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.

Dans ce travail, nous avons développé le modèle de rat ayant subi une lésion néonatale de l'hippocampe ventral (rat LNHV), un outil reconnu pour l'étude de la schizophrénie. <br />Nous avons validé l'utilisation de l'imagerie anatomique (IRM) comme outil permettant une détection fiable et précoce (avant la puberté) de la taille et la cible des lésions, pour remplacer l'évaluation post mortem.<br />Au niveau comportemental, nous avons montré que le nombre des anomalies qui sont associées à des troubles de la transmission dopaminergique augmente du stade juvénile à l'âge adulte chez les animaux LNHV. <br />Au niveau neurochimique, nous avons montré que les animaux LNHV présentent des altérations des efflux de dopamine au niveau mésocorticolimbique (noyau accumbens et cortex préfrontal) qui n'apparaissent qu'après la puberté. <br />Nos résultats confirment l'implication majeure de la dopamine dans la physiopathologie de la schizophrénie et soulignent l'importance des facteurs qui pourraient précipiter l'émergence de la maladie au moment de l'adolescence.

To test the hypothesis that the hypothalamic tuberoinfundibular dopaminergic neuronal system plays a role in the dynamic regulation of pituitary prolactin secretion, its activity was correlated with experimentally-induced prolactin secretory episodes in the male rat. Direct estimates of tuberoinfundibular neuronal activity were made by measuring its rates of dopamine and norepinephrine synthesis or release. Prolactin secretion was assessed in vivo by measuring radioimmunoassayable prolactin levels in peripheral blood and the pituitary and in vitro by measuring prolactin concentrations released into incubation media. The anesthetic urethane and a substance isolated from the pineal gland were both demonstrated to inhibit prolactin secretion. Significant elevations of newly synthesized tuberoinfundibular dopamine were observed concomitant with this decreased prolactin secretion suggesting that acute increases in tuberoinfundibular dopaminergic neuronal activity were perhaps causally related to acute decreases in prolactin secretion since these substances were without a direct effect on the pituitary in vitro. Conversely, acute decreases in tuberoinfundibular neuronal activity induced by dopamine biosynthesis inhibition or mimicked by pituitary receptor blockade induced acute increases in prolactin secretion. As another prerequisite for its involvement in the dynamic regulation of prolactin secretion, the tuberoinfundibular neuronal system was demonstrated to be involved in the negative feedback control of prolactin over its own secretion. Elevated circulating prolactin levels produced by pituitary homografts transplanted beneath the kidney capsule accelerated tuberoinfundibular dopaminergic neuronal activity. In two unrelated experimental conditions, rats rendered blind and anosmic or hyperprolactinemic, the chronic inhibition of prolactin secretion was not associated with the maintenance of an increased tuberoinfundibular neuronal activity, but rather with a supersensitivity of the anterior pituitary to the prolactin-release-inhibitory action of dopamine. Long-lasting alterations in tuberoinfundibular dopaminergic neuronal activity appeared to induce this pituitary supersensitivity to dopamine. The tuberoinfundibular neuronal system appears to have the capacity to modulate prolactin secretory episodes via the alteration of its dopaminergic activity. Long-lasting alterations in this activity may induce changes in anterior pituitary sensitivity to dopamine essential for the chronic inhibition of pituitary prolactin secretion.

