Rozprawy doktorskie na temat „Dopamine”

Neuro-computational models allow to study the brain mechanisms involved in intelligent behavior and extract essential computational principles which can be implemented in cognitive systems. They are a promising solution to achieve a brain-like artificial intelligence that can compete with natural intelligence on realistic behaviors. A crucial property of intelligent behavior is motivation, defined as the incentive to interact with the world in order to achieve specific goals, either extrinsic (obtaining rewards such as food or money, or avoiding pain) or intrinsic (satisfying one’s curiosity, fun). In the human brain, motivated or goal-directed behavior depends on a network of different structures, including the prefrontal cortex, the basal ganglia and the limbic system. Dopamine, a neurotransmitter associated with reward processing, plays a central role in coordinating the activity of this network. It structures processing in high-level cognitive areas along a limbic-associative-motor gradient and impacts the learning capabilities of the whole system. In this habilitation thesis, I present biologically-constrained neuro-computational models which investigate the role of dopamine in visual object categorization and memory retrieval (Vitay and Hamker, 2008), reinforcement learning and action selection (Vitay and Hamker, 2010), the updating, learning and maintenance of working memory (Schroll et al., 2012) and timing processes (Vitay and Hamker, 2014). These models outline the many mechanisms by which the dopaminergic system regulates cognitive and emotional behavior: bistable processing modes in the cerebral cortex, modulation of synaptic transmission and plasticity, allocation of cognitive resources and signaling of relevant events. Finally, I present a neural simulator able to simulate a variety of neuro-computational models efficiently on parallel architectures (Vitay et al., 2015)
Neuronale Modelle nach dem Vorbild des Gehirns bieten die Möglichkeit intelligente, kognitive Prozesse nicht nur besser zu verstehen, sondern sie stellen auch eine vielversprechende Lösung dar, um eine Gehirn-ähnliche künstliche Intelligenz für Wahrnehmung und Verhaltensweisen zu erreichen, die mit natürlicher Intelligenz konkurrieren kann. Eine entscheidende Eigenschaft von intelligentem Verhalten ist Motivation, definiert als der Anreiz mit der Welt zu interagieren, um bestimmte Ziele zu erreichen, sei es extrinsisch (Belohnungen wie Nahrung oder Geld zu erhalten oder die Vermeidung von Schmerzen) oder intrinsisch (die Neugier zu befriedigen, Spaß zu haben). Im menschlichen Gehirn basiert motiviertes oder zielgerichtetes Verhalten auf einem Netzwerk von verschiedenen Strukturen, einschließlich des präfrontalen Cortex, der Basalganglien und des limbischen Systems. Dopamin, ein Neurotransmitter, welcher der Belohnungsverarbeitung zugeordnet wird, spielt eine zentrale Rolle bei der Koordination der Aktivität in diesem Netzwerk. Es strukturiert die Verarbeitung in High-Level-kognitiven Bereichen entlang eines limbischen-assoziativ-motor Gradienten und beinflusst die Lernfähigkeit des gesamten Systems. In dieser Habilitation, präsentiere ich biologisch motivierte neuronale Modelle, die die Rolle von Dopamin in der visuellen Objektkategorisierung und Gedächtnisabruf (Vitay and Hamker, 2008), Reinforcement Lernen und Aktionsauswahl (Vitay and Hamker, 2010), Aktualisierung, Lernen und Aufrechterhaltung von Arbeitsgedächtnis (Schroll et al., 2012) und Timing Prozessen (Vitay and Hamker, 2014) untersuchen. Diese Modelle beschreiben Mechanismen, durch die das dopaminerge System kognitives und emotionales Verhalten reguliert: bistabile Verarbeitungsmodi in der Hirnrinde, Plastizität und Modulation der synaptischen Übertragung, Zuweisung von kognitiven Ressourcen und Signalisierung von relevanten Ereignissen. Schließlich beschreibe ich einen neuronalen Simulator, der in in der Lage ist, eine Vielzahl von neuronalen Modellen effizient auf parallelen Architekturen zu simulieren (Vitay et al., 2015)

The primary focus of the present thesis is the exploration of factors that influence the dopamine (DA) neuron by examining the expression of the dopamine transporter (DAT), a marker of the DA neuron. The secondary focus of this thesis is on the serotonin neuron and in particular the serotonin transporter (SERT), a marker of the serotonin neuron. To this end three distinct and separate models have been employed. The goals of this thesis were: (1) to test the hypothesis that monoamine oxidase inhibition during development alters serotonergic innervation in the cortex and raphe, while not affecting relative DA innervation of nigrostriatal pathway, (2) to test the hypothesis that elevated brain levels of hypoxanthine (Hx) deleteriously affect the DA neuron, and (3) to test the hypothesis that densities of DAT and SERT in brainstem cell body regions distinguish alcohol-preferring vervet monkeys with different behavioral patterns of ethanol consumption.
Alterations in the activity of monoamine oxidase (MAO), a degradative enzyme that plays an important role in regulating levels of monoamine transmitters, may have a profound effect on brain development. The present study investigates relative DA and serotonin innervation of cortical and subcortical areas, measured by DAT and SERT densities, following MAO inhibition (A or B or A+B) in mice throughout gestation and early post-natal development. DAT binding was unaltered within the nigrostriatal pathway. The most significant finding reported here is that the combined MAO-A+B inhibition significantly reduced SERT binding by 25% in both the cortex and raphe nucleus. Lower levels of SERT binding were apparent during the early post-natal period (PND 14), a period during which pups were still exposed to MAO inhibitors in the dam's milk, but also persisted into later life (PND's 35 and 90) after inhibitors were no longer being administered. Persistent effects were restricted to cortex and raphe, suggesting a relative vulnerability of these regions to alterations in monoamine transmitter levels during development.
The second study presents data demonstrating that Hx delivered intracerebroventricularly significantly reduces the number of tyrosine hydroxylase immunoreactive cells (TH-ir) in the substantia nigra by 22% and 30%, at 7 and 21 days, respectively. After 3 days of Hx administration, striatal DA and serotonin were elevated over control levels by 22% and 25%, respectively, but returned to control levels by 7 days. The serotonin metabolite 5-HIAA was elevated after 3 days of Hx, but levels of DA metabolites were not different from control. Locomotion, a behavior thought to be related to DA transmission, was elevated following Hx treatment, as were presynaptic markers of the DA system such as DAT and TH protein levels. The persistent reduction in TH positive cell numbers suggests that Hx damages or kills DA neurons. The increase in intracellular DA at early time points suggests that Hx might interfere with DA release, possibly by temporarily inactivating DA neurons. These findings are consistent with the hypothesis that Hx, a purine significantly elevated in blood and CSF of Lesch-Nyhan patients, maybe involved in DA dysfunction.
Studies on alcohol abuse have focused on the mesolimbic DA pathway and the serotonergic influence within this pathway. Here we report that abstinent binge-drinking monkeys have significant reductions of SERT binding, and to a lesser extent, DAT binding in the midbrain region, while abstinent heavy-drinking subjects have elevated levels of DAT binding, as compared to controls. Both mesolimbic and nigrostriatal pathways are affected. CSF levels of both HVA and 5-HIAA substantiate the neuroanatomical differences between binge- and heavy-drinking vervets. Taken together, these findings provide a neurochemical profile with which to further distinguish subtypes of alcohol-preferring vervet monkeys.

Today there is a growing awareness that in Parkinson's disease can occur motor and non motor disorders, in particular substances dependency syndromes and behavioral addiction. Objectives of the study. Objective of the study is to determine any changes frontal circuits potentially involved in the development and maintenance of "behaviour addiction", by neuroimaging and neurophysiological methods in patients with Parkinson's disease with impulses control disorder. Materials and methods. In the study of 8 patients with Parkinson's disease with at least an impulse control disorder, particularly 8 pathological gambler , and 8 patients with non-demented Parkinson disease with comparable clinical and demographic characteristics who had not demonstrated behavioural abnormalities . Patients were subjected to a magnetic resonance imaging with conventional sequences and DWIs in 15 space directions (diffusion tensor imaging) and T1 weighted sequences volumetric, determination of P 300 neurophysiological and neuropsychological test battery for exploring the cognitive functions. Results. The two groups have had no statistically significant differences evaluated using t-Student test, although patients with ICDS have shown average levels of FA tend to higher; in assessing NP the only statistically significant differences have emerged the WCST and NPI. However, it should be pointed out as ICD patients, compared to Parkinsoniani without compulsive disorder, P3a monsters lower latency than the P3b, as evident in healthy subjects.

A procedure was developed to determine the effect of a dopamine (DA) antagonist (haloperidol) and a DA agonist (d-amphetamine) on rats' perceptions of the hedonic value of food. Eighteen rats were trained to discriminate between two quantities of sweet food pellets (1 and 4), in a forced-choice two-lever successive discrimination procedure. To control for non-specific perceptual effects of the treatments, the rats were also trained to discriminate between 1 and 4 tones. It was established that rats attended to the value of food, as well as the proportional differences in quantity, when discriminating food quantities. This was accomplished by altering the value of the food in two ways. Firstly, "hunger" was altered by changing the degree of food deprivation during testing. Secondly, unsweetened food pellets were introduced as probe cues. These two methods of altering the value of food pellets were utilized while quantity generalization gradients were determined, by presenting animals with 1,2, 3 and 4 numbers of stimuli as probe cues. Two measures were derived from these generalization gradients: the point of subjective equality (PSE), which is the calculated number of stimuli that would maintain responses equally distributed between the two levers, and the slope of the gradient. The PSE primarily reflects perceptual processes, while the slopes of the gradients provide an index of performance impairment. It was observed that decreasing the value of food by either decreasing food deprivation or reducing the sweetness of the food pellets resulted in the rats perceiving a given quantity of food as larger than before these treatments (decreased the food PSE). Neither altering food deprivation nor introducing novel tone probes had an effect on the numerical attributes of tones, as reflected by the tone PSE. Haloperidol (0.030, 0.50 and 0.083 mg/kg, i.p.) produced a statistically significant, but slight dose-dependent performance deficit, as reflected by the slope of the generalization gradients. It did not affect the perception of food pellet quantities at any dose, as reflected by the food PSE. Haloperidol decreased the number of tones a given quantity was perceived as by rats (increased the tone PSE). Amphetamine (0.25, 0.50 and 1.0 mg/kg, i.p.) decreased the perception of a given quantity of food (increased the food PSE) in a dose-dependent manner, without a significant effect on performance. Thus, amphetamine enhanced the hedonic value of food. Amphetamine also increased rats' perceptions of a given number of tones (decreased the tone PSE). It therefore appears that while d-amphetamine can enhance the perceived hedonic value of food, haloperidol has no effect on rats' perceptions of the hedonic value of food. Furthermore, evidence that DA systems are involved in the mechanism of an "internal clock" or "counter" was obtained.
Medicine, Faculty of
Graduate

