcc and NAcSh, NMDA receptor and DAT expression did not differ between high and low impulsive rats. Furthermore, intra-NAcc administration of NDMA and DA receptor ligands did not significantly alter impulsive choice. These results suggest that Glu and DA systems within NAcc do not directly mediate impulsive decision making. Future work is needed to determine the precise role of NAcc in mediating impulsive choice.

Le récepteur de l'inositol 1,4,5-trisphosphate (IP?R), un canal calcique, est un des principaux régulateurs de la mobilisation de Ca[indice supérieur 2+] intracellulaire. En regardant globalement les mécanismes de régulation des IP?Rs, on peut se rendre compte que cette régulation se fait bien souvent dans le contexte précis d'une ou de plusieurs voies de signalisation. Bien que de nombreux modulateurs de l'activité calcique des IP?Rs soient déjà connus, encore aujourd'hui beaucoup de questions sont en suspens. En fait, un nombre croissant de preuves et d'observations suggèrent que la régulation spécifique des mécanismes de signalisation calcique peut être accomplie par l'activation simultanée de plusieurs voies de signalisation ce qui permet de soutenir la diversité d’action du Ca[indice supérieur 2+]. Une de ces voies, la voie de croissance et de prolifération cellulaire PI3K-AKT-mTOR fut d’abord investiguée dans la première partie de cette thèse afin de comprendre l’importance de l’activité calcique des IP?R dans ces phénomènes d’importance majeure. Nous avons montré que mTOR interagit et phosphoryle l'IP?R-3 dans les cellules RINm5F puis déterminé que le niveau d’activité de mTOR affectait les relâches calciques médiées par l'IP?R-3 de ces cellules. Sachant que mTOR contrôle la prolifération et l'homéostasie cellulaire et que le Ca[indice supérieur 2+] joue aussi un rôle clé dans ces deux phénomènes, il est logique de croire que mTOR facilite les relâches calciques dépendantes des IP?Rs afin de fournir le Ca[indice supérieur 2+] nécessaire aux actions physiologiques de mTOR. La deuxième section de cette thèse a exploré l’interaction entre la voie dopaminergique et la voie calcique dépendante des IP?Rs. Nos résultats suggèrent que calcyon, une protéine liant le Ca [indice supérieur 2+] surexprimée dans plusieurs neuropathologies, est impliquée dans l'internalisation des récepteurs D2 par un mécanisme encore inconnu qui cause par le fait même une relâche calcique tardive dans les cellules PC-12. Nous avons montré que cette protéine affecte aussi l’activité calcique des IP?Rs. Nos résultats suggèrent que calcyon est la protéine reliant les voies de signalisation calcique et dopaminergique et que son implication est d’importance majeure afin d’assurer l’homéostasie calcique neuronale. L’ensemble de ces travaux montre toute l’étendue de la régulation calcique et de l’interaction entre les voies de signalisation.