Thèses sur le sujet « Basal ganglia model »

This thesis presents a computational model of the basal ganglia that is able to learn sequences and perform action selection. The basal ganglia is a set of structures in the human brain involved in everything from action selection to reinforcement learning, inspiring research in psychology, neuroscience and computer science. Two temporal difference models of the basal ganglia based on previous work have been reimplemented. Several experiments and analyses help understand and describe the original works. This uncovered flaws and problems that is addressed.

2010 - 2011
Our research activity investigates the computational processes underlying the execution of complex sequences of movements and aims at understanding how different levels of the nervous system interact and contribute to the gradual improvement of motor performance during learning. Many research areas, from neuroscience to engineering, investigate, from different perspectives and for diverse purposes, the processes that allow humans to efficiently perform skilled movements. From a biological point of view, the execution of voluntary movements requires the interaction between nervous and musculoskeletal systems, involving several areas, from the higher cortical centers to motor circuits in the spinal cord. Understanding these interactions could provide important insights for many research fields, from machine learning to medicine, from the design of robotic limbs to the development of new treatments for movement disorders, such as Parkinson’s disease. This goal could be achieved by finding an answer to the following questions: · How does the central nervous system control and coordinate natural voluntary movements? · Which brain areas are involved in learning a new motor skill? What are the changes that happen in these neural structures? What are the aspects of the movement memorized? · Which is the process that allows people to perform a skilled task, such as playing an instrument, being apparently unaware of the movements they are performing? · What happen when a neurodegenerative disease affects the brain areas involved in executing movements? These questions have been addressed from different perspectives and levels of analysis, from the exploration of the anatomical structure of the neural systems thought to be involved in motor learning (such as the basal ganglia, cerebellum and hippocampus) to the investigation of their neural interaction; from the analysis of the activation of these systems in executing a motor task to the specific activation of a single or a small group of neurons within them. In seeking to understand all the breadth and facets of motor learning, many researchers have used different approaches and methods, such as genetic analysis, neuroimaging techniques (such as fMRI, PET and EEG), animal models and clinical treatments (e.g. drugs administration and brain stimulation). These studies have provided a large body of knowledge that has led to several theories related to the role of the central nervous system in controlling and learning simple and complex movements. These theories envisage the interaction among multiple brain regions, whose cooperation leads to the execution of skilled movements. How can we test these interactions for the purpose of evaluating a theory? Our answer to this question is investigating these interactions through computational models, which provide a valuable complement to the experimental brain research, especially in evaluating the interactions within and among multiple neural systems. Based on these concepts arises our research, which addresses the questions previously pointed out and aims at understanding the computational processes performed by two neural circuits, the Basal Ganglia and Cerebellum, in motor learning. We propose a new hypothesis about the neural processes occurring during acquisition and retention of novel motor skills. According to our hypothesis, a sequence of movements is stored in the nervous system in the form of a spatial sequence of points (composing the trajectory plan associated to the motor sequence) and a sequence of motor commands. We propose that learning novel motor skills requires two phases, in which two different processes take place. Early in learning, when movements are slower, less accurate, and attention demanding, the motor sequence is performed by converting the sequence of target points into the appropriate sequence of motor commands. During this phase, the trajectory plan is acquired and the movements rely on the information provided by the visuo-proprioceptive feedback, which allows to correct the sequence of movements so that the actual trajectory plan corresponds to the desired one and the lowest energy is spent by the muscular subsystem involved. During the late learning phase, when the sequence of movements is performed faster and automatically, with little or no cognitive resources needed to complete it, and is characterized by anticipatory movements, the sequence of motor commands is acquired and thus, the sequence of movements comes to be executed as a single behavior. We suggest that the Basal Ganglia and Cerebellum are involved in learning novel motor sequences, although their role is crucial in different stages of learning. Accordingly, we propose a neural scheme for procedural motor learning, comprising the basal ganglia, cerebellum and cortex, which envisages that the basal ganglia, interacting with the cortex, select the sequence of target points to reach (composing the trajectory plan), whereas the cerebellum, interacting with the cortex, is responsible for converting the trajectory plan into the appropriate sequence of motor commands. Consequently, we suggest that early in learning, task performance is more dependent on the procedural knowledge maintained by the cortex-basal ganglia system, while after a long-term practice, when the sequence of motor commands is acquired within the cerebellum, task performance is more dependent on the motor command sequence maintained by the cortexcerebellar system. We tested the neural scheme (and the hypothesis behind it) through a computational model that incorporates the key anatomical, physiological and biological features of these brain areas in an integrated functional network. Analyzing the behavior of the network in learning novel motor tasks and executing well-known motor tasks, both in terms of the neural activations and motor response provided, we found that the results obtained fit those reported by many neuroimaging and experimental studies presented in the literature. We also carried out further experiments, simulating neurodegenerative disorders (Parkinson's and Huntington disease, which affect the basal ganglia) and cerebellar damages. Results obtained by these experiments validates the proposed hypothesis, showing that the basal ganglia play a key role during the early stage of learning, whereas the cerebellum is crucial for motor skill retention. Our model provides some insights about the learning mechanisms occurring within the cerebellum and gains further understanding of the functional dynamics of information processing within the basal ganglia and cerebellum in normal as well as in diseased brains. Therefore the model provides novel predictions about the role of basal ganglia and cerebellum in motor learning, motivating further investigations of their interactions. [edited by author]
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Contemporary reinforcement learning (RL) theory suggests that choices can be evaluated either by the model-free (MF) strategy of learning their past worth or the model-based (MB) strategy of predicting their likely consequences based on learning how decision states eventually transition to outcomes. Statistical and computational considerations argue that these strategies should ideally be combined. This thesis aimed to investigate the neural implementation of these two RL strategies and the mechanisms of their interactions. Two non-human primates performed a two-stage decision task designed to elicit and discriminate the use of both MF and MB-RL, while single-neuron activity was recorded from the prefrontal cortex (frontal pole, FP; anterior cingulate cortex, ACC; dorsolateral prefrontal cortex) and striatum (caudate and putamen). Logistic regression analysis revealed that the structure of the task (of MB importance) and the reward history (of MF and MB importance) significantly influenced choice. A trial-by-trial computational analysis also confirmed that choices were made according to a weighted combination of MF and MB- RL, with the influence of the latter approaching 90%. Furthermore, the valuations of both learning methods also influenced response vigour and pupil response. Neural correlates of key elements for MF and MB learning were observed across all brain areas, but functional segregation was also in evidence. Neurons in ACC encoded features of both MF and MB, suggesting a possible role in the arbitration between both strategies. Striatal activity was consistent with a role in value updating by encoding reward prediction errors. Finally, novel neurophysiological evidence was found in favour of the role of the FP in counterfactual processing. In conclusion, this thesis provides insight into the neural implementation of MF and MB-RL computations and their various effects on diverse aspects of behaviour. It supports the parallel operation and integration of the two approaches, while revealing unexpected intricacies.