IntroductionThe mesolimbic dopamine (DA) system is thought to be important for reward-based decision-making. Dysfunction of this system is implicated in impulsivity. Many neuropsychiatric disorders, such as drug addiction, obesity and pathological gambling, are characterized by a relative oversensitivity to reward and insensitivity to negative outcomes, as well as impulsivity. The administration of DA agonists in some patients with Parkinson's disease (PD) can trigger unusually risky behavior, such as pathological gambling and compulsive sexual behavior, which are impulse control disorders (ICD). Interestingly the risk factors predisposing PD patients to ICDs closely resemble those associated with drug addiction in the general population. PD patients who develop ICDs have risk factors similar to non-PD patients who develop addictions, possibly due to a similar underlying neurobiological mechanism. We asked two questions. 1) Are there underlying abnormalities in the neural processing of risky decision-making in PD, specifically those who develop ICDs? 2) What role does DA plays in risky decision-making in young healthy subjects: without the limiting factor of age, disease or pathology.MethodsExperiment 1 used functional magnetic resonance imaging (fMRI) to look at differences in neural activation during gambling in PD patients who had a history of ICDs due to DA agonist treatment (n=11, PD-ICD) compared to their disease (n=12, PD-CON) and age-matched controls (n=11, AM-CON). Experiment 2 tested the relationship between DA level and performance on a gambling and probabilistic selection task, using the acute phenylalanine/tyrosine depletion (APTD) method, fMRI and [11C]raclopride positron emission tomography (PET), in healthy subjects 18- 30 years of age (n=17). ResultsExperiment 1: (1) Individual predisposition to ICDs is evidenced by differences in personality, betting strategies and evaluation of financial risk. (2) The PD-ICD group showed dysfunction in the weighting of losses relative to gains both behaviourally and neurally. (3) The ventral striatal (VStr) reward prediction error signal is not necessary to develop and maintain behavioural addictions. And (4) Abnormality in the insular cortex's evaluation of risk and reward, and perhaps that of the mesocorticolimbic network in general, may be the main neural correlate mediating susceptibility for the development of an ICD in PD.Experiment 2: (1) APTD-induced lowering of brain DA led to an increase in loss aversion and an increased ability to learn from negative outcomes; the greater the depletion, the greater the improvement in punishment accuracy. (2) High impulsivity scores were correlated with high baseline D2 receptor levels, which may index low tonic DA and susceptibility to high phasic DA neuron firing. (3) The BOLD signal was correlated with the expected value and reward prediction error in the VStr bilaterally, however, this correlation was significantly increased after DA depletion, suggesting that the striatal BOLD signal in that area is not a direct result of DA release. Conclusion: The combination of both experiments resulted in four main conclusions: (1) the BOLD signal in the VStr may not be as DA driven as previously thought, (2) risk and loss aversion can be directly controlled by DA levels, (3) impulsivity, a risk factor for addiction and ICDs, is caused by an elevated phasic DA response to potential rewards, and (4) learning to avoid maladaptive choices is sensitive to acute changes in DA levels, possibly D2 receptor driven. The result of the present work provides a framework for understanding the reward and loss aversion systems that control risk taking, and impulsivity. Together, our results support a model in which impulsivity, a risk factor for addiction, is caused by an elevated phasic DA response to potential rewards, while learning to avoid maladaptive choices is sensitive to acute changes in DA levels, possibly D2 receptor driven.
IntroductionLe système dopaminergique mésolimbique est connu pour son importance dans la prise de décision, lié au mécanisme de la récompense. Un disfonctionnement de ce système influe sur l'impulsivité, qui peut être considéré comme une forme anormale de la prise de décisions. De nombreux désordres neuropsychiatriques, tel que l'addiction aux drogues, l'obésité et le jeu compulsif, sont caractérisés par une sur-sensibilité à la récompense et une insensibilité aux conséquences négatives.Le traitement des patients atteint de la maladie Parkinson, par des substituts en Dopamine peut entrainer, de manière inhabituelle, des comportements à risques, tel que des pathologies liées aux jeux d'argent ou des impulsions sexuelles. Nous posons deux questions : 1) Existent-ils des anormalités sous-jacentes dans un procédé neuronal de prise de décisions chez les patients atteints de Parkinson qui développent des désordres impulsifs ? 2) Quel rôle joue la Dopamine dans la prise de décisions à risque chez des jeunes sujets sains.MéthodesL'Expérience 1 : Nous avons utilisé l'Imagerie par Résonance Magnétique Fonctionnelle chez des patients atteints de Parkinson, avec désordre impulsif (n=11), comparé a deux groupes témoins : Groupe de patients parkinsoniens sans troubles compulsif, (n=12) et sujets-control non-atteint du même âge (n=11).Expérience 2 : Nous avons utilisé la méthode "Acute phenylalanine/tyrosine depletion (APTD)" [pour réduire le niveau Dopamine], l'Imagerie par Résonance Magnétique Fonctionnelle et la Tomographie par Emission de Positons chez des sujets sains âgés de 18 a 30 ans (n=17) ainsi que des tests d'impulsivité et d'apprentissage.RésultatsExpérience 1 : (1) La prédisposition aux désordres impulsifs est relié a des différences de personnalité, et dans l'évaluation du risque financier. (2) Le groupe PD-avec troubles compulsifs révèle un disfonctionnement dans la pondération des pertes relative aux gains, de manière comportementale et neuronale. (3) L'erreur dans la prédiction de la récompense n'est pas nécessaire dans le développement ou maintien des addictions comportementales. Les irrégularités dans l'évaluation du risque et de la récompense par le cortex insulaire seraient à l'origine du développement d'un désordre impulsif dans la maladie de Parkinson.Expérience 2 : (1) La diminution de la Dopamine dans le cerveau a augmenté l'aversion à la perte, ainsi que l'aptitude à apprendre les conséquences négatives; plus la diminution de la Dopamine (déplétion) est importante, telle que mesurée par la TEP, plus cela améliore l'évaluation de la punition (2). Les scores élevés d'impulsivité corrèlent avec le niveau de récepteurs D2, qui pourraient indiquer une faible réponse tonique des neurones dopaminergiques et une tendance vers une haute réponse phasique de ces neurones. (3) Le signal BOLD corrèle avec la valeur attendue et l'erreur de prédiction de la récompense, de façon bilatérale dans le striatum ventral. Cette corrélation augmente de manière significative après la réduction de la Dopamine, suggérant que le signal BOLD dans cette même région ne résulte pas directement de la libération de Dopamine.Conclusion: Il y a quatre conclusions principales: (1) le signal BOLD dans le striatum ventral peut être due à d'autres neurotransmetteurs que la dopamine, (2) le risque et l'aversion à la perte dependent du niveau de Dopamine, (3) l'impulsivité est causée par une haute réponse phasique de Dopamine, enfin (4) apprendre à éviter des choix mal-adaptés depend des niveaux de Dopamine et des récepteurs D2. Ce projet fournit les bases d'une meilleure compréhension de la récompense et de l'aversion à la perte qui contrôle la prise de risque, l'apprentissage de la récompense et l'impulsivité.

Stress activates dopamine (DA) transmission in the basolateral amygdala (BLA), medial prefrontal cortex (mPFC) and nucleus accumbens (NAc), and DA transmission in these regions mediates different aspects of the behavioural response to stress. Evidence indicates that a functional interdependence exists between the DA projections to these areas. mPFC DA exerts an inhibitory influence on stress-induced NAc DA release. Similarly, BLA DA modulates mPFC and NAc DA function, although it is unknown if this is also the case in response to stress. Thus, we examined whether BLA DA modulates NAc and mPFC DA release in response to stress. We first determined the effects of BLA DA depletion on stress-induced NAc and mPFC DA release. BLA DA depletion potentiated the NAc and attenuated the mPFC DA responses to stress. We then examined the effects of intra-BLA D1 and D2/D3 receptor antagonists on the NAc and mPFC DA responses to stress. BLA D1, but not D2/D3, receptor antagonism potentiated and attenuated stress-induced NAc and mPFC DA release, respectively. Co-administration of D1 antagonist into BLA and DA agonists into mPFC abolished the potentiation of stress-induced NAc DA release, indicating that BLA D1 receptor modulation of the NAc DA stress response occurs indirectly by modulating stress-induced mPFC DA release. We then turned to examining the influence of BLA DA on behavioural measures of information processing which are themselves modulated by NAc and mPFC DA function. The effects of BLA D1 and D2/D3 receptor blockade on prepulse inhibition (PPI) and latent inhibition (LI) were examined. BLA D1 receptor antagonist enhanced and D2/D3 receptor antagonist reduced PPI. Conversely, neither BLA DA receptor antagonist had an effect on LI. Finally, we determined the effects of BLA D1 and D2/D3 receptor antagonists on acoustic startle habituation. Although BLA DA receptor antagonism had no effect on this measure, both D1 and D2/D3 receptor blockade reduc

The Herrnstein matching law was used to dissociate motoric from motivational drug-induced performance changes. The effects of neuroleptic drugs were compatible with, at low doses, a reduction in reinforcer efficacy, and at higher doses, an additional motor impairment However, the Herrnstein matching law was found to be prone to artifactual error; in particular, under reinforcement-lean conditions reductions in reinforcer efficacy were time-dependent These problems compromised the use of the Herrnstein law to assess drug-induced performance changes. Raclopride-induced time-dependent reductions in response rate occurred in the absence of both primary and secondary reinforcement. Within-session decrements in both operant and consummatory behaviour were observed following administration of sulpiride to the anterodorsal striatum, but not following administration of sulpiride to the nucleus accumbens. The implications of this finding are discussed in relation to the internal organisation of behaviour, and Parkinson's disease. Consumption of sucrose and operant responding maintained by sucrose pellets follows an inverted-U-shaped concentration-intake function. Systemic administration of raclopride shifted the curve to the right. It is argued that this curve shift reflects an impairment in the primary reward process. Effects of intracranial administration of sulpiride on sucrose consumption were restricted to the nucleus accumbens at a low concentration of sucrose, but were also observed within the anterodorsal striatum and basolateral amygdala at higher concentrations. These findings are discussed in relation to the neuroanatomical substrates for the guidance of behaviour by external cues, and for reward processes.