Posttraumatic stress disorder (PTSD) is a chronic disabling condition that results from exposure to traumatic stress. However, although trauma is fairly common, PTSD will only occur in a small proportion of people. This suggests that resilience is a common response to trauma. The neurobiology underlying this adaptive response is thought to involve reward related areas as well as reward functions. This dissertation proposes that reward and reward-related areas have a role in anxiety disorders such as PTSD. This hypothesis was explored using an animal model of PTSD as well as a human mode of psychosical stress. The hypothesis that the ventral tegmental area (VTA), crucial for reward processing, is part of the neural circuitry involved in the symptomatology of PTSD was explored. To assess the role of VTA in PTSD, cells in this area were reversibly inactivated during a single exposure to inescapable foot-shock in a rodent model. Animals that underwent inactivation of VTA neurons decreased avoidance and lowered long-term anxiety-like behaviors in comparison with control groups. To assess short- and long-term electrophysiological effects of trauma on VTA cells, in vivo extracellular recordings were conducted. Results showed that the firing frequency of VTA cells changed both in the short- and long-term, following shock procedures. A human model of psychosical stress was used to test the hypothesis that the ability to respond appropriately to positive stimuli is important for the preservation of positive emotions following stressful events. The results show a positive correlation between trait resilience and trait reward sensitivity. To investigate the link between resilience and reward sensitivity further, the empirical portion of this study used a Monetary Incentive Delay Task (MID) to measure reward sensitivity before and after exposure to a psychosocial stressor. Moreover, behavioral reward sensitivity (as measured by MID and self-report satisfaction after the reward task) also correlated positively with trait and behaviorally measured resilience. The results shown in this dissertation suggest that the neural circuits involved in reward processing and reward function may be involved in resilient responses to stress.

Recent approaches in depression therapeutics include triple reuptake inhibitors, drugs that target three monoamine systems. Using in vivo electrophysiological and microdialysis techniques, the effects of 2- and 14-day treatments of escitalopram, nomifensine and the co-administration of these two drugs (TRI) were examined in male Sprague-Dawley rats. Short- and long-term TRI administration decreased NE firing and had no effect on DA neurons. Normal 5-HT firing rates were maintained after 2-day TRI administration compared to the robust inhibitory action of selective serotonin reuptake inhibitors (SSRIs). Escitalopram treatment enhanced the tonic activation of the 5-HT1A receptors given the increase in firing observed following WAY100635 administration. Nomifensine treatment enhanced tonic activation of the α2–adrenoceptors following idazoxan administration. TRI treatment caused a robust increase in extracellular DA levels that was in part mediated by a serotonergic contribution. Therapeutic effects of the drugs examined in this study may be due to the enhancement of 5-HT, NE and/or DA neurotransmission.

The prefrontal cortex (PFC) is critically involved in many higher cognitive functions such as goaldirected behaviour, affective behaviour and especially working memory. In vivo extracellular recordings of PFC neural activity during working memory tasks show high variety in observed spiking patterns. These complex dynamics are critically shaped by intrinsic, synaptic and structural parameters of respective prefrontal networks. Moreover, dopamine (DA) is crucial for correct functioning of the PFC during working memory tasks. DA modulates a number of synaptic and intrinsic biophysical properties of single neurons, in particular deep layer pyramidal cells, which represent the major output neurons of the PFC. Despite a high variability of cortical pyramidal cell firing patterns, and somatodendritic morphology, no study has yet systematically examined correlations between intrinsic properties, morphological features and dopaminergic modulation of intrinsic properties. This study investigated properties of deep layer pyramidal cells through whole cell patch clamp in acute brain slices of the adult rat PFC. Cells were characterised physiologically through a variety of stimulation protocols surveying different time scales and wide intensity ranges, while all fast synaptic transmission was blocked. Furthermore the same catalogue of stimuli was recorded whilst applying specific DA receptor agonists to elucidate effects of DA receptor activation on intrinsic properties. All recorded cells were injected with biocytin and dendritic morphology was reconstructed from confocal image stacks of fluorescently labelled neurons. From the resulting data a set of characteristic variables were defined and a combination of principal components analysis and hierarchical cluster analysis was used to identify similarity between recorded cells in different parameter spaces spanned by intrinsic properties, intrinsic properties under dopaminergic modulation and morphology, respectively. The analysis presents evidence for distinct subpopulations within prefrontal deep layer pyramidal cells, as seen by clustering of recorded cells in these high dimensional parameter spaces. These subpopulations also show distinct input-output relationships, bearing implications for computational functions of these subpopulations. Furthermore, this study presents for the first time evidence of subpopulation specific DA effects in deep layer pyramidal cells. The quantitative analysis of somatodendritic morphology confirms physiological subpopulations and identifies characteristic morphological features of deep layer pyramidal cells. Moreover, cluster observed in different parameter spaces overlap, leading to a definition of subpopulations that concurs with previously described prefrontal pyramidal cell types. In conclusion, the results presented provide some deeper insight into fundamental principles of information processing in prefrontal pyramidal cells under the influence of dopamine.