The basal ganglia (BG) is a complex set of heavily interconnected nuclei located in the central part of the brain that receives inputs from the several areas of the cortex and projects via the thalamus back to the prefrontal and motor cortical areas. Despite playing a significant part in multiple brain functions, the physiology of the BG and associated disorders like dystonia remain poorly understood. Dystonia is a devastating condition characterized by ineffective, twisting movements, prolonged co-contractions and contorted postures. Evidences suggest that it occurs due to abnormal discharge patterning in BG-thalamocortocal (BGTC) circuitry. The central purpose of this study was to understand the electrophysiology of BGTC circuitry and its role in motor control and dystonia. Toward this goal, an advanced multi-target multi-unit recording and analysis system was utilized, which allows simultaneous collection and analysis of multiple neuronal units from multiple brain nuclei. Over the cause of this work, neuronal data from the globus pallidus (GP), subthalamic nucleus (STN), entopenduncular nucleus (EP), pallidal receiving thalamus (VL) and motor cortex (MC) was collected from normal, lesioned and dystonic rats under awake, head restrained conditions. The results have shown that the neuronal population in BG nuclei (GP, STN and EP) were characterized by a dichotomy of firing patterns in normal rats which remains preserved in dystonic rats. Unlike normals, neurons in dystonic rat exhibit reduced mean firing rate, increased irregularity and burstiness at resting state. The chaotic changes that occurs in BG leads to inadequate hyperpolarization levels within the VL thalamic neurons resulting in a shift from the normal bursting mode to an abnormal tonic firing pattern. During movement, the dystonic EP generates abnormally synchronized and elongated burst duration which further corrupts the VL motor signals. It was finally concluded that the loss of specificity and temporal misalignment between motor neurons leads to corrupted signaling to the muscles resulting in dystonic behavior. Furthermore, this study reveals the importance of EP output in controlling firing modes occurring in the VL thalamus.

Nous avons étudié les récepteurs GABAA dans un modèle de la maladie de Huntington. En combinant des approches biochimiques, moléculaires, électrophysiologiques et de l’imagerie haute résolution, nous avons montré une modification de la neurotransmission GABAergique chez des animaux à des stades pre- et post-symptomatiques. Nos études montrent une diminution de de la neurotransmission GABAergique dans le globus pallidus des souris Huntington qui pourrait conduire à une modification des noyaux de sortie des ganglions de la base et de l’activité motrice. L’ensemble de nos résultats permet de définir le rôle de différents types de récepteurs GABAA dans le cerveau dans des conditions physiologiques et pathologiques
We explored GABAergic neurotransmission in a mouse model of Huntington's disease. Combining molecular, imaging and electrophysiologicaltechniques, we showed changes of GABAergic neurotransmission in presymptomatic and symptomatic R6/1 mice. Our data demonstrated a decreased GABAergic inhibition in the globus pallidus of R6/1 mice, which could result in an alteration of basal ganglia output nuclei and motor activity. Taken together, our results will help to define the contribution of receptor subtypes to inhibitory transmission throughout the brain in physiological and pathophysiological states

Les noyaux gris centraux (ganglions de la base en anglais) sont un réseau de structures sous-corticales dont la persistance dans l'ensemble des vertébrés plaide en faveur d'une fonction clef au cours de l'évolution. Comme ce fut remarqué dès le 18ème siècle, ils ont l'unique particularité de concentrer des afférences de l'entièreté de la surface corticale. Cette position centrale et l'analyse de l'anatomie du réseau leur ont valu le rôle d'arbitre central du cerveau, réglant les conflits entre processus neuronaux concomitants bien qu'incompatibles. Au sein du réseau, le noyau subthalamique jouit d'une notoriété particulière. Ce noyau, sur la base de ses afférences corticales, et en vertu de ses projections sur le soma des neurones pallidaux, aurait pour fonction de filtrer les programmes comportementaux codés par le striatum et concourant pour leur expression. Rapporté aux théories de la prise de décision, le noyau subthalamique fixerait le seuil décisionnel, ou la quantité d'information à accumuler en faveur d'une option comportementale afin qu'elle soit exprimée. Mais si ce petit noyau est devenu si célèbre, c'est surtout qu'il est la cible d'une procédure chirurgicale spectaculaire: la stimulation cérébrale profonde. Cette opération du cerveau est le dernier recours pour les patients souffrant d'une maladie de Parkinson ou d'un trouble obsessionnel compulsif sévère. Elle parvient même parfois à faire disparaître leurs symptômes. Malgré cette efficacité remarquable, les mécanismes de la stimulation cérébrale profonde restent inconnus. Il faut, entre autres, blâmer l'obscurité qui règne encore sur le noyau subthalamique, car les fonctions mentionnées ci-dessus restent des conjectures théoriques en manque de validation expérimentale. La première étape de ce travail a été d'en valider les bases anatomiques. En effet, l'existence d'une voie fronto-subthalamique - nécessaire au modèle - n'était connue que sur la base d'études menées chez le rat. Nous avons démontré, par des méthodes de traçage axonal, l'existence de cette connexion chez le primate. En sus, cette connexion aura permis de redéfinir les frontières médiales du noyau subthalamique avec les conséquences cliniques qui peuvent en être tirées. Le deuxième objectif global de cette thèse était de tester la validité fonctionnelle du modèle, la stimulation cérébrale profonde offrant un accès rare aux activités du noyau subthalamique. Cependant, il était d'abord nécessaire de caractériser la population étudiée, à savoir des patients souffrants d'un trouble obsessionnel compulsif. Grâce à l'imagerie de diffusion nous démontrons une diminution ainsi qu'une désorganisation des connexions cortico-sous corticales, se traduisant probablement par un défaut de contrôle conscient sur le processus de sélection. Une étude de magnétoencéphalographie est en cours pour approfondir les changements d'activité corticale. Pour tester le rôle du noyau subthalamique dans l'établissement du seuil décisionnel nous avons enregistré son activité électrophysiologique pendant que les patients effectuaient une tâche de prise de décision perceptuelle. Nous démontrons que les neurones du noyau subthalamique ont une réponse multimodale, concordant en cela avec nos données anatomiques qui montrent une convergence d'informations au niveau du noyau subthalamique. De plus, une augmentation de l'activité est retrouvée dans les conditions attendues
The basal ganglia are a network of subcortical structures of which the invariant architecture throughout vertebrate evolution suggests a key function in evolution. As was noted as early as the 18th century, they have the unique characteristic of concentrating afferences from the entire cortical surgace. Given this central position and the internal architecture of the network, they could provide a centralised selection mechanism in the brain, arbitrating between any two conflicting processes. Among the basal ganglia, the subthalamic nucleus has become of particular interest as it is the target of deep brain stimulation, a neurosurgical procedure used to treat severe Parkinson’s disease and obsessive-compulsive disorder. It would have for function to integrate contextual information from its cortical inputs to filter behavioural programs encoded by the striatum. Within the framework of decision-making models, this filtering function is akin to setting the decision threshold, or the amount of evidence required before selecting a program. However, this considerations remain hypothetical as they are lacking experimental support. The first objective of this work was to validate the anatomical basis of these assumptions. Indeed, the existence of a prefrontal-subthalamic pathway, necessary to expand the decision models to every type of decision, had only been demonstrated in rodents. We demonstrated its existence in the primate using anterograde axonal tracing. In addition, this projection will have allowed us to redefine the medial border of the subthalamic nucleus with the clinical consequences that that may have. The second objective of this thesis was to test the functional validity of the models, and specifically the role of the subthalamic nucleus in setting decision thresholds. Deep brain stimulation offers a rare access to the electrophysiology of this structure; however, it is a patient population, here obsessive-compulsive disorder patients. A first step was, therefore, to characterise this population, anatomically and behaviourally, to understand how it might be of use as a model of decision-making in the basal ganglia. We demonstrated a reduction in the strength of cortico-subcortical anatomical connections. We suggest that this prevents accurate conscious control over decision mechanisms. Behaviourally, patients displayed a pathologically low confidence levels in their decisions and we hypothesised that this would lead to an increase of the decision threshold and matching subthalamic activity. To test this, we recorded the activity of the subthalamic nucleus during a decision-making task. We demonstrate that subthalamic neurons have a multimodal activity, consistent with our demonstration of convergent cortical inputs. However, we were unable to demonstrate a link between subthalamic activity and decision threshold, although this may be due to technical considerations…