Alcohol use disorder is a debilitating disorder affecting nearly 5% of people in the United States. Despite the prevalence of alcohol use disorder few affected individuals seek treatment and of those who do many will relapse. This highlights a need to develop new treatments for alcohol use disorder that are both more accessible and more effective. This dissertation characterizes a novel pathway involved in ethanol enhancement of dopamine levels in the nucleus accumbens as well as investigating alterations in dopamine 2 receptor expression and function following an acute dose of ethanol. This was done by using microdialysis to measure dopamine levels in the nucleus accumbens, single-unit recordings of dopamine neurons in the ventral tegmental area to measure dopamine neuron activity and place conditioning to measure the rewarding properties of the intravenous dopamine and ethanol. It was found that activation of peripheral dopamine 2 receptors by intravenous dopamine enhanced dopamine levels in the nucleus accumbens and dopamine neuron firing rate in the ventral tegmental area. Additionally, intravenous dopamine produced a modest conditioned place preference. Domperidone, a peripheral dopamine 2 receptor antagonist blocked each of these effects. Further, domperidone blocked ethanol enhancement of dopamine release in the nucleus accumbens and bidirectionally modulated the sedating effects of ethanol depending on the dose of ethanol administered. The involvement of peripheral dopamine 2 receptors in ethanol reward could not be ascertained in these studies as domperidone produced a weak conditioned place aversion. Finally, acute ethanol was found to enhance dopamine 2 receptor expression in the nucleus accumbens and medial prefrontal cortex while also enhancing dopamine 2 receptor expression on NK and B cells. Additionally, ethanol was found to reduce desensitization of dopamine 2 receptors in the ventral tegmental area. These results demonstrate that activation of peripheral dopamine 2 receptors can enhance dopamine levels in the nucleus accumbens and that this effect has relevance in understanding the effects of ethanol on dopamine release in the mesolimbic pathway. These results also provide evidence for transient upregulation of dopamine 2 receptors in the brain and on leukocytes suggesting that dopamine 2 receptor levels on leukocytes may be a useful biomarker for central dopamine function.

Thesis (Ph. D.)--Illinois State University, 2002.
Title from title page screen, viewed February 3, 2006. Dissertation Committee: Maarten E.A. Reith (chair), Hou Tak Cheung, Paul A. Garris, Stephen M. Lasley, Brian J. Wilkinson. Includes bibliographical references and abstract. Also available in print.

Thesis (Ph. D.)--Illinois State University, 1996.
Title from title page screen, viewed May 30, 2006. Dissertation Committee: Maarten E. A. Reith (chair), Hou Tak Cheung, John W. Dailey, Robert L. Preston, Brian J. Wilkinson. Includes bibliographical references and abstract. Also available in print.

Neuromelanin (NM) is a dark polymer pigment present in specific populations of catecholaminergic neurons in the brain. Interest in this pigment has rekindled in recent years because of a hypothesised link between NM and the especial vulnerability of NM-containing neurons to cell death in Parkinson???s disease (PD). Many aspects of the biology of NM are yet to be characterised. It is not known if NM like the similar melanin of the skin is synthesised via an enzymatic pathway or solely through autoxidation as has traditionally been thought. Examination of the ultrastructure of NM granules showed that in contrast to peripheral melanosomes, an electron-lucent lipid component was present that represented 30% of pigment volume. The identity of the lipid component of NM has remained unclear since it was first suggested that NM contained lipid in the 1960???s. NM lipid was biochemically isolated from the substantia nigra of 32 human brains. Using reversed-phase high performance liquid chromatography, atmospheric pressure chemical ionisation mass spectrometry and 1H- and 13C NMR techniques, it was shown for the first time that the NM lipid is the polyisoprenoid dolichol. The age-related development and regulation of NM has not previously been described. Optical density and area measurements of unstained NM in ventral substantia nigra neurons spanning the ages of 24 weeks to 95 years old demonstrated three developmental phases. NM was not present at birth and initiation of pigmentation began at approximately 3 years of age, followed by a period of increasing pigment granule number and colouration until age 20. In PD brain, the ultrastructure of NM demonstrated that the amount of lipid did not change. However, filipin staining showed a reduction of cholesterol in PD NM containing neurons. In addition, immunogold staining of ??-synuclein demonstrated that this protein redistributed to the NM lipid in PD brain. The finding of phases in the development of NM, and the identification of lipid species in NM suggest that NM biology is regulated. This thesis has also demonstrated changes in the lipid and associated proteins in PD, suggesting NM???s chemical composition alters which may have functional consequences that contribute to PD.

The mesolimbic dopamine (DA) system projects from the ventral tegmental area (VTA) to structures associated with the limbic system, primarily the nucleus accumbens (NAc). This system has been implicated in the rewarding effects of drugs of abuse. Many drugs of abuse act in the VTA, the NAc, or both. Dopamine neurons in the VTA that project to the NAc, and the GABA neurons that inhibit DA neurons locally in the VTA or project to the NAc, play an important role in mediating addiction to various drugs of abuse, in particular alcohol. There is a growing body of evidence of co-dependence of nicotine and ethanol drug abuse. Given this evidence, it is possible that both ethanol and nicotine target similar receptors in the NAc. The GABA-A and GABA-B receptors have also been implicated in the modulation of ethanol's reinforcing properties (Anstrom, Cromwell, Markowski, & Woodward, 2003; Besheer, Lepoutre, & Hodge, 2004; Colombo et al., 2000; Moore & Boehm, 2009; Stromberg, 2004; Walker & Koob, 2007). Thus, there is a growing literature suggesting that GABA receptors are implicated in ethanol reward. In these studies, we evaluated the possibility of co-dependence of nicotine and ethanol by activity on a similar receptor in the NAc. In addition, we evaluated the role of GABA modulation of DA release, in particular GABA-A receptors and GABA-B receptors, in modulating DA release in the NAc with acute ethanol exposure. The rationale for this study was predicated on the belief that advancement in the understanding of the brain mechanisms underlying the recreational use and abuse potential of alcohol will pave the way for more effective treatment strategies that could reverse alcohol dependence and co-dependence and save lives and resources throughout the world.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
No presente trabalho, foi avaliado a interaÃÃo entre os sistemas dopaminÃrgico e colinÃrgico atravÃs do estudo dos efeitos comportamentais (campo aberto e catalepsia) e neuroquÃmicos (densidade de receptores dopaminÃrgicos (D1 e D2-sÃmile) e muscarÃnicos (M1+M2-sÃmile) em corpo estriado de rato. As seguintes drogas foram utilizadas: mazindol (agonista dopaminÃrgico indireto), apomorfina (agonista dopaminÃrgico D1-sÃmile e D2-sÃmile), pimozida (antagonista dopaminÃrgico D2-sÃmile), SCH 23390 (antagonista dopaminÃrgico D1-sÃmile), pilocarpina (agonista muscarÃnico M1-sÃmile), carbacol (agonista muscarÃnico M2-sÃmile), pirenzepina (antagonista muscarÃnico M1-sÃmile), atropina (antagonista muscarÃnico M1 e M2 nÃo seletivo), clozapina (neurolÃptico atÃpico). Os resultados mostraram que a pimozida e o carbacol, sozinhos ou associados, causaram um aumento da resposta catalÃptica e uma diminuiÃÃo da atividade locomotora. O mazindol tambÃm aumentou a atividade locomotora. Carbacol, nas menores doses, e o mazindol aumentaram a densidade de receptores D1-sÃmile. A pimozida e a atropina, isoladamente, aumentaram a densidade de receptores D1-sÃmile no corpo estriado enquanto que a atropina causou uma diminuiÃÃo dos receptores D2-sÃmile e uma upregulation dos receptores muscarÃnicos. O mazindol aumentou o binding de 3H-NMS no corpo estriado. O presente trabalho sugere, de maneira geral, que existe uma relaÃÃo entre os receptores muscarÃnicos M1 e M2 com os receptores dopaminÃrgicos D1 e D2, sendo que esta relaÃÃo pode ocorrer de maneira positiva ou negativa, dependendo da seletividade e da dose das drogas utilizadas
In the study, the interaction between the dopaminergic and cholinergic systems through the study of behavioral (open field and catalepsy) and neurochemical (density of dopaminergic receptor (D1 and D2-like) and muscarinic (M1+M2-like)) effect in striatum rat was evaluated. The following drugs were used: mazindol (indirect dopaminergic agonist), apomorphine (D1-like and D2-like dopaminergic agonist), pilocarpine (M1-like muscarnic aginist), carbachol (M-2like agonist muscarinc), pirenzepine (M1-like antagonist muscarinic), atropine (non-selective M1 and M2 antagonist muscarinic), clozapina(non-typical neuroleptic). The results showed that the pimozida and carbachol, alone or associated, caused increase in the cataleptic response and reduction in the motor activity. The mazindol also increased the motor activity. In small dosage, the carbachol and mazindol increased the density of D1-like receptors. Isolated, the pimozida and atropine increased the density of the D1-like receptors in striatum whereas the atropine caused a reduction of D2-like receptors and upregulation of muscarinic receptors. This work suggests a relationship, between muscarinc receptors M1 and M2 and dopaminergic receptors D1 and D2, and that this relationship can occur in a positive and negative manner, depending on the selectivity and the dose of the used drug