Neuroscience makes use of many metaphors in its attempt to explain the relationship between our brain and our behaviour. In this thesis I contrast the most commonly used metaphor - that of computation driven by neuron action potentials - with an alternative view which seeks to understand the brain in terms of an agent learning from the reward signalled by neuromodulators. To explore this reinforcement learning model I construct computational models to assess one of its key claims — that the neurotransmitter dopamine signals unexpected reward, and that this signal is used by the brain to learn control of our movements and drive goal-directed behaviour. In this thesis I develop a selection of computational models that are motivated by either theoretical concepts or experimental data relating to the effects of dopamine. The first model implements a published dopamine-modulated spike timing-dependent plasticity mechanism but is unable to correctly solve the distal reward problem. I analyse why this model fails and suggest solutions. The second model, more closely linked to the empirical data attempts to investigate the relative contributions of firing rate and synaptic conductances to synaptic plasticity. I use experimental data to estimate how model neurons will be affected by dopamine modulation, and use the resulting computational model to predict the effect of dopamine on synaptic plasticity. The results suggest that dopamine modulation of synaptic conductances is more significant than modulation of excitability. The third model demonstrates how simple assumptions about the anatomy of the basal ganglia, and the electrophysiological effects of dopamine modulation can lead to reinforcement learning like behaviour. The model makes the novel prediction that working memory is an emergent feature of a reinforcement learning process. In the course of producing these models I find that both theoretically and empirically based models suffer from methodological problems that make it difficult to adequately support such fundamental claims as the reinforcement learning hypothesis. The conclusion that I draw from the modelling work is that it is neither possible, nor desirable to falsify the theoretical models used in neuroscience. Instead I argue that models and metaphors can be valued by how useful they are, independently of their truth. As a result I suggest that we ought to encourage a plurality of models and metaphors in neuroscience. In Chapter 7 I attempt to put this into practice by reviewing the other transmitter systems that modulate dopamine release, and use this as a basis for exploring the context of dopamine modulation and reward-driven behaviour. I draw on evidence to suggest that dopamine modulation can be seen as part of an extended stress response, and that the function of dopamine is to encourage the individual to engage in behaviours that take it away from homeostasis. I also propose that the function of dopamine can be interpreted in terms of behaviourally defining self and non-self, much in the same way as inflammation and antibody responses are said to do in immunology.

For some behavior there are consistent differences between individuals within a population, which is called animal personality. Across species, ranging from insects to mammals, personality has been described along behavioral gradients like activity, exploration, boldness and aggression. Monoamines such as dopamine have been shown to be essential for modulating animal behavior and could therefore be important also in explaining variation in animal personality. Supporting this, the dopaminergic system affect activity (in Confused flour beetles), and aggression (in Mediterranean field crickets). However, the causality and effect of dopamine on these behaviors, and also other behavioral traits used to describe personality is currently less explored. This study experimentally investigated how increased level of dopamine affects activity, boldness, exploration and aggression in Mediterranean field crickets (Gryllus bimaculatus). I show that dopamine manipulation had no effects on measured behavior. These results indicate that increased dopamine levels do not affect the scored personality traits in Mediterranean field crickets. The causality and generality of the relationship between dopamine and behavior used to score variation in personality is thus not clear in this species.