Dopamine replacement therapy is the main method of treating Parkinson’s Disease (PD), however over time this treatment causes increasingly abnormal, involuntary movements. This symptom, known as Levodopa-Induced-Dyskinesia (LID) is associated with aberrant, high frequency oscillations (HFOs) in the motor cortex and basal ganglia, as demonstrated with implanted electrodes in human Parkinson’s patients as well as in a rat model of Parkinson’s Disease. However, despite efforts to determine if the same high frequency oscillations are also present during dyskinesia in the widespread 6-OHDA mouse model of Parkinson’s Disease, studies have been unable to do so. By building and implanting a 64-channel multi-electrode array into a unilateral 6-OHDA lesioned mouse, we were able to record HFOs at 80Hz and >100Hz in the motor cortex, basal ganglia and thalamus in the lesioned hemisphere during LID. We also recorded bilateral HFOs at >100Hz in the intact hemisphere. With this work we show that the same HFOs that are present in the motor cortex and basal ganglia of rats and humans are also present in mice during dyskinesia. This work will act to further validate the 6-OHDA PD-model in mice and provide opportunities to investigate new treatments for Parkinson’s Disease, dyskinesia and other neurological conditions. It will also serve as a model to study a purposed mechanism underlying the information processing in populations of neurons.
Dopaminbehandling är den mest förekommande metoden för att behandla Parkinsons sjukdom men detta orsakar dessvärre en bieffekt i form av gradvis förvärrande ofrivilliga rörelser. Detta beteendemönster kallas för Levodopa-Inducerad-Dyskinesi (LID) och med hjälp av elektrodimplantat i hjärnan, på parkinsonpatienter och djurmodeller av parkinsons, har man kunnat se att beteendet är förknippat med högfrekventa oscilleringar (HFO) av hjärnaktivitet i motorcortex och basala ganglierna. Trots försök att kartlägga om dessa högfrekventa oscilleringar också är närvarande i den populära 6-OHDA musmodellen av Parkinsons sjukdom, så har man hittills inte lyckats demonstrera detta. Genom att bygga och implantera ett elektrodimplantat med 64 kanaler i en ensidigt-leisonerad 6-OHDA musmodell av Parkinsons sjukdom så kunde vi åskådliggöra HFO i motor cortex, basala ganglierna och thalamus i den lesionerade hjärnhalvan under LID. Vi kunde också påvisa HFO som sträckte sig över till den intakta hjärnhalvan, med frekvenser över 100 Hz. Denna forskning ger stöd att 6-OHDA modellen för Parkinsons i möss är valid och ger möjlighet till nya metoder att utforska och behandla Parkinsons, dyskinesi och andra neurologiska åkommor. Studien lägger också grunden för framtida studier som ämnar att undersöka föreslagna mekanismer bakom sättet populationer av neuroner bearbetar information.
ingår i ett projekt finansierat av Vetenskapsrådet #2018-02717

L'objectiu general d'aquest treball és aprofundir en l'estudi de la fisiopatologia de les malalties degeneratives, un dels objectius principals per a la farmacologia actual degut a l'increment de la seva incidència en les últimes dècades. Mitjançant la utilització de models experimentals d 'aquestes patologies s'han plantejat diferents objectius més concrets: A.- Induir la malaltia de Parkinson experimental a través de la injecció de l'MPP+ en la substància negra de rata. A1.-Estudiar la resposta glial en el nucli estriat després de La degeneració anterograda de les neurones dopaminèrgiques causada per la injecció de l'MPP+ en la substància negra. Implicació en el mecanisme de mort neuronal i de regeneració. B.- Induir la malaltia de Huntington experimental a través de la injecció d'aminoàcids excitadors en el nucli estriat de rata. B1.-Caracteritzar la resposta endògena tròfica a l'excitotoxicitat, valorant els canvis en l 'expressió del BDNF i l'NT-3 així com la dels seus receptors, TrkB i TrkC, en l'escorça cerebral després de la injecció de diferents agonistes del receptor del glutamat en el nucli estriat. B2.-Estudiar la regulació endògena dels nivells de BDNF en Ja substància negra de rata després de la lesió estriatal induïda per l'àcid kaínic, així com la possible implicació d'aquesta neurotrofina en la supervivència de les neurones de la substància negra front la lesió excitotòxica en el nucli estriat. 3. Estudiar el possible efecte neuroprotector de les neurotrofines BDNF, NT-3 iNT-4/5, sobre les diferents poblacions neuronals de projecció del nucli estriat, en el model excitotòxic de l'àcid quinolínic. Es van implantar en l 'estriat de rata adulta línies cel·lulars establertes que secreten alts nivells de BDNF, NT-3 i NT-4/5 recombinant abans de la injecció de l'aminoàcid excitador.

The aim of this thesis is to improve our understanding of the neural systems involved in schizophrenia by suggesting possible avenues for future computational modelling in an attempt to make sense of the vast number of studies relating to the symptoms and cognitive deficits relating to the disorder. This multidisciplinary research has covered three different levels of analysis: abnormalities in the microscopic brain structure, dopamine dysfunction at a neurochemical level, and interactions between cortical and subcortical brain areas, connected by cortico-basal ganglia circuit loops; and has culminated in the production of five models that provide useful clarification in this difficult field. My thesis comprises three major relevant modelling themes. Firstly, in Chapter 3 I looked at an existing neural network model addressing the Neurodevelopmental Hypothesis of Schizophrenia by Hoffman and McGlashan (1997). However, it soon became clear that such models were overly simplistic and brittle when it came to replication. While they focused on hallucinations and connectivity in the frontal lobes they ignored other symptoms and the evidence of reductions in volume of the temporal lobes in schizophrenia. No mention was made of the considerable evidence of dysfunction of the dopamine system and associated areas, such as the basal ganglia. This led to my second line of reasoning: dopamine dysfunction. Initially I helped create a novel model of dopamine neuron firing based on the Computational Substrate for Incentive Salience by McClure, Daw and Montague (2003), incorporating temporal difference (TD) reward prediction errors (Chapter 5). I adapted this model in Chapter 6 to address the ongoing debate as to whether or not dopamine encodes uncertainty in the delay period between presentation of a conditioned stimulus and receipt of a reward, as demonstrated by sustained activation seen in single dopamine neuron recordings (Fiorillo, Tobler & Schultz 2003). An answer to this question could result in a better understanding of the nature of dopamine signaling, with implications for the psychopathology of cognitive disorders, like schizophrenia, for which dopamine is commonly regarded as having a primary role. Computational modelling enabled me to suggest that while sustained activation is common in single trials, there is the possibility that it increases with increasing probability, in which case dopamine may not be encoding uncertainty in this manner. Importantly, these predictions can be tested and verified by experimental data. My third modelling theme arose as a result of the limitations to using TD alone to account for a reinforcement learning account of action control in the brain. In Chapter 8 I introduce a dual weighted artificial neural network, originally designed by Hinton and Plaut (1987) to address the problem of catastrophic forgetting in multilayer artificial neural networks. I suggest an alternative use for a model with fast and slow weights to address the problem of arbitration between two systems of control. This novel approach is capable of combining the benefits of model free and model based learning in one simple model, without need for a homunculus and may have important implications in addressing how both goal directed and stimulus response learning may coexist. Modelling cortical-subcortical loops offers the potential of incorporating both the symptoms and cognitive deficits associated with schizophrenia by taking into account the interactions between midbrain/striatum and cortical areas.