Este trabalho tem como objetivo principal a utilização da técnica “bottom-up” para a construção de nanoestruturas de ouro por meio de técnicas voltamétricas sobre uma superfície de Carbono Vítreo. Estas estruturas foram caracterizadas por métodos microscópicos e voltamétricos. A dimensão crítica das nanoestruturas foi obtida por meio de uma curva Gausseana de distribuição de alturas e diâmetros, fornecendo um valor médio de 35 nm por 150 nm de diâmetro. As nanoestruturas obtidas foram modificadas pela deposição de uma camada automontada de cistamina (um tiól), resultando num substrato adequado para futuras aplicações como a imobilização de enzimas. Os comportamentos eletroquímicos da dopamina e do ácido ascórbico, sobre eletrodos de ouro, Carbono Vítreo e Nanoestruturado, foram avaliados para a caracterização das modificações dos eletrodos. A diferença dos potenciais de pico de oxidação do AA sobre eletrodos de Carbono Vítreo e de ouro, de cerca de 300 mV, possibilitou avaliar o recobrimento do Carbono Vítreo com nanoestruturas pela diminuição da corrente de pico. Já a dopamina foi utilizada neste trabalho para determinar a presença da camada automontada de cistamina sobre a superfície das nanoestruturas de ouro, uma vez que sua resposta voltamétrica mostra um deslocamento do potencial de pico quando realizada sobre Au e sobre Au modificado com a camada automontada.
The subject of this work is focused in the utilization of a “bottom-up” approach to develop an Au nanostructured electrodeposits on the glassy carbon surface by a voltammetric procedure. These Au nanostructures have been characterized either by microscopic and voltammetric techniques. The critical dimensions of nanostructures has been evaluated by a Gausean normal data distribution curve and presented, as results a mean height of 35 nm and a mean diameter of 150 nm. The Au nanostructures were further modified by the deposition of a cystamine (thiol) self-assembled monolayer, yielding a suitable substrate for enzyme immobilization, as a possible future application. The electrochemical behaviour of dopamine and ascorbic acid on gold, Glassy Carbon and nanostructures was studied aiming to characterize the modified electrode. The 300 mV shift in the oxidation peak potential for ascorbic acid on gold in relation to Glassy Carbon allows to calculate the nanostructure coverage factor by the peak current inhibition as 43%. Finally, dopamine was employed to detect the self assembled monolayer formation on the Au nanostructure through its voltammetric behaviour. The variation in the oxidation potential for DA on gold and self assembled monolayer was used as an indicator for such modification.

Thesis (Ph. D.)--Illinois State University, 1999.
Title from title page screen, viewed July 31, 2006. Dissertation Committee: Paul A. Garris (chair), Maarten E.A. Reith, Anthony J. Otsuka, Robert L. Preston, David L. Williams. Includes bibliographical references (leaves 221-242) and abstract. Also available in print.

In Parkinson’s disease (PD) the loss of the neurotransmitter dopamine (DA) results in abnormal oscillations of the cortico-basal ganglia network, the emergence of which correlate with symptoms. Increased oscillatory power in the primary motor cortex (M1) is reduced by dopamine replacement therapy and by targeted stimulation, suggesting that M1 plays an important role in the pathology of PD. In this study we have investigated, using pharmacology, the mechanisms by which oscillatory activity in rat M1 is generated and determined the power changes associated with DA depletion and DA receptor modulation. Extracellular local field potential recordings were made in brain slices of M1 which were prepared using a modified protocol to improve viability. Co-application of carbachol (5 μM) and kainic acid (100 nM) elicited simultaneous theta (4-8 Hz) and gamma (30-40 Hz) oscillations in layer V of M1. These oscillations displayed phase-amplitude coupling; the first report of such findings in vitro. These oscillations were found to be pharmacologically distinct with theta oscillations generated by intrinsic non-synaptic mechanisms while gamma oscillations required contributing excitatory and inhibitory networks. Following successful unilateral lesions using 6-hydroxydopamine (6-OHDA), as determined by the adjusting step test, DA-depleted (ipsilateral) and DA-intact (contralateral) slices were obtained. Although no difference in the oscillatory profile of M1 ipsilateral, contralateral or age-matched control (AMC) slices was found, bath application of DA reduced gamma power only in the ipsilateral slices and amphetamine only decreased gamma power in contralateral slices. Furthermore, D2-like receptor activation consistently increased both theta and gamma power in contralateral and AMC slices, while only theta power was increased in ipsilateral slices. Overall, these data suggest that DA, through action at multiple sites, differentially modulates the power of both theta and gamma oscillations in M1. Using the 6-OHDA model, the oscillatory activity of M1 in vivo was investigated. Successful lesions were determined by using the rotometer, the locomotor activity and the adjusting stepping tests at 2-4 weeks post-surgery. Further testing at 22 weeks post-surgery indicated the long-term stability of the lesions. Using depth electrode and EEG recordings, oscillatory activity in the 2-10 Hz range was found in the ipsilateral and contralateral hemispheres of both lesioned and sham animals. However, only in the ipsilateral hemisphere of DA-depleted animals did we detect a 30-40 Hz oscillatory peak, which was localised to layer V of M1. In EEG recordings this led to a significant increase in the interhemispheric ratio. Using depth electrode recordings, the ipsilateral 30-40 Hz oscillation (but not 2-10 Hz oscillation) was reduced by the administration of L-DOPA (6 mg/kg) with a reduction in interhemispheric ratio. However, administration of zolpidem (0.3 mg/kg), which previously reduced abnormal beta oscillatory activity in vivo and in vitro resulting in the rebalancing of interhemispheric beta power (Hall et al., 2014; Prokic et al., 2015), was without effect. Overall, these studies demonstrate that M1 alone can generate multiple, pharmacologically distinct, but interacting oscillations, which contribute to pathological activity in the DA-depleted state.

La regulació dopaminèrgica presinàptica és important per mantenir un equilibri homeostàtic dels nivells emmagatzemats de dopamina i el seu alliberament. Els canvis en la neurotransmissió de dopamina contribueixen a trastorns neurològics i psiquiàtrics. Treballs recents del nostre grup (Ma et al., 2015; González-Sepúlveda et al.,-presentada) van descriure importants efectes de diverses classes de fàrmacs dopaminèrgics sobre la síntesi de dopamina, inclosa la L-DOPA (emprada en Parkinson), la tetrabenazina (Huntington) i aripiprazol (esquizofrènia). En aquest estudi, vam confirmar i ampliar aquestes troballes i vam comparar els efectes dels antipsicòtics agonistes parcials D2R cariprazina i brexpiprazol, els pricostimulants amfetamina i metilfenidat i diversos altres compostos selectius i experimentals. L’estriat cerebral de rata va ser trocejat i incubat ex-vivo en presència o absència d’aquests fàrmacs a diferents concentracions. De manera espontània, la dopamina i la serotonina es van acumular al llarg del temps i van assolir nivells d’emmagatzematge gairebé màxims. Aquest enfocament experimental ens va permetre avaluar la seva síntesi i dinàmica d’emmagatzematge sota la influència d’agents farmacològics escollits. Els nostres resultats podrien ser útils per comprendre els mecanismes d’acció dels antipsicòtics, i podrien facilitar més investigacions amb models animals i assajos clínics mitjançant nous agents dopaminèrgics.
La regulación dopaminérgica presináptica es importante para mantener un equilibrio homeostático de los niveles almacenados y liberación de dopamina. Los cambios en la neurotransmisión de dopamina contribuyen a los trastornos neurológicos y psiquiátricos. Hallazgos recientes de nuestro grupo (Ma et al., 2015; González-Sepúlveda et al., presentado) describieron los fuertes efectos de varias clases de medicamentos dopaminérgicos en la síntesis de dopamina, incluida L-DOPA (utilizada en Parkinson), tetrabenazina (Huntington) y aripiprazol (esquizofrenia). En este estudio, confirmamos y ampliamos esos hallazgos y comparamos los efectos de los antipsicóticos agonistas parciales D2R cariprazina y brexpiprazol, las psicoestimulantes anfetamina y metilfenidato varios otros compuestos selectivos y experimentales. El estriado cerebral de rata fue troceado e incubado ex-vivo en presencia o ausencia de estos fármacos a diferentes concentraciones. Espontáneamente, la dopamina y la serotonina se acumularon con el tiempo alcanzando niveles de almacenamiento casi máximos. Este enfoque experimental nos permitió evaluar su dinámica de síntesis y almacenamiento bajo la influencia de los agentes farmacológicos elegidos. Nuestros resultados podrían ser útiles para comprender los mecanismos de acción de los antipsicóticos, y podrían facilitar la investigación futura con modelos animales y ensayos clínicos utilizando nuevos agentes dopaminérgicos.
Presynaptic dopaminergic regulation is important to maintain a homeostatic balance of dopamine stored levels and release. Changes in dopamine neurotransmission contribute to neurological and psychiatric disorders. Recent findings from our group (Ma et al., 2015; González-Sepúlveda et al.,-submitted) describe strong effects of several classes of dopaminergic drugs on dopamine synthesis, including L-DOPA (used in Parkinson), tetrabenazine (Huntington) and aripiprazole (schizophrenia). In this study, I confirm and extend those findings and compare the effects of D2R partial agonist antipsychotics cariprazine and brexpiprazole, the psychostimulants amphetamine and methylphenidate, and several other selective and experimental compounds. Rat brain striatum was minced and incubated ex-vivo in the presence or absence of these drugs at different concentrations. Spontaneously, dopamine and serotonin accumulated over time reaching near-maximal storage levels. This experimental approach allowed me to evaluate their synthesis and storage dynamics under the influence of chosen pharmacological agents. My results could be useful to understand the mechanisms of action of antipsychotics, and they could facilitate further research with animal models and clinical trials using new dopaminergic agents.

Orientadores: Susanne Rath, Jarbas Jose Rodrigues Rohwedder
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica
Made available in DSpace on 2018-08-09T10:51:51Z (GMT). No. of bitstreams: 1 Winter_Eduardo_D.pdf: 3366512 bytes, checksum: 6c8baea3dd6502c19761ad449683bb95 (MD5) Previous issue date: 2007
Resumo: Muitas doenças neurodegenerativas são associadas com disfunções de neurotransmissores, em particular catecolaminas, no cérebro. Numerosas pesquisas têm indicado que íons metálicos podem induzir estresse oxidativo - dependente da neurodegeneração de dopamina e são responsáveis pelo aparecimento de doenças neurodegenerativas. Em estudos prévios foi verificado que alguns carboxilatos diminuem a velocidade de oxidação de catecolaminas e inibem a passivação de eletrodos sólidos durante a análise voltamétrica destes compostos fenólicos. Este trabalho teve por objetivo estudar a influência de carboxilatos (EDTA, NTA, EGTA, DTPA, acetato, citrato e oxalato) e íons metálicos (Ce(IV), Fe(III) e Hg(II)) durante a oxidação de neurotransmissores (dopamina, serotonina, epinefrina, norepinefrina e L-dopa) no intuito de estabelecer mecanismos de reações que possam contribuir no esclarecimento do papel destes compostos no processo de degeneração dos neurotransmissores, assim como compreender como os carboxilatos inibem o envenenamento do eletrodo durante a varredura de potencial. Para eses propósitos foram empregadas as técnicas de espectrofotometria, voltametria e espectroeletroquímica. A cela espectroeletroquímica de camada delgada desenvolvida incorporou um sistema de três eletrodos, sendo o eletrodo de trabalho uma minigrade de Pt. O sistema foi caracterizado usando o-tolidina e K4[Fe(CN)6]/ K3[Fe(CN)6] e permitiu o monitoramento das reações in situ. Os resultados obtidos mostraram que os carboxilatos desprotonados interagem com os produtos intermediários formados durante a oxidação das catecolaminas por meio de ligações de hidrogênio, sendo estas interações dependentes do pH do meio, estruturas do carboxilato e do próprio neurotransmissor. Foi proposto um mecanismo eletroquímico para a oxidação de aminas biogênicas na presença de carboxilatos no eletrodo de platina. A estabilização dos produtos intermediários formados inibe a formação de compostos poliméricos que são responsáveis pelo envenenamento do eletrodo. Do mesmo modo, os carboxilatos retardam ou inibem a oxidação química de algumas aminas biogênicas por íons metálicos
Abstract: Several neurological disorders are associated with improper catechoalmine regulation in the brain. Numerous researches have indicated that metallic ions can induce oxidative stress-dependent neurodegeneration of dopamine, and are responsible for the induction of neurodegenerative diseases. In previous work was verified that some carboxylates diminishes the oxidation rate of catecholamines and inhibit the well known solid state electrode passivation during voltammetric analysis of these phenolic compounds. The aim of this work was to study the influence of carboxylates (EDTA, NTA, EGTA, DTPA, acetate, oxalate and citrate) and metallic ions (Ce(IV), Fe(III) and Hg(II)) during the oxidation of neurotransmitters (dopamine, serotonin, epinephrine, norepinephrine and L-dopa) in order to establish putative reaction mechanism which could contribute to understand the role of these compounds in neurodegenerative processes, as well as comprehend how the carboxylates inhibit the electrode fouling during potential scan. For these purposes, the studies were carried out using spectrophotometric, voltammetric and spectroelectrochemical techniques. The spectroelectrochemical thin layer cell developed incorporated a three electrode system, using a Pt- minigrade as working electrode. The system was characterized employing o-tolidine and K4[Fe(CN)6]/ K3[Fe(CN)6] and allowed monitoring the electrode reactions in situ. The results obtained showed that the deprotonated carboxylates interacts with the intermediates formed at the electrochemical oxidation of catecholamines by hydrogen bonds. These interactions are dependent on the pH of the medium, as well as on the chemical structures of the carboxylates and neurotransmitters itself. An electrochemical mechanism for the oxidation of biogenic amines in the presence of carboxylates at the platinum electrode is proposed. The stabilization of the intermediates formed inhibits the formation of polymeric compounds that are responsible for the electrode fouling. In the same manner, the carbolxylates retards or inhibit the chemical oxidation of some biogenic amines by metallic ions by the same reaction pathway
Doutorado
Quimica Analitica
Doutor em Ciências

Thesis (Ph. D.)--University of Kentucky, 2005.
Title from document title page (viewed on March 2, 2006). Document formatted into pages; contains vii, 264 p. : ill. Includes abstract and vita. Includes bibliographical references (p. 196-260).

Abstract A large body of animal studies indicates that reinforcement from alcohol is associated with dopaminergic neurotransmission in the mesocorticolimbic pathway. However, as most psychiatric phenomena cannot be studied with animals, human studies are needed. Furthermore, because of the fluctuating nature of phenomena regarding the status of abuse and withdrawal, repeated observations of the same study subjects under different situations can elucidate a variety of pathophysiological mechanisms. In this study 42 alcoholics were monitored during withdrawal and 30 alcoholics after four weeks of abstinence. 123I-β-CIT SPET was used as a method for the semi quantification of their striatal dopamine transporter (DAT) densities reflecting the function and structure of the dopaminergic system. DAT density was markedly lower during withdrawal among alcoholics as compared to control subjects, but it elevated during abstinence to the level of healthy volunteers. This increases in DAT density during withdrawal and afterwards correlated with the depression scores of alcoholics. DAT density correlated with the Novelty Seeking (NS) personality trait, especially among abstinent alcoholics. After four weeks of controlled abstinence alcoholics with an A1 allele of dopamine receptor D2 were found to have higher DAT densities than alcoholics without it. The results indicate that striatal DAT density is associated with mood, personality, A1 genotype and the length of the abstinence period after heavy alcohol drinking.

This study investigated the involvement of the neurotransmitter dopamine in feeding behaviours. A conditioned feeding paradigm was used to study incentive responses. After conditioning rats responded to a conditional stimulus (CS+) by approaching a feeding site. Approach responses were attenuated by 0.4 or 0.6mg/kg of the dopamine antagonist pimozide. Neurochemical investigation revealed that exposure to the CS+ increased dopamine turnover in the forebrain. Thus, dopamine appears to be actively involved in the initiation of appetitive responses. In contrast, another experiment indicated that consumption of a liquid diet was not altered by up to 0.6mg/kg pimozide. These data were interpreted as supporting an "incentive-response hypothesis" of dopamine function, which states that "When an animal observes an incentive stimulus, the release of dopamine in the forebrain is increased, resulting in approach to the stimulus by the animal. Once the animal is in contact with a goal object, consummatory reactions occur which are not mediated by dopamine systems". A final experiment investigated the activity of dopamine systems following ingestion. After one hour during which food pellets or liquid diet were available to rats, dopamine turnover was increased in the n. accumbens and the striatum, relative to non-fed animals. No increase was observed in the brains of rats which had consumed similar quantities of saccharin solution. Thus, the increase observed following consumption of pellets or liquid diet could not be attributed to motor or "reward" effects. It was concluded that in addition to their involvement in incentive-responding, dopamine systems are also affected by the ingestion of nutrients.
Arts, Faculty of
Psychology, Department of
Graduate

Oligodendrocyte progenitors are highly sensitive to oxidative insults. Among the factors postulated to contribute to this susceptibility are high levels of intracellular iron and low antioxidant content. During ischemia, the neurotransmitter dopamine (DA) is released and may contribute to oxidative stress and oligodendrocyte injury in the hypomyelinating disorder, periventricular leucomalacia (PVL). In this thesis, I investigated the role of iron in DA-induced toxicity in primary cultures of oligodendrocyte progenitors, and assessed the contribution of the antioxidant defenses (glutathione (GSH), glutathione peroxidase (GPx) and superoxide dismutase (SOD)) and other survival factors (heat shock proteins and the protein kinase Akt) in determining the response of the cells to DA.
Addition of iron to cultures increased DA-induced expression of the stress protein heme oxygenase-1 (HO-1), and toxicity as assessed by mitochondrial activity, cellular release of lactate dehydrogenase, nuclear condensation and caspase-3 activation. In contrast, an iron chelator reduced these events. Furthermore, DA induced accumulation of superoxide, which was also reduced by the iron chelator. Surprisingly, a mimetic of the superoxide detoxifying enzyme, SOD potentiated DA toxicity, suggesting that generation of hydrogen peroxide via superoxide dismutation may be contributing to toxicity. Both a mimetic of the peroxide-scavenging enzyme, GPx and a GSH analog blocked DA-induced superoxide accumulation, HO-1 expression and caspase-3 activation. In addition, the GPx mimetic blocked caspase-3 activation induced by the combination of DA with iron. In contrast, an inhibitor of glutathione synthesis potentiated DA-induced HO-1 expression and cell death.
Finally, in further examining the cellular defense mechanisms, I found that various heat shock proteins increased in expression levels during oligodendroglial differentiation, however only heat shock protein-90 (HSP-90) was detected in oligodendrocyte progenitors. An HSP-90 inhibitor decreased activated Akt levels, induced caspase-3 activation, increased nuclear condensation, reduced oligodendrocyte progenitor viability, and potentiated DA-induced apoptosis. In addition, an Akt inhibitor alone exacerbated DA toxicity and in combination with the HSP-90 inhibitor caused synergistic potentiation of DA toxicity by enhancing caspase-3 activation.
In conclusion, elevated levels of iron, superoxide, deficient detoxification of peroxides by glutathione peroxidase and inadequate defense by glutathione contribute to the susceptibility of oligodendrocyte progenitors to DA-induced toxicity. On the other hand, HSP-90 alone or in concert with Akt play important roles in oligodendrocyte progenitors survival following an insult that produces oxidative stress.

The current project examined the ability of nicotine to modulate dopamine transporter (DAT) function. Initial experiments determined the dose-response for nicotine to modulate dopamine (DA) clearance in rat striatum and medial prefrontal cortex (MPFC) using in vivo voltammetry and determined if this effect was mediated by nicotinic receptors (nAChRs). In both striatum and MPFC, nicotine increased DA clearance in a mecamylamine-sensitive manner, indicating nAChR-mediation. The effect of acute nornicotine on DAT function was also determined. In contrast to nicotine, nornicotine in a dose-related manner decreased striatal DA clearance in a mecamylamine-sensitive manner, indicating nAChR mediation. To determine if tolerance developed to the nicotine effect nicotine, separate groups of rats were injected once daily for 5 days with nicotine or saline. DA clearance in striatum and MPFC was determined 24 hrs after the last injection. Nicotine increased DA clearance only 10-15% in the group repeatedly administered nicotine, demonstrating that tolerance developed. To determine if nicotine altered striatal DAT efficiency, following nicotine injection, DAT density and maximal velocity of [3H]DA uptake was determined using [3H]GBR12935 binding and saturation analysis of [3H]DA uptake in rat striatum, respectively. Nicotine did not alter the Bmax or Kd of maximal binding of [3H]GBR12935 binding. However, an increase in Vmax was observed at 10 and 40 min following nicotine injection, suggesting that nicotine increases DAT efficiency. To determine if systemic nicotine enhanced DAT function via an action at nAChRs on striatal DA terminals, [3H]DA uptake was determined in striatum in vitro in the absence or presence of nicotine in the buffer. Nicotine did not alter the Vmax for [3H]DA uptake in vitro, suggesting that the nicotine-induced increase in DAT function observed in vivo is mediated by nAChRs on DA cell bodies or another site which indirectly alters DAT function. To determine if the increase in DAT efficiency was due to increased surface expression of striatal DAT, biotinylation and Western blot analyses were performed. Nicotine did not alter striatal DAT, suggesting that the nicotine-induced increase in DA clearance in vivo and DAT efficiency in vitro is not the result of increased trafficking of this protein to the cell surface.

Both centrally and systemically administered dopamine agonists and antagonists decrease ingestive behaviour. The aim of this thesis was to examine whether drugs acting at different receptor subtypes decreased intake in different ways. A microstructural analysis was used to examine dopaminergic drug effects on licking behaviour. A dopamine D3 receptor agonist, 7-OH-DPAT, a dopamine D2 receptor antagonist raclopride and a mixed dopamine D2/D3 agonist quinpirole were compared in this paradigm. These drugs reduced the number of licks by differentially decreasing parameters which are thought to reflect the palatability of the stimulus such as mean bout duration of licking and the initial rate of licking. Follow-up experiments were conducted to further examine the possibility that motor deficits were underlying decreases in licking parameters. The effects of raclopride and 7-OH-DPAT were compared to the effects of a dopamine Dl antagonist SCH- 23390 and were analysed using a brief contact licking test. Again, the behavioural expression of anorexia induced by these drugs seemed to rely on a differential decrease in mean bout duration. Results also revealed that the three drugs used differed in the extent to which they produced a motoric deficit Attempts to block the effects of 7-OH-DPAT on licking (wameters were made by using the putative D3 receptor antagonists PNU-99194A and amisulpride. In addition, the effects of these drugs alone on licking behaviour were examined PNU-99194A failed to block the effects of 7-OH-DPAT and was relatively ineffective in producing changes in licking behaviour when administered alone. Amisulpride blocked the effects of 7-OH-DPAT only at high doses and when injected alone produced an increase in intake through an increase in mean bout duration of licking. Results from Chapters 4,5 and 6 suggested that 7-OH-DPAT was having an effect on palatability. Therefore, Chapter 7 presents an experiment which examines the effect of 7-OH-DPAT on the licking behaviour of rats which encounter a devaluation of reward (successive negative contrast). 7-OH-DPAT reduced successive negative contrast leading to the proposal that D3 receptors may mediate relative as well as absolute reinforcer value. These results bear important implications for understanding the role of dopamine receptor subtypes in components of food reward and appetitive behaviour in general and may well have implications for the treatment of eating disorders.

Dopamine is heavily implicated in learning and memory, but the timescale of dopaminergic action in human memory consolidation is not yet clearly mapped, and is the focus of this thesis. A series of behavioural experiments were run with Parkinson's disease (PD) patients to examine the effects of dopaminergic medication withdrawal and administration at different times during reinforcement learning (RL) and episodic memory. Episodic memory was impaired in PD, and dopaminergic medication during learning increased this impairment, while, in contrast, medication overnight (8-24 hours after learning) improved recall at 24 hours. Dopamine during learning also led to a more liberal response bias 24 hours later, for which computational modelling suggested consolidation mechanisms were a plausible explanation. The period between 1 and 7 hours after learning was also explored but no effects of dopamine on RL and episodic memory consolidation were found, in contradiction to animal literature. Surprisingly, in neither experiment did dopamine affect RL behaviour, but participants did develop a bias towards avoiding the negatively reinforced stimuli when a 24 hour gap between learning and recall was introduced. Finally, separate effects of PD and dopamine therapy on working memory were demonstrated, with PD impairing elements that required manipulation of items held in memory, but dopamine affecting phonological storage of items. While the above results all relate to accuracy of performance, there were also unexpected effects of PD and dopamine on reaction times; for example, PD patient responded more quickly than healthy participants in procedural tasks. Altogether, this thesis demonstrates that the timing of dopamine is only one of the factors influencing the way memories are stored over time. Many of the results were unexpected, generating new hypotheses to be tested in future endeavours.

This study investigates renal dopamine production via the desulphation of dopamine sulphate in a sample cohort during normal unregulated dietary sodium intake and following a low sodium regimen. After dietary salt restriction urinary dopamine sulphate levels were significantly increased, indicating that dopamine sulphate is indeed a physiological reservoir of active free dopamine, the necessity for which is reduced during self depletion. This confirmed the dopamine/dopamine sulphate pathway as one which may be relevant to the maintenance of sodium homeostasis. The activity of urinary ASA was investigated in diabetes mellitus as an example of a sodium-retaining state, and compared with that in a matched normal control group. A decreased ASA activity was anticipated, given the blunted dopamine excretion observed in many sodium-retaining states, however an unexpected increase in activity in the diabetic group was observed. Enzyme kinetic analysis of ASA showed that this was not due to the existence of an isoform having an altered affinity for dopamine sulphate. This rather paradoxical situation, that urinary-dopamine is decreased while ASA activity is increased, may be explained by the sequestering of free dopamine by autoxidation to 6-hydroxydopamine as has been hypothesised recently to occur in diabetes mellitus. To confirm the homogeneity of ASA in the normal and diabetic groups, four amplicons spanning the 3637bp intronic and exonic regions of the gene were generated by PCR. These were sequence utilising a fluorescent-dye terminator reaction using the forward PCR primer as sequencing primer. Although single nucleotide polymorphisms were observed between the two groups these occurred either in intronic regions or, when exonic, generated silent mutations, supporting the enzyme kinetic data. The expression of ASA was investigated to determine the basis of the increased activity observed in diabetes mellitus. Although a validated comparative RT-PCR assay was developed for amplification of arsa transcripts from fresh blood samples, expression analysis from archived paraffin-embedded renal tissue was complicated by the low yield and degradation of unprotected mRNA. Suggestions for the development of this work using renal cell-culture are discussed.

Low doses of dopamine autoreceptor (DA) agonists are presumed to act by stimulating DA autoreceptors on the sona/dendrites and axon terminals of DA neurons. Low doses of apomorphine reduced food Intake, in a microstructural analysis paradigm, by reducing both the time spent feeding and the rate of food ingestion. The reduction of eating time was shown to result from the stimulation of DA autoreceptors located on the cell bodies and dendrites of the mesolimbic DA system. The reduction of eating rate however, appeared to result from the activation of axon terminal DA autoreceptors. The significance of this dissociation is discussed in relation to the mechanisms through which presynaptic DA receptors on the same neuron may subserve different behavioural functions. The observation that apomorphine administration resulted in a selective manipulation of the microstructural parameters of feeding, was then used to assess the action of antidepressant drugs on DA autoreceptor function. In both normal and chronically stressed rats, chronic antidepressant treatment failed to alter the sensitivity of DA autoreceptors. However, on withdrawal, the sensitivity of cell body DA autoreceptors appeared reduced, as apomorphine no longer in any way influenced the time spent feeding in the microstructural paradigm. The Implications of these findings are discussed in relation to the hypothesis that antidepressant drugs increase DA function by reducing the sensitivity of presynaptic DA receptors.

Parkinson’s disease is a widespread neurodegenerative disorder that affect 2% of the population above the age of 60. The hallmark of the pathology is the preferential degeneration of the dopaminergic neurons in the substantia nigra pars compacta of the midbrain, and the presence of proteinaceous inclusions called Lewy bodies in the surviving neurons (Braak 2004). In 10% of the cases, the disease is linked to mutation on several genes, among them α-synuclein, DJ-1, PARKIN, PINK1 and LRRK2, but in the vast majority of the cases the aetiology is still unknown (sporadic PD) (Gwinn-Hardy 2002). Post mortem studies and in vitro and in vivo PD model have revealed a possible interconnection between genetic and sporadic PD, which involves both mitochondrial dysfunction and oxidative stress as central players in the pathogenesis of the disease (Gilgun-Sherki Y. et al. 2001, Mythri R. B. et al. 2011). Oxidative stress is a condition characterized by the inability of the cellular antioxidant defences to cope with the production of reactive oxygen species (ROS). This condition of unbalance between the production and the clearance of ROS causes irreversible damage to cellular components such as lipids, proteins and DNA, leading eventually to cell death (Lotharius et al. 2002). Among the enzymes implicated in the detoxification of ROS, are superoxide dismutases (SODs) that catalyze the dismutation of superoxide anion into molecular oxygen and hydrogen peroxide (Fridovich 1995). Since oxidative stress does not explain alone the selectivity death of dopaminergic neurons, the main working hypothesis is that dopamine itself could have a central role. Under physiological conditions, dopamine is synthesized in the cytosol and stored in synaptic vesicles by the action of Vesicular Monoamine Transporter (VMAT2) where it is stabilized by the low pH (Erickson, J. D 1992). If the amount of cytosolic DA exceeds the physiological concentration, DA is metabolized to the non-toxic metabolite 3,4-dihydroxyphenylacetic acid and hydrogen peroxide by the action of monoamine oxidase (MAO) and aldehyde dehydrogenase, or sequestered into lysosomes where it can auto-oxidize to form neuromelanin (NM). If not buffered by these pathways, cytosolic DA can be oxidized to DA-quinone (DAQs) (spontaneously or enzymatically), (Sulzer, D., 2000, Elsworth, J. D. 1997), a reaction that also leads to the formation of ROS. On these premise, we evaluated two line of research using a cellular model for PD (SH SY5Y cell line): one concerning about the effect of dopamine and its oxidized forms on cellular viability, the second one on the potential role of superoxide dismutases (1 and 2) over expression. From the use of different techniques we started to evaluate which kind of cell death pathway was activated by dopamine and DAQs. Looking for the presence of nuclear fragmentation, that is one of the later stages of apoptosis, we determined that both dopamine and DAQs induce cell death via apoptosis but the dopamine toxicity depends on its internalization by the action of the dopamine transporter (DAT), since the pre-treatment of cells with GBR 12909 (a DAT inhibitor) had a rescue effect. To confirm the apoptotic pathway we also evaluated another hallmark of apoptosis (one of the former stages of the apoptotic cascade): phosphatidil-serine externalization (PS) using ANNEXIN-V-FLUOS; a specific probe for PS. Using flow cytometry we confirm that both dopamine and DAQs induce cell death via apoptosis. Next we wanted to evaluate if dopamine and DAQs exert their toxicity from extracellular environment or they are required to enter in the cells. Treating cells with GBR12909, we demonstrate that dopamine needs to enter cells to exert its toxicity (since the treatment with the DAT inhibitor rescues cells from DA toxicity) while DAQs toxicity was not affected by this treatment leading to cell death. Since oxidative stress is one of the mechanisms that have been implicated in the pathogenesis of PD, and the chemistry of dopamine (auto-oxidation and enzyme-mediated oxidation) leads to the production of ROS, we evaluate the production of mitochondrial superoxide anion using a specific probe. The data demonstrate that only the auto oxidation of dopamine leads to the production of superoxide anion and dopamine is required to enter cell to exert its effect. To dissect more in depth the toxicity mechanism of both dopamine and DAQs, and since only cytosolic dopamine led to the production of mitochondrial superoxide anion, we asked if this two different oxidation processes activated different cell death pathways (the major are the mitochondrial one and the one mediated by death receptor) or not. From preliminary data we observed a marked difference in the activation of caspase 3 and the subsequent cleavage and inactivation of Poly (ADP) ribose polymerase (PARP) due to DAQs treatment convincing us to proceed in the investigation of the possible differences between this different oxidation processes. The second part of the work was focused on the role for superoxide dismutases 1 and 2 against dopamine and DAQs cytotoxicity since previous data demonstrated a role in superoxide anion production and induction of cell death in the case of cytosolic dopamine. Over expression of both SOD1 and SOD2 revealed a protective effect against dopamine cytotoxity, while they were not able to counteract DAQs-induced cell death. In the present work the main working hypothesis was that oxidative stress induced by dopamine and its oxidized forms accumulation could have a central role in the specific dopaminergic cell loss in Parkinson’s disease. The data obtained so far seems to highlights that dopamine and DAQs activates different apoptotic pathway that are superoxide anion-dependent for DA and superoxide anion-independent for DAQs. Since oxidative stress is considered one of the mechanism that interconnect genetic form and sporadic forms of the pathology and dopamine, in its oxidative chemistry, leads to the production of ROS, understanding which cell death pathways are activated and to which extent, is crucial to develop a therapy to counteract the start and the progression of the pathology. Data from the over expression of SODs demonstrate that compounds that counteract the production of superoxide anion (like SOD-mimetics that are currently used for other diseases) could have a protective role against the oxidative stress and the subsequent cell death condition induced by dopamine. Also compounds that block the activation of the apoptotic cascade induced by dopamine and DAQs could rescue cells from dying in this neurodegenerative disease
La malattia di Parkinson è una diffusa sindrome neurodegenerativa che affligge circa il 2% della popolazione oltre l’età dei 60 anni. La caratteristica principale della patologia è la preferenziale morte dei neuroni dopaminergici della substantia nigra pars compacta del mesencefalo, e la presenza di inclusioni proteinacee chiamate Lewy body nei neuroni sopravvissuti. Nel 10% dei casi, la malattia è collegata a mutazioni su diversi geni, tra i quali α-synucleina, DJ-1, PARKIN, PINK1 e LRRK2, ma nella maggior parte dei casi (Parkinson sporadico) (Gwinn-Hardy 2002) l’eziologia è ancora sconosciuta. Da studi post mortem e da modelli in vitro e in vivo per il PD è stata rilevata una possibile connessione tra le forme genetiche e quelle sporadiche che implica sia la disfunzione mitocondriale e lo stress ossidativo come fattori centrali nella patogenesi della malattia (Gilgun-Sherki Y. et al. 2001, Mythri R. B. et al. 2011). Lo stress ossidativo è una condizione in cui le capacità antiossidanti della cellula non sono in grado di sopperire alla produzione di specie reattive dell’ossigeno (ROS). Questa condizione di sbilanciamento tra la produzione e la detossificazione dei ROS causa danni irreversibili ai componenti cellulari come lipidi, proteine e DNA, portando alla morte cellulare (Lotharius et al. 2002). Tra gli enzimi coinvolti nella difesa antiossidante delle cellule, le superossido dismutasi giocano un ruolo fondamentale poiché catalizzano la dismutazione dell’anione superossido in ossigeno molecolare e perossido di idrogeno (Fridovich 1995). Poiché lo stress ossidativo non spiega da solo la selettiva morte dei neuroni dopaminerigici, l’ipotesi è che la dopamina stessa abbia un ruolo chiave. In condizioni fisiologiche, la dopamina viene metabolizzata nel citosol e stoccata all’interno delle vescicole sinaptiche grazie all’azione del trasportatore vescicolare delle monoamine 2 (VMAT2) dove è stabilizzata dal basso pH (Erickson, J. D 1992). Se la concentrazione citosolica di dopamina supera quella fisiologica, essa viene metabolizzata ad acido 3,4-diidrofenilacetico e perossido di idrogeno grazie all’azione dell’enzima monoamina ossidasi (MAO) e aldeide deidrogenasi, o sequestrata nei lisosomi dove può auto ossidarsi a formare neuromelanina. Se non è metabolizzata in questi pathway, la dopamina può essere ossidata a dopamino-chinoni (DAQs) (spontaneamente o enzimaticamente) (Sulzer, D., 2000, Elsworth, J. D. 1997), una reazione che produce anche specie reattive dell’ossigeno (ROS). Con queste premesse, abbiamo valutato due linee di ricerca usando un modello in vitro per il PD (la linea cellulare SH SY5Y): la prima riguardante l’effetto della dopamina e le sue forme di ossidazione sulla vitalità cellulare, la seconda sul potenziale ruolo dell’over espressione delle superossido dismutasi (1 e 2). Utilizzando differenti tecniche abbiamo iniziato a valutare quale pathway di morte cellulare veniva attivato dalla dopamina e dalle sue forme ossidate. Andando a valutare la frammentazione nucleare, uno degli ultimi stadi dell’apoptosi, abbiamo determinato che sia la dopamina sia i chinoni inducono l’attivazione di questo processo di morte. Per confermare il processo apoptotico, abbiamo valutato un altro marker: l’esternalizzazione delle fosfatidil-serine (PS): uno dei primi stadi di attivazione del processo apoptotico. con l’utilizzo di ANNEXIN-V-FLUOS, una sonda specifica per le PS. Mediante la tecnica di citoflurimetria abbiamo confermato che sia la dopamina che i chinoni attivano l’apoptosi. Successivamente abbiamo voluto valutare se la dopamina e i chinoni avevano effetti tossici a livello extracellulare o necessitavano di entrare nelle cellule. Pre-trattando le cellule con l’inibitore specifico del trasportatore della dopamina (GBR 12909) abbiamo dimostrato che la tossicità della dopamina è dipendente dal suo trasporto all’interno della cellula (in quanto il trattamento con l’inibitore ha avuto un effetto protettivo per le cellule), mentre i chinoni non lo necessitano. Poichè lo stress ossidativo è stato proposto come possibile meccanismo implicato nella patogenesi della malattia, e la chimica ossidativa della dopamina (auto ossidazione e ossidazione mediata da enzimi) porta alla produzione di specie radicaliche, abbiamo valutato la produzione dell’anione superossido (la prima specie radicalica prodotta soprattutto a livello del mitocondrio) usando una sonda specifica. I dati dimostrano che solo il processo auto ossidativo della dopamina porta alla produzione di anione superossido e che questo processo è dipendente dall’internalizzazione della dopamina a livello del trasportatore. Per comprendere maggiormente il meccanismo alla base della tossicità del processo di ossidazione e ossidazione mediate da enzima della dopamina abbiamo valutato altri marker di apoptosi. Poiché dai dati precedenti è stato visto che solo la dopamina citosolica era in grado di indurre la produzione dell’anione superossido a livello del mitocondrio, abbiamo voluto verificare se i due processi ossidativi della dopamina, attivassero differenti pathway apoptotici (i prevalenti comprendo la via mediata dai recettori di morte, l’altro quella mitocondriale). Dati preliminari dimostrano una marcata attivazione della caspasi 3 e la conseguente inattivazione della poli (ADP) ribosio polimerasi in conseguenza al trattamento con i chinoni suggerendoci di investigare maggiormente sulle possibili differenze tra i diversi processi ossidativi della dopamina. La seconda parte del progetto si è focalizzata sul possibile ruolo delle superossido dismutasi 1 e 2 contro la tossicità indotta dalla dopamina e dai suoi prodotti di ossidazione. L’over espressione sia della SOD1 che della SOD2 hanno rivelato un effetto protettivo contro la produzione di anione superossido indotto dalla dopamina, mentre non hanno presentato alcun effetto contro la tossicità indotta dai chinoni rimarcando il ruolo dell’anione superossido nella tossicità indotta dalla dopamina. Nel presente progetto di dottorato, abbiamo valutato l’ipotesi che lo stress ossidativo indotto dalla dopamina e dalle sue specie ossidate possa avere un ruolo chiave nella specifica degenerazione dei neuroni dopaminergici caratteristici della malattia di Parkinson. I dati ottenuti sembrano dare indicazioni sulla differente attivazione di pathway di morte cellulare indotta dalla dopamina e dalle sue forme ossidate con un differente ruolo nella produzione dell’anione superossido. Poichè lo stress ossidativo è considerate uno dei meccanismi che collegano il Parkinson sporadico a quello genetico e la dopamina, nella sua chimica ossidativa, porta alla produzione di specie radicaliche, riuscire a capire quale pathway di morte cellulare è attivato e in quale misura, è cruciale per lo sviluppo di terapie per impedire l’inizio e la progressione della malattia. I dati derivanti dall’over espressione delle superossido dismutasi dimostrano che composti che impediscono la formazione dell’anione superossido (come SOD-mimetici che sono attualmente usati per altre patologie) potrebbero avere un ruolo protettivo contro lo stress ossidativo e la conseguente morte cellulare indotta. Inoltre, anche composti in grado ci bloccare la cascata apoptotica indotta da dopamina e chinoni potrebbe proteggere dalla morte le cellule affette in questa malattia neurodegenerativa

Dopamine is a neurotransmitter found in central nerve system which has a vital role for human health. Dopamine oxidation in body is an important issue since it may form reactive metabolites which can be toxic to the cell. Surface-enhanced Raman scattering (SERS) is currently recognized as one of the most sensitive spectroscopic tools, which can be exploited for ultrasensitive chemical and biological detection, addition to providing structural information on the systems of interest. SERS of dopamine displays three strong bands at 1269, 1331 and 1479 cm-1. These bands are the signature of dopamine molecule. The most intense band at 1479 cm-1 is contributed mainly from stretching of the carbon-carbon bond to which the oxygens are attached. A bidentate silver-dopamine complex or in general bidentate metal-dopamine complex formation is required for the SERS detection of dopamine and other catecholamines. In other words, for acquiring the characteristic dopamine SERS signature, both of the catechol oxygens should take a part in the adsorption of dopamine to the silver metal surface which is used as a SERS substrate. Therefore, the reactivity of different oxidation forms of dopamine for the formation of bidentate silver-dopamine complex was investigated by obtaining their SERS spectra and following the characteristic C-C ring vibration at 1479 cm-1. Dopamine oxidation was carried out electrochemically, utilizing platinum and silver electrodes as working electrode. Oxidation products formed were identified with UV-vis Spectrometer. Also, silver metal ions were used to oxidize dopamine, leading to formation silver nanoparticles. Dopamine functionalized silver nanoparticles were characterized by Scanning Electron Microscope, Transmission Electron Microscope, UV-vis Spectrometer. Surface- enhanced Raman spectra of polydopamine on the surface of synthesized silver nanoparticles and the electrodeposited dopamine on the porous surface of silver electrode were also obtained.

The project concentrated on characterising the effect of D4 receptor activation in the context of an animal model relevant to schizophrenia (phencyclidine pretreatment) and elucidating the mechanisms involved. Behavioural studies measured selective attention to motivational stimuli, through measurement of latent inhibition of conditioned learning (LI), and episodic memory measured by novel object recognition (NOR), behaviours which are dysfunctional in schizophrenia. Subchronic PCP pre-treatment for five days disrupted LI and induced behavioural deficits in NOR, replicating previous findings using seven days pre-treatment. A412997 reversed deficit in NOR but not in LI. However, the neural the mechanisms these processes are as yet unclear. A better understanding of the physiology of cortical-limbic circuits is important in elucidating the neurophysiological mechanisms underlying dopamine-mediated processes which are vital for normal behaviour, and which may be abnormal in schizophrenia. The project examined the neuropharmacology underlying these behavioural processes, both in normal animals, and in animals pretreated with phencyclidine. Focusing on the role of D4 receptors. To achieve these aims fast-scan cyclic voltammetry was used to measure electrically stimulated dopamine release in nucleus accumbens, in rat brain slices in vitro. The selective dopamine receptor agonist A412997 caused a decrease in electrically stimulated dopamine release which was abolished in animals pretreated with PCP. This inhibitory effect of A412997 was blocked by D4 specific antagonist L-741,742. Gene expression of dopamine D4 receptors, as well as in other dopamine receptors (D1, D2, D3, D5) in response to sub-chronically pre-treated with PCP was significantly changed in different regions of rat brain, as well as these pre-treatment as modelling relevant to schizophrenia produced changes on the basal level of dopamine and its metabolites in the same brain areas. Taken together with behavioural data this demonstrated changes in D4-receptor mediated regulation of accumbal dopamine function after PCP pretreatment, suggesting a role for these receptors in schizophrenia.

La maladie de Parkinson (MP) est la seconde maladie neurodégénérative la plus fréquente. Il n’y a pas de traitement curatif de la maladie. Les traitements symptomatiques s’appuient principalement sur le remplacement de la dopamine (DA). Le traitement par L-DOPA, particulièrement efficace initialement, se complique à long terme par des fluctuations et dyskinésies. Les mécanismes de la plasticité striatale anormale sous-tendant l’apparition de ces dyskinésies sont mal compris. Le but de ce projet était d’identifier les anomalies des voies de signalisation dans les neurones de projection du striatum en l’absence de DA. Nous avons utilisé chez des souris avec lésion ou non des neurones DA par la 6-OHDA, des biosenseurs permettant l’étude de voies de signalisation en imagerie cellulaire multiphotonique de neurones vivants dans des tranches corticostriatales. Nous avons d’abord mis au point ce modèle combinant injection stéréotaxique de toxine et de vecteur viral chez des souris adultes. Dans certaines expériences nous avons étudié spécifiquement les réponses des neurones de projection striataux des voies directe (NPSd) ou indirecte (NPSi) en utilisant des biosenseurs activés par la recombinase Cre et des lignées transgéniques exprimant spécifiquement cette enzyme dans l’une ou l’autre population. Nous avons utilisé des biosenseurs FRET pour mesurer l’activité de la kinase dépendante de l’AMPc (PKA, sonde AKAR3) ou ERK (extracellular signal-regulated kinase, sonde EKAR-EV) et le senseur calcique GcAMP6S pour le Ca2+ libre cytosolique avec une bonne résolution spatiale et temporelle. Nous avons modulé pharmacologiquement les récepteurs de la DA, du glutamate et de l’adénosine, ainsi que les activités des kinases et phosphodiestérases. Nous avons observé que la lésion augmentait les réponses ERK à la stimulation des récepteurs D1 de la DA dans les NPSd. Nous avons montré une augmentation des réponses PKA dans ces neurones pouvant être liée à une augmentation de la protéine G stimulatrice d’adénylyle cyclase, Gαolf, ainsi qu’à une inhibition des phosphodiestérases. L’imagerie calcique a mis en évidence une augmentation de l’activité spontanée des NPSd et, de manière inattendue, de la sensibilité à la stimulation des récepteurs AMPA du glutamate des NPSi. En conclusion notre travail utilise pour la première fois l’imagerie biphotonique par biosenseurs dans le striatum dépourvu de DA de souris adulte. Il met en évidence des déficits multiples et distincts de la signalisation dans les deux populations de neurones de projection du striatum et suggère des mécanismes possibles de ces altérations
Parkinson’s disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s disease. There is currently no cure for PD. Symptomatic drug therapy essentially relies on dopamine (DA) replacement therapy. The spectacular antiparkinsonian effect of levodopa in PD is however hampered by long-term complications, motor fluctuations and dyskinesia in all patients at some time during the disease course. The mechanisms of the maladaptive striatal plasticity leading to dyskinesia are not well understood. The aim of this project was to identify the dysregulations of signaling pathways in striatal projection neurons (SPN) in the absence of dopamine. We used a mouse model of lesion of DA neurons with 6-OHDA and virally transduced biosensors to monitor signaling pathways in live neurons with two-photon imaging of corticostriatal slices. We focused our attention on extracellular signal-regulated kinase (ERK), cAMP-dependent protein kinase (PKA) and Ca2+ which are known to be altered in the absence of DA. We first set up a reliable experimental model in adult mice, successfully combining 6-OHDA and viral vector in the same unilateral stereotactic injection into the dorsal striatum. In some experiments we targeted the biosensor expression to specific neuronal populations using Cre-dependent “flexed” biosensors. We used mice expressing Cre under the control of the D1 DA receptor (D1R) promoter to target specifically striatal projection neurons of the direct pathway (dSPNs) or the adenosine A2A receptor (A2AR) to target SPNs of the indirect pathway (iSPNs). We used fluorescence resonance energy transfer (FRET)-based biosensors EKAR-EV and AKAR-3 to monitor ERK and PKA activities, respectively. We also monitored cytosolic free Ca2+ with the genetically encoded calcium indicator GCaMP6S. We used pharmacological tools to modulate glutamate, DA, and adenosine receptors as well as phosphodiesterases (PDE) and kinases activities. We observed that the DA lesion increased ERK responsiveness to stimulation of D1R. Since ERK activation depends on both cAMP and Ca2+ signals, we then investigated these two pathways. We observed an increased activation of PKA in response to D1R but not A2AR. We explored the mechanism of this increased sensitivity using mice deficient for Gαolf, the G protein that couples striatal receptors to adenylyl cyclase. We provided evidence that increased levels of Gαolf contributed to enhanced D1 responses after 6-OHDA lesions and identified a deficit in PDE activity in D1 neurons that was likely to amplify this effect. By monitoring Ca2+ signals we showed an increased spontaneous activity of D1 neurons in lesioned mice. However, unexpectedly the Ca2+ responses to stimulation of AMPA glutamate receptors were increased in iSPNs and not dSPNs. In conclusion, our work using for the first time 2-photon biosensor imaging in the DA-depleted striatum of adult mice confirms and extends previous observations on signaling dysregulations in the absence of DA. It reveals distinct cell type-specific alterations of cAMP, Ca2+ and ERK responses in the two populations of SPNs and suggests possible mechanisms for these alterations

A study has been made of the secretory response and the electrical reponse (a hyperpolarization followed by a depolarization) mediated by dopamine receptors of the cockroach (Nauphoeta cinerea Olivier) salivary gland in-vitro. Although domperidone did not inhibit the electrical response to dopamine, three other actions were observed: one, post-synaptic, led to the potentiation of the hyperpolarization; this action was shared by (±)sulpiride. A separate post-synaptic action resulted in the inhibition of the depolarizing phase of the response. Finally a pre-synaptic action led to the abolition of the response to nerve stimulation.Effects of the calmodulin antagonists W7 and calmidazolium. In an attempt to investigate the role of calmodulin in stimulus-secretion coupling with the salivary gland, the actions of two calmodulin antagonists, W7 and calmidazolium, were studied. In high concentrations, but within the range in which they are known to inhibit calmodulin, W7 and calmidazolium were found to inhibit dopamine-induced secretion and hyperpolarize the acinar cells. The hyperpolarization was not inhibited by SGH23390, and resulted from an increase in cytosolic free calcium, released from the same source as that accessed by dopamine. Lower concentrations of these two antagonists caused submaximal secretion and potentiated dopamine-induced hyperpolarizations. An interpretation of these results is that calmodulin promotes secretion, and exerts a negative control on cytosolic free calcium by an independent process which can be selectively inhibited.