Mes travaux de thèse concernent le rôle du récepteur D1 de la dopamine dans les dyskinésies L-dopa induites, effets secondaires extrêmement handicapants du traitement de la maladie de Parkinson. En condition de dénervation striatale mimant l’environnement de la maladie de Parkinson, le traitement chronique par la L-dopa entraine des altérations majeures du trafic intraneuronal et de la signalisation du récepteur D1 de la dopamine dans les principaux neurones cibles de la dopamine, les neurones épineux de taille moyenne du striatum. Il existe en particulier une hypersensibilisation des récepteurs D1 dans les neurones striataux, avec une abondance accrue à la membrane plasmique et une diminution du niveau d’expression de la protéine GRK6 (Protéine kinase des Récepteurs Couplés aux Protéines G 6), un des acteurs clefs des phénomènes de désensibilisation, en relation directe avec l’apparition des dyskinésies.C’est dans ce contexte que se situe mon travail de thèse qui a eu pour objectif de mettre à profit et/ou de développer différents modèles expérimentaux et outils « in vivo » et « in vitro ». Nous avons associé des techniques d’imagerie cellulaire et tissulaire à des approches comportementales, afin d’explorer certains des événements cellulaires et moléculaires à l’échelle du neurone striatal et des réseaux neuronaux, reliant le niveau d’expression du récepteur D1, sa compartimentation cellulaire, son trafic intraneuronal et les dyskinésies ou des conditions pharmacologiques équivalentes.Nous avons confirmé dans le modèle du rat lésé unilatéralement à la 6-OHDA, traité par la L-dopa et développant des mouvements anormaux analogues aux dyskinésies chez l’homme, que le récepteur D1 est anormalement abondant à la membrane plasmique des neurones du striatum, alors qu’il devrait être internalisé après stimulation par son ligand naturel, la dopamine. Nous avons mis en évidence que les mécanismes d’internalisation après stimulation par un agoniste restent néanmoins fonctionnels. Après administration de l’agoniste D1, chez les animaux dyskinétiques, l’abondance des récepteurs D1 augmente dans les compartiments notamment impliqués dans les mécanismes d’internalisation et de transport (vésicules) et de dégradation (corps multivésiculaires). Nous avons apporté une explication possible à cette abondance anormale et à ce défaut d’internalisation, en montrant qu’ils pourraient être dus à une hétérodimérisation entre les récepteurs D1 et D3. La co-activation des récepteurs D1 et D3 par la L-dopa favoriserait l’ancrage du récepteur D1 à la membrane plasmique des neurones striataux.Dans ce cadre, l’abord de l’étude de l’implication du protéasome dans la régulation de l’expression du récepteur D1 de la dopamine nous a semblé particulièrement important, sur la base des premières études soulignant l’implication de ce système catalytique dans le contrôle de l’activité et du métabolisme des récepteurs aux neurotransmetteurs. Nous avons révélé pour la première fois des liens entre l’activité catalytique du protéasome et la dynamique intraneuronale du récepteur D1 et plus particulièrement nous avons montré que son activité chymotrypsine-like est réduite de façon spécifique dans le striatum d’animaux dyskinétiques, comme une conséquence directe d’une déplétion en dopamine associée à une hyperstimulation dopaminergique.Nous avons testé en situation expérimentale une stratégie « thérapeutique » nouvelle en restaurant le mécanisme de désensibilisation homologue du récepteur D1 de la dopamine, par correction du déficit de la kinase GRK6 par transfert du gène correspondant via l’injection intrastriatale d’un vecteur lentiviral. Nous avons montré que cette approche permet de réduire considérablement la sévérité des dyskinésies dans les modèles rat et primate non-humain, analogues des dyskinésies chez l’homme et qu’elle restaure les effets thérapeutiques de la L-dopa. Ces effets sont la conséquence de la restauration des mécanismes de désensibilisation homologue : la surexpression de GRK6 entraîne l’internalisation spécifique des récepteurs D1. L’ensemble de nos résultats s’inscrit dans une démarche de recherche translationnelle menée depuis plusieurs années au laboratoire allant de la cellule au patient, avec pour but de transposer la compréhension des données expérimentales concernant les anomalies de l’expression du récepteur D1 de la dopamine en stratégies thérapeutiques dans les dyskinésies L-dopa induites. Nos investigations montrent qu’il est possible d’agir sur l’expression du récepteur D1 à la membrane plasmique des neurones striataux de manière indirecte, en manipulant trois co-activateurs de son métabolisme, pour espérer réduire « in fine » la sévérité des dyskinésies. / In my thesis work, I studied the role of dopamine D1 receptor in L-dopa induced dyskinesia, a debilitating complication of Parkinson's disease’s treatment. In condition of striatal denervation, that mimics the Parkinson's disease environment, chronic treatment with L-dopa leads to major alterations of intraneuronal trafficking and dopamine D1 receptor signaling in the major target of dopamine neurons, the striatal medium spiny neurons. In particularly, there is a D1 receptor hypersensitivity in striatal neurons, with an increased abundance of D1 receptor at the plasma membrane and a decreased level of GRK6 protein expression, a key actor in desensitization mechanism, directly related with the apparition of dyskinesia.In this context, I used different in vitro and in vivo experimental models and tools. I have associated cell and tissue imaging techniques and behavioural approaches in order to explore cellular and molecular events in striatal neuron and neuronal networks, linking the D1 receptor expression level, its cellular compartmentalization, its intraneuronal trafficking and the dyskinesia behaviour or equivalent pharmacological conditions.We confirmed in the rat analog of L-dopa-induced dyskinesia, i.e., the L-dopa-induced abnormal involuntary movements in unilaterally 6-hydroxydopamine (6-OHDA)-lesioned animals, that D1 receptor is abnormally abundant in the plasma membrane of neurons in the striatum, whereas it should be internalized after stimulation by its natural ligand, the dopamine. We showed that nevertheless the internalization mechanisms after agonist stimulation remains functional. After D1 agonist administration in dyskinetic animals, D1 receptor abundance increases in the cytoplasmic compartments involved in the internalization and transport (vesicles) and degradation (multivesicular bodies) mechanisms. Based on D3 receptor antagonist experiment, we propose that this abnormal abundance and this lack of internalization could be due to heterodimerization between the D1 and D3 receptors. D1 and D3 receptors co-activation by L-dopa might anchor D1 receptor at the plasma membrane of striatal neurons.In this context, analysis of proteasome involvement in the regulation of dopamine D1 receptor expression seemed particularly important, on the basis of the first studies underlying proteasome involvement in the activity and metabolism of neurotransmitter receptors. We demonstrated for the first time links between the proteasomal catalytic activity and D1 receptor intraneuronal dynamics and more particularly we showed that the proteasome chymotrypsin-like activity is reduced specifically in the striatum of dyskinetic animals, as a direct consequence of dopamine depletion associated with dopaminergic hyperstimulation.We tested in experimental condition, a new "therapeutic"strategy in order to restore the dopamine D1 receptor homologous desensitization mechanism, correcting the GRK6 kinase deficit by gene transfer through the intrastriatal injection of a lentiviral vector. We showed that this approach reduces significantly the dyskinesia severity in rat and non-human primate models and restores the L-dopa therapeutic effects. These effects are a consequence of the homologous desensitization mechanisms restoration : indeed GRK6 overexpression provokes specific D1 receptor internalization.Our results are part of a translational research conducted over several years in the laboratory from cell to patient, in order to translate our increased understanding of D1 receptor function abnormalities into therapeutic strategies for L-dopa induced dyskinesia. Our investigations show that it is possible to act on D1 receptor expression at the plasma membrane of striatal neurons via various routes, all resulting into diminished dyskinesia severity.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.