The model of basal ganglia function proposed by Albin, Young and Penney (1989) describes two anatomically independent motor pathways, the direct and indirect. However, under normal conditions striatal dopamine (DA) is required for the expression of motor behavior, and DAergic control of the two pathways (via D1 and D2 receptors, respectively) is dependent on co-activation. We tested for a possible breakdown of D1/D2 synergism using transgenic R6/1 mice bearing the human huntingtin allele (Htt). Motor stereotypy, observed prior to the onset of HD-related symptoms, was rated on a 5-point scale following activation of: A) D1 receptors alone, B) D2 receptors alone, and C) stimulation of both D1 and D2 receptors. Results revealed that mice with the HD allele, like their WT litter mates, depend on the co-activation of the indirect and direct motor pathways to facilitate deliberate behavior.

The motor nervous system is one of the main systems of the body and is our principle means ofbehavior. Some of the most debilitating and wide spread disorders are motor systempathologies. In particular the basal ganglia are complex networks of the brain that control someaspects of movement in all vertebrates. Although these networks have been extensively studied,lack of proper methods to study them on a system level has hindered the process ofunderstanding what they do and how they do it. In order to facilitate this process I have usedcomputational models as an approach that can faithfully take into account many aspects of ahigh dimensional multi faceted system.In order to minimize the complexity of the system, I first took agnathan fish and amphibians asmodeling animals. These animals have rather simple neuronal networks and have been wellstudied so that developing their biologically plausible models is more feasible. I developedmodels of sensory motor transformation centers that are capable of generating basic behaviorsof approach, avoidance and escape. The networks in these models used a similar layeredstructure having a sensory map in one layer and a motor map on other layers. The visualinformation was received as place coded information, but was converted into population codedand ultimately into rate coded signals usable for muscle contractions.In parallel to developing models of visuomotor centers, I developed a novel model of the basalganglia. The model suggests that a subsystem of the basal ganglia is in charge of resolvingconflicts between motor programs suggested by different motor centers in the nervous system.This subsystem that is composed of the subthalamic nucleus and pallidum is called thearbitration system. Another subsystem of the basal ganglia called the extension system which iscomposed of the striatum and pallidum can bias decisions made by an animal towards theactions leading to lower cost and higher outcome by learning to associate proper actions todifferent states. Such states are generally complex states and the novel hypothesis I developedsuggests that the extension system is capable of learning such complex states and linking themto appropriate actions. In this framework, striatal neurons play the role of conjunction (BooleanAND) neurons while pallidal neurons can be envisioned as disjunction (Boolean OR) neurons.In the next set of experiments I tried to take the idea of basal ganglia subsystems to a new levelby dividing the rodent arbitration system into two functional subunits. A rostral group of ratpallidal neurons form dense local inhibition among themselves and even send inhibitoryprojections to the caudal segment. The caudal segment does not project back to its rostralcounterpart, but both segments send inhibitory projections to the output nuclei of the rat basalganglia i.e. the entopeduncular nucleus and substantia nigra. The rostral subsystems is capableof precisely detecting one (or several) components of a rudimentary action and suppress othercomponents. The components that are reinforced are those which lead to rewarding stateswhereas those that are suppressed are those which do not. The hypothesis explains neuronalmechanisms involved in this process and suggests that this subsystem is a means of generatingsimple but precise movements (such as using a single digit) from innate crude actions that theanimal can perform even at birth (such as general movement of the whole limb). In this way, therostral subsystem may play important role in exploration based learning.In an attempt to more precisely describe the relation between the arbitration and extensionsystems, we investigated the effect of dynamic synapses between subthalamic, pallidal andstriatal neurons and output neurons of the basal ganglia. The results imply that output neuronsare sensitive to striatal bursts and pallidal irregular firing. They also suggest that few striatalneurons are enough to fully suppress output neurons. Finally the results show that the globuspallidus exerts its effect on output neurons by direct inhibition rather than indirect influence viathe subthalamic nucleus.

QC 20131209

Neural activity in dopaminergic areas such as the ventral tegmental area is influenced by timing processes, in particular by the temporal expectation of rewards during Pavlovian conditioning. Receipt of a reward at the expected time allows to compute reward-prediction errors which can drive learning in motor or cognitive structures. Reciprocally, dopamine plays an important role in the timing of external events. Several models of the dopaminergic system exist, but the substrate of temporal learning is rather unclear. In this article, we propose a neuro-computational model of the afferent network to the ventral tegmental area, including the lateral hypothalamus, the pedunculopontine nucleus, the amygdala, the ventromedial prefrontal cortex, the ventral basal ganglia (including the nucleus accumbens and the ventral pallidum), as well as the lateral habenula and the rostromedial tegmental nucleus. Based on a plausible connectivity and realistic learning rules, this neuro-computational model reproduces several experimental observations, such as the progressive cancelation of dopaminergic bursts at reward delivery, the appearance of bursts at the onset of reward-predicting cues or the influence of reward magnitude on activity in the amygdala and ventral tegmental area. While associative learning occurs primarily in the amygdala, learning of the temporal relationship between the cue and the associated reward is implemented as a dopamine-modulated coincidence detection mechanism in the nucleus accumbens.

Nous avons étudié les ganglions de la base (GB) sous un angle théorique, au travers de modèles conçus avec des algorithmes évolutionnistes multiobjectifs. Dans un premier temps, nous avons caractérisé les possibles corrélats neuronaux de la sélection supposément opérés par les GB, via l’étude de deux modèles: le CBG (Girard et al. 2008) et le GPR (Gurney et al. 2001). Les voies directe et indirecte sont importantes; la boucle thalamique, indifférente; la projection GPe → GPi/SNr, antagoniste à la sélection. La connexion GPe → MSN et le caractère diffus de GPe → GPi/SNr expliquent la meilleure sélectivité dans le CBG. Nous avons aussi conçu des modèles des GB respectant un corpus de contraintes anato-électrophysiologiques issues du primate. Ces contraintes sont respectées sous l’hypothèse que la projection GPe → GPi/SNr est faiblement inhibitrice. En outre, nous montrons que ces modèles plausibles opèrent une sélection parmi leurs entrées d’une façon compatible avec les données électrophysiologiques. La projection GPe → GPi/SNr permet une meilleure sélection lorsqu’elle est diffuse. Enfin, nous avons étudié avec ces modèles plausibles les origines potentielles des oscillations associés à la maladie de Parkinson. Une modélisation du manque de dopamine via une augmentation modérée de l’efficacité des potentiels d’action au niveau du STN, GPe et GPi/SNr, se révèle suffisante pour entraîner des oscillations dans la bande β. De plus, la fréquence de ces oscillations dépend des délais de la boucle GPe ↔ STN, qui ne semble pas être compatible avec des oscillations dans la bande θ
Our work brings contributions to the field of computational models of the basal ganglia(BG) with the use of multi-objective evolutionary algorithms. We first characterized what underlies the hypothesized selection capability in the BG structure with two models : the CBG (Girard et al. 2008) and the GPR (Gurney et al. 2001). The direct/indirect pathway were found to be important; those of the thalamic loop, indifferent; the GPe → GPi/SNr projection, antagonist to selection. Two pathways explain the better selectivity in CBG: the GPe → MSN connection and the diffuse pattern of GPe → GPi/SNr. We also build plausible BG models which respect a collection of constraints issued from a review of anatomical and electrophysiological primate literature. Our first result is that anatomical and electrophysiological data are consistent if we suppose a GPe → GPi/SNr projection that is weakly inhibitory. Our second result is that the plausible models perform selection, with electrophysiological activities that are furthermore plausible. We finally studied the pattern of projection of the GPe → GPi/SNr projection, and found that a diffuse pattern is more efficient for selection. Finally, we studied with the plausible models the origin of the oscillations occurring in Parkinson’s disease. We first established delays matching the timing data from stimulation experiments. Modeling the dopamine depletion by a moderate plausible increase in single spike efficiency in STN, GPe and GPi/SNr is enough to trigger oscillatory regimens in the β-band. The oscillations frequency is highly dependent of the GPe ⇋ STN delays, which could not plausibly support - band oscillations in our models

Les crises d'épilepsie proviennent d'une synchronisation pathologique de réseaux neuronaux du cortex. Les crises motrices, générées à partir du cortex moteur primaire, sont souvent pharmaco-résistantes. La résection neurochirurgicale du foyer épileptique est rarement l'option thérapeutique de choix au regard des risques de deficits moteurs potentiellement induits par la résection. Les ganglions de la base ont un rôle important dans la propagation des crises. Des enregistrements par micro-électrode réalisés dans une précédente étude ont montré que les activités des structures d'entrée des ganglions de la base telles que le Putamen, le noyau caudé et le noyau sous-thalamique (NST) sont fortement modifiées pendant des crises motrices. Le taux de décharge moyen des neurones du NST et du Putamen augmente et le pourcentage de neurones oscillants synchronisés avec l'EEG durant la période ictale est plus élevé durant les crises que pendant la période inter-ictale. Des études pilotes chez l'humain ont montré un effet bénéfique potentiel de la stimulation cérébrale profonde (SCP) chronique du NST pour traiter les crises motrices pharmaco-résistantes. Le but de notre étude est d'évaluer les effets thérapeutiques de la SCP des structures d'entrée des ganglions de la base. Nous avons dans un premier temps développé un modèle primate de crise d'épilepsie motrice focale stable et reproductible par injection intra-corticale de pénicilline. Nous avons ensuite caractérisé la pharmaco-résistance du modèle. Nous avons implanté stéréotactiquement des électrodes de SCP dans le NST et le Putamen. Le stimulateur a été placé sous la peau dans le dos de l'animal. Un protocole de stimulation à 130 Hz à un voltage inférieur à l'apparition d'effets secondaires a été réalisé dans le NST. Le stimulateur était mis en marche au moment de l'injection de la pénicilline. Un protocole de stimulation à 0 volt a été réalisé comme condition contrôle. Chaque primate étant son propre contrôle. L'apparition des crises, leur nombre et leur durée ont été comparés par période de 1 heure entre la condition stimulée et non stimulée. Chaque session expérimentale a été menée sur une durée de plus de six heures. Nous avons évalué l'effet préventif de la SCP à haute fréquence (130 Hz) du NST sur les crises motrices. Nous avons également étudié l'effet préventif de la SCP à basse fréquence (5-20 Hz) du Putamen sur ce même modèle. Enfin, sur un autre primate, nous avons étudié l'effet combiné de la SCP du NST à haute fréquence et du Putamen à basse fréquence sur les crises motrices. Résultats : Les effets de la SCP chronique du NST à haute fréquence ont été analysés à partir de 1572 crises apparues au cours de 30 sessions expérimentales chez 3 primates. Les effets de la SCP préventive du NST ont été évalués sur 454 crises motrices durant 10 sessions expérimentales chez un primate. L'effet de la SCP du Putamen à basse fréquence a été analysé sur 289 crises durant 14 sessions chez 2 primates. Enfin l'effet combiné de la SCP du NST et du Putamen a été évalué sur 477 crises durant 10 sessions. Les meilleurs résultats ont été obtenus par SCP chronique du NST. L'apparition de la première crise était significativement retardée lorsque le primate était stimulé. Le temps total passé en situation de crise motrice était diminué en moyenne d'environ 69 % (p ≤0.05) par rapport à la condition non-stimulé au regard de la diminution significative du nombre de crises particulièrement durant les 3 heures après le début de la stimulation. La durée de chaque crise était modérément réduite. Les modes de stimulation mono-polaire ou bi-polaire avaient une efficacité similaire. La SCP préventive du NST n'a pas eu d'effet supérieur à la stimulation chronique du NST. La SCP chronique du Putamen à basse fréquence avait un effet positif mais principalement durant les deux premières heures de stimulation. L'effet combiné de la SCP du NST et du Putamen était inférieur à la SCP chronique du NST ou du Putamen.

The motor symptoms of Parkinson's disease (PD) have been linked to the emergence of exaggerated oscillatory activity in the 13 - 35 Hz beta range in recordings of the basal ganglia (BG) thalamocortical circuit of PD patients and animal models. PD patients and animal models also express dopamine-dependent cognitive impairments, implying effects of dopamine loss on the function of the anterior cingulate cortex (ACC). This thesis examines the electrophysiological behavior of the BG thalamocortical circuit in PD and dopamine-normal states during cognitive and motor activity. In vivo recordings in the BG of PD and dystonic patients were used to study the influence of dopamine during a test of executive function. Normal executive function was also investigated in the dopamine-healthy ACC of chronic pain patients. Both the BG and ACC exhibited lateralized electrophysiological responses to feedback valence. The BG also exhibited dopamine-sensitive event-related behavior. In additional experiments, chronically implanted recording electrodes in awake, behaving hemiparkinsonian rats were used to examine the transmission of synchronized oscillatory activity from the BG, through the ventral medial (VM) thalamus, to the ACC. Modulation of subthalamic nucleus, VM thalamus, and ACC activity during a simple cognitive/movement task was also investigated in hemiparkinsonian rats. Findings in the rat model suggest that ACC-mediated executive function is dopamine-sensitive and is reflected in the region's electrophysiology. These results may provide further insight into the significance of excessive oscillatory activity in PD and its influence on cognitive systems.

Synucleins are highly conserved presynaptic proteins with unknown function. α-synuclein plays a key role regulating dopamine homeostasis and is intimately involved in Parkinson’s disease (PD) pathogenesis. However, the normal/pathological role of α-synuclein remains unidentified. Studies exploring its function are limited as current transgenic mouse models do not fully recapitulate PD pathology. This thesis reports the functional characterisation of two novel synuclein-based mouse models. I report the molecular and functional characterisation of transgenic mouse lines with wild-type or A30P-mutant human α-synuclein genomic locus carried within a bacterial artificial chromosome. SNCA-A30P⁺Snca-/- mice exhibited a highly physiologically relevant expression pattern of the transgene, including expression in the substantia nigra pars compacta (SNpc) and a specific, age-related loss of TH⁺ cells in the SNpc, the key region of preferential cell loss in PD, compared with non-transgenic Snca -/- littermate controls. Analysis of dopamine signalling using fast-scan cyclic voltammetry (FCV) showed young adult SNCA-A30P⁺Snca-/- mice had an approximately 20% lower evoked extracellular dopamine concentration ([DA]o) compared with non-transgenic Snca -/- littermate controls, a decrease specific to the dorsal striatum. This difference diminished with age and could not be attributed to changes in dopamine reuptake/content. I detail the behavioural and neurochemical phenotype in mice lacking all three synucleins (α/β/γ). Functional compensation between synucleins emphasises the importance of studying their effects by removing all three proteins simultaneously. Triple-null mice exhibited hyperactivity in a novel environment reminiscent of a hyperdopaminergic-like phenotype, but showed no phenotype in anxiety or motor related tests. FCV revealed synuclein triple-null mice had a two-fold increase in [DA]o, specific to the dorsal striatum and not attributable to changes in dopamine reuptake/content, changes in striatal nicotinic receptor activity nor calcium-dependent changes in dopamine exocytosis. Together, the analysis from these two novel mouse models reveal synucleins play an important role in altering synaptic function in the dorsal striatum (the region selectively affected in PD) and contributes to growing evidence suggesting synucleins are negative regulators of synaptic dopamine release.

The striatum represents the main input nucleus of the basal ganglia, a system of subcortical nuclei critically involved into motor control and motivational processes and altered in several conditions such as Parkinson’s diseases or drug addiction. The projection neurons of the striatum are GABAergic (γ-aminobutyric acid) medium-sized spiny neurons (MSNs), and account for the large majority of striatal neurons, while interneurons represent about 10% of striatal cells. The MSNs are subdivided into two subpopulations that form two main efferent pathways: the striatonigral and striatopallidal neurons. The striatonigral MSNs project to the entopeduncular nucleus (EP) and substancia nigra pars reticulata (SNr) (direct pathway) and co-express dopamine D1 receptors (D1R) and substance P neuropeptide (SP). On the other hand, striatopallidal MSNs project to the lateral globus pallidus (LGP) (indirect pathway) and co-express dopamine D2 receptor (D2R), adenosine A2A receptor (A2AR) and enkephalin (Enk). The D1R striatonigral and D2R striatopallidal MSNs are equal in number and shape and are mosaically distributed through all the striatum. The dorsal striatum is mainly involved in motor control and learning while the ventral striatum is crucial for motivational processes. In view of the still debating respective functions of projection D2R-striatopallidal and D1R-striatonigral neurons and striatal interneurons, both in motor control and learning of skills and habits but also in more cognitive processes such as motivation, we were interested in the development of models allowing the removal of selective striatum neuronal populations in adult animal brain. Because of the mosaical organisation of the striatum, a targeting of specific neuronal type, with techniques such as chemical lesions or surgery, is still impossible. Taking advantage of new transgenic approaches, the goal of the present work was to generate and/or to initiate the characterization of genetic models in which a selective subtype of striatal neuron can be ablated in an inducible way. We used a transgenic approach in which mice express a monkey diphtheria toxin (DT) receptor (DTR) in D2R-striatopallidal or D1R-striatonigral neurons. Local stereotactic injections of DT can then induce selective neuronal ablation in functionally different striatal areas.

We first investigated functions of D2R-striatopallidal neurons in motor control and drug reinforcement by their selective ablation in the entire striatum or restricted to the ventral striatum. This DTR strategy produced selective D2R striatopallidal MSN ablation with integrity of the other striatal neurons as well as the striatal dopaminergic function. D2R MSN ablation in the entire striatum induced permanent hyperlocomotion while ventral striatum-restricted ablation increased amphetamine place preference.

We next compared respective roles of D2R-striatopallidal and D1R-striatonigral neurons in motor control and skill learning in functionally different striatum subregions.

Finally, to target nitrergic interneurons of the striatum, we developed a bacterial artificial chromosome genetic strain in which the cre-recombinase expression is under the control of the neuronal nitric oxide gene promoter.

Altogether, those results show that DTR expression and DT local injections is an efficient and flexible strategy to ablate selective striatum neuronal types with spatial resolution. We provide the first direct experimental evidences that D2R striatopallidal neurons inhibit both locomotor and drug-reinforcement processes and that D2R and D1R MSNs in different striatum subregions have distinct functions in motor control and motor skill learning. Those results strongly support a cell-type and topographic functional organization of the striatum and underscore the need for characterization of the specific cellular and molecular modifications that are induced in D2R and D1R MSNs during drug-reinforcement or procedural learning.

Doctorat en Sciences médicales
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The experiment tested the hypothesis that 192 IgG-saporin lesions of the nucleus basalis magnocellularis (NBM) in rats would impair performance in a biconditional visual discrimination task, which requires configural association learning. Experiment used 22 male Long-Evan rats (Harlan Sprague-Dawley). Behavioral testing was conducted in two identical T-mazes. Rats were randomly assigned to either a bilateral 192 IgG-saporin lesion group (n = 10) or to a control group (n = 12). Results support the hypothesis that NBM is critically involved in configural but not simple association learning and suggest that NBM may be involved more generally in cognitive flexibility.

Decision-making is necessary to adapt to the variable environment in everyday life. During this process, our goal is to select the most beneficial course of action in order to obtain the best outcome, to develop efficient choice strategies. That is, estimating the probability to obtain any of the available outcomes as well as their value. Moreover, poor decision-making ability is a common symptom to several psychiatric disorders, such as pathological gambling, depression, schizophrenia and bipolar disorder.The cognitive and emotional mechanisms controlling decision-making processes depend, among others, on the striatum, Basal Ganglia’s main input nucleus. The striatum is divided into the dorsal striatum, responsible for motor and cognitive control that initiate actions (Dorsomedial Striatum, DMS) and generate habits (Dorsolateral Striatum, DLS), and Nucleus Accumbens (NAc) which manages reward and the influence of motivation on motor behavior. A2A-expressing and D1-expressing medium spiny neurons (iMSNs and dMSNs, respectively), accounting for 95% of striatal neurons act in coordination to generate adaptive behavioral responses. It has been shown that imbalanced activity between these two populations leads to abnormal behaviors: overactivation of striatonigral neurons promotes an increased locomotion as well as a higher sensitivity for reward, whereas overactivation of striatopallidal neurons produces the exact opposite effects. However, the specific contributions to decision-making of these two populations in each striatal territory remains unclear. Here, we made use of a chemogenetic (DREADD) tool to manipulate striatal projection neurons’ activity within each specific striatal area and tested their role in a decision-making operant protocol. To do so, we used two different mouse models that allowed us to target specifically iMSNs (A2A-Cre mice) or dMSNs (D1-Cre mice) and induce neuronal-specific expression of the hM3Dq DREADD receptor. CNO-mediated activation of these receptors led to neuronal activation. Then, we tested DREADD-dependent activation of MSNs during the Iowa Gambling Task (IGT), a test used to assess the influence of different rewards on choice and to evaluate the ability of mice to develop advantageous choice strategies. We found an exclusive role of DMS’ dMSNs in controlling choice preference, as DREADD-induced activation of these neurons produced a loss of preference. Manipulations of MSNs in other striatal areas led to altered task performance without affecting choice preference.These results contribute to a better understanding of the role of the striatum on decision-making and moreover, suggest the existence of a high level of functional specialization in this area, a fact that could be explained by the local circuits in which each MSN population is involved.
Doctorat en Sciences biomédicales et pharmaceutiques (Médecine)
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Μελέτη της υπόθεσης ότι τα βασικά γάγγλια, μία κεντρική δομή του εγκεφάλου, συμμετέχει ενεργά στην επιλογή της κατάλληλης δράσης. Προσπάθεια μοντελοποίησης της λειτουργίας των βασικών γαγγλίων κια προσομοίωση παθολογικών καταστάσεων του ανθρώπινου εγκεφάλου.
Stydy of the hypothesis that the basal ganglia, a major structure in the mamalian brain, plays a crusial role in the selection of the appropriate action. In this direction we try to simulate the functionality of the basal ganglia as well the pathophysiological conditions (Parkinson, Huntington disease) related to them.

Deep brain stimulation (DBS) is used to treat the motor symptoms of advanced Parkinson's disease (PD). Although this therapy has been widely applied, the mechanisms of action underlying its effectiveness remain unclear. The goal of this dissertation was to investigate the mechanisms underlying the effectiveness of subthalamic nucleus (STN) DBS by quantifying changes in neuronal activity in the basal ganglia during both effective and ineffective DBS.

Two different approaches were adopted in this study. The first approach was the unilateral 6-hydroxydopamine (6-OHDA) lesioned rat model. Using this animal model, we developed behavioral tests that were used to quantify the effectiveness of DBS with various frequencies and temporal patterns. These changes in behavior were correlated with changes in the activity of multiple single neurons recorded from the globus pallidus externa (GPe) and substantia nigra reticulata (SNr). The second approach was a computational model of the basal ganglia-thalamic network. The output of the model was quantified using an error index that measured the fidelity of transmission of information in model thalamic neurons. We quantified changes in error index as well as neural activity within the model GPe and globus pallidus interna (GPi, equivalent to the SNr in rats).

Using these two approaches, we first quantified the effects of different frequencies of STN DBS. High frequency stimulation was more effective than low frequency stimulation at reducing motor symptoms in the rat, as well as improving the error index of the computational model. In both the GPe and SNr/GPi from the rat and computational model, pathological low frequency oscillations were present. These low frequency oscillations were suppressed during effective high frequency DBS but not low frequency DBS. Furthermore, effective high frequency DBS generated oscillations in neural firing at the same frequency of stimulation. Such changes in neuronal firing patterns were independent of changes in firing rates.

Next, we investigated the effects of different temporal patterns of high frequency stimulation. Stimulus trains with the same number of pulses per second but different coefficients of variation (CVs) were delivered to the PD rat as well as PD model. 130 Hz regular DBS was more effective than irregular DBS at alleviating motor symptoms of the PD rat and improving error index in the computational model. However, the most irregular stimulation pattern was still more effective than low frequency stimulation. All patterns of DBS were able to suppress the pathological low frequency oscillations present in the GPe and SNr/GPi, but only 130 Hz stimulation increased high frequency 130 Hz oscillations. Therefore, the suppression of pathological low frequency neural oscillations was necessary but not sufficient to produce the maximum benefits of DBS.

The effectiveness of regular high frequency STN DBS was associated with a decrease in pathological low frequency oscillations and an increase in high frequency oscillations. These observations indicate that the effects of DBS are not only mediated by changes in firing rate, but also involve changes in neuronal firing patterns within the basal ganglia. The shift in neural oscillations from low to high frequency during effective STN DBS suggests that high frequency regular DBS suppresses pathological firing by entraining neurons to the stimulus pulses.

Therefore, results from this dissertation support the hypothesis that the underlying mechanism of effective DBS is its ability to entrain and regularize neuronal firing, therefore disrupting pathological patterns of activity within the basal ganglia.

Dissertation

Dissertação de Mestrado em Bioquímica apresentada à Faculdade de Ciências e Tecnologia
A doença de Willis-Ekbom, também conhecida como síndrome da perna inquieta, é uma doença neurológica caracterizada pelos seus sintomas. Os portadores experienciam uma inquietante necessidade de estar em movimento, mesmo quando se encontram em repouso sendo que, há um agravamento dos sintomas no período noturno. Outro aspeto clínico é o experienciamento de pequenos episódios de despertares durante o sono, que normalmente precedem o início do movimento das pernas. Dado o estado de excitamento, acredita-se que a nível neurofisiológico exista um hiper-excitamento do sistema nervoso, mais concretamente, associado a uma hiperexcitabilidade dos terminais glutamatérgicos no estriado. A condição mais bem estudada e mais associada a WED é a deficiência de ferro que, pode não ser evidente sistemicamente e ocorrer localmente, como no cérebro. Quer a deficiência de ferro (e a anemia consequente) quer a WED são mais prevalentes em mulheres, que sublinha a necessidade de comparar o efeito de deficiência de ferro no estriado de roedores laboratoriais de ambos os generos. O estriado é a principal estrutura de entrada de estímulos excitatórios dos gânglios da base e encontra-se envolvido tanto em processos motores como cognitivos. A neurotransmissão de glutamato é modulada por vários neurotransmissores como o GABA e neuromoduladores como a dopamina, adenosina, opioides, acetilcolina e endocanabinóides. A existência de uma perturbação nestes sistemas de modulação pode levar a uma variedade de doenças neurológicas. Muitos estudos apontam para a existência de alterações a nível do sistema adenosinérgico em pacientes e modelos de BID. Recentemente foi descrito, pelos nossos colaboradores, um estado hipoadenosinérgico causada por um aumento na densidade dos recetores de adenosina do tipo A2A e/ou uma diminuição dos recetores A1. Assim, recorrendo a um modelo animal (foram alvo de estudo ambos machos e fêmeas, de modo a avaliar se existe um fenótipo específico entre géneros) sujeito a uma deficiência de ferro através da comida. Primeiramente, os animais foram sujeitos a testes comportamentais para avaliar a atividade locomotora, ansiedade e memória. Mais tarde, o cérebro foi recolhido e realizou-se a purificação de sinaptossomas do estriado para realizar o objetivo desta tese: avaliar alterações de densidade dos recetores de adenosina, tal como a percentagem de co-localização entre ambos, em terminais gutamatérgicos cortico-estriatais e tálamo-estriatais e, em terminais colinérgicos, recorrendo à técnica de “sinaptometria” de fluxo. Para além destes, também foram avaliadas alterações dos recetores µ-opióides (MOR), uma vez que agonistas do MOR são eficazes no controlo de sintómas sensorimotórios dos pacientes. e canabinóide do tipo 1 (CB1R), o recetor que é capaz de modular o funcionamento tal dos receptores adenosinérgicos como do MOR via heteromerização, tal como a sua percentagem de co-localização.Uma vez que a deficiência de ferro cerebral (DFC) foi induzido no dia 21 pós-natal, ou seja, depois de maioritariamente concluir o desenvolvimento cerebral, já não foram observadas alterações na locomoção e na habituação dos animais com BID no teste “open field”, tal como no comportamento ansioso realizado no “elevated-plus maze”.No entanto, a técnica da sinaptometria de fluxo revelou fortes tendências para uma redução na densidade dos terminais positivos para MOR e um aumento nos terminais equipados com o recetor facilitatório A2A. Tudo junto, observou-se pela primeira vez um disbalanço no controlo inibitório e facilitatório no nível sináptico em terminais nervosos individualmente identificados, que certamente contribuirá ao nosso entendimento sobre o patomecanismo e a farmacoterapia adequada da WED.
Willis-Ekbom's disease, also known as restless leg syndrome, is a neurological disease characterized by an unsettling need of the patients to move their legs and sometimes arms, even when they are at rest. Nocturnal aggravation of symptoms strongly impar sleep quality. It is believed that at the neurophysiological level, there is a hyperarousal of the nervous system, which is associated with a hyperexcitability of the corticostriatal glutamatergic terminals in the striatum. Iron deficiency is as a major comorbidity for WED has been recognized. Iron deficiency may be restricted to the nervous tissue, which is called brain iron deficiency (BID). Both iron deficiency (and consequent anemia) and WED are more prevalent in women, which underlines the need to compare the effect of iron deficiency on the striatum of laboratory rodents of both genders.The striatum is the main input structure for excitatory stimuli from the basal ganglia and is involved in both motor and cognitive processes. Glutamatergic corticostriatal neurotransmission is modulated by several neurotransmitters such as GABA and neuromodulators including dopamine, adenosine, opioids, acetylcholine and endocannabinoids. Impaired neuromodulation at corticostriatal synapses can lead to a variety of neurological diseases. Many studies point to the existence of changes in the adenosinergic system in patients and models of BID. Recently, a hypoadenosinergic state was described by our collaborators, caused by an increase in the density of type A2A adenosine receptors and a decrease in A1 receptor density, or both.Thus, here I studied the possible presynaptic changes in striatal glutamatergic as well as cholinergic nerve terminals, isolated from young adult rats, after completing a four-week diet with iron poor chow (BID rats), or normal chow (controls). Both males and females were studied in order to purported gender-specific phenotypes.First, the animals were subjected to behavioral tests to evaluate locomotor activity, anxiety and memory. Subsequently, the animals were euthanized, and their brains were collected to prepare purified striatal synaptosomes. My aims were to evaluate changes in the density of glutamatergic and cholinergic nerve terminals endowed with adenosine receptors, as well as µ-opioid receptors (MOR) – since MOR agonists are efficacious in controlling patients' sensorimotor symptoms, and finally, cannabinoid CB1 receptors, because it is capable of modulating the functioning of both adenosine receptors and MOR via heteromerization.Since BID was induced on the 21st postnatal day, that is, after that the brain development is largely completed, overt behavioral changes in locomotion, habituation and anxiety-like behaviour of BID rats were not observed in the open field and elevated-plus maze tests. Spatial working memory in the Y-maze spontaneous alternation test also remained intact in the BID rats. However, the technique “flow synaptometry” revealed strong tendencies towards a reduction in the density of MOR-positive terminals and an increase in A2AR-positive terminals. Alltogether, for the first time an imbalance has been documented in inhibitory and facilitatory neuromodulation at the synaptic level in individually identified nerve terminals, which will certainly contribute to our understanding of the pathomechanism and the adequate pharmacotherapy of WED.

Indiana University-Purdue University Indianapolis (IUPUI)
Physical and biological phenomena that involve oscillations on multiple time scales attract attention of mathematicians because resulting equations include a small parameter that allows for decomposing a three- or higher-dimensional dynamical system into fast/slow subsystems of lower dimensionality and analyzing them independently using geometric singular perturbation theory and other techniques. However, in most life sciences applications observed dynamics is extremely complex, no small parameter exists and this approach fails. Nevertheless, it is still desirable to gain insight into behavior of these mathematical models using the only viable alternative – ad hoc computational analysis. Current dissertation is devoted to this latter approach. Neural networks in the region of the brain called basal ganglia (BG) are capable of producing rich activity patterns. For example, burst firing, i.e. a train of action potentials followed by a period of quiescence in neurons of the subthalamic nucleus (STN) in BG was shown to be related to involuntary shaking of limbs in Parkinson’s disease called tremor. The origin of tremor remains unknown; however, a few hypotheses of tremor-generation were proposed recently. The first project of this dissertation examines the BG-thalamo-cortical loop hypothesis for tremor generation by building physiologically-relevant mathematical model of tremor-related circuits with negative delayed feedback. The dynamics of the model is explored under variation of connection strength and delay parameters in the feedback loop using computational methods and data analysis techniques. The model is shown to qualitatively reproduce the transition from irregular physiological activity to pathological synchronous dynamics with varying parameters that are affected in Parkinson’s disease. Thus, the proposed model provides an explanation for the basal ganglia-thalamo-cortical loop mechanism of tremor generation. Besides tremor-related bursting activity BG structures in Parkinson’s disease also show increased synchronized activity in the beta-band (10-30Hz) that ultimately causes other parkinsonian symptoms like slowness of movement, rigidity etc. Suppression of excessively synchronous beta-band oscillatory activity is believed to suppress hypokinetic motor symptoms in Parkinson’s disease. Recently, a lot of interest has been devoted to desynchronizing delayed feedback deep brain stimulation (DBS). This type of synchrony control was shown to destabilize synchronized state in networks of simple model oscillators as well as in networks of coupled model neurons. However, the dynamics of the neural activity in Parkinson’s disease exhibits complex intermittent synchronous patterns, far from the idealized synchronized dynamics used to study the delayed feedback stimulation. The second project of this dissertation explores the action of delayed feedback stimulation on partially synchronous oscillatory dynamics, similar to what one observes experimentally in parkinsonian patients. We employ a computational model of the basal ganglia networks which reproduces the fine temporal structure of the synchronous dynamics observed experimentally. Modeling results suggest that delayed feedback DBS in Parkinson’s disease may boost rather than suppresses synchronization and is therefore unlikely to be clinically successful. Single neuron dynamics may also have important physiological meaning. For instance, bistability – coexistence of two stable solutions observed experimentally in many neurons is thought to be involved in some short-term memory tasks. Bistability that occurs at the depolarization block, i.e. a silent depolarized state a neuron enters with excessive excitatory input was proposed to play a role in improving robustness of oscillations in pacemaker-type neurons. The third project of this dissertation studies what parameters control bistability at the depolarization block in the three-dimensional conductance-based neuronal model by comparing the reduced dopaminergic neuron model to the Hodgkin-Huxley model of the squid giant axon. Bifurcation analysis and parameter variations revealed that bistability is mainly characterized by the inactivation of the Na+ current, while the activation characteristics of the Na+ and the delayed rectifier K+ currents do not account for the difference in bistability in the two models.

Η παρούσα διατριβή ασχολήθηκε με τη μελέτη της έκφρασης των υπομονάδων των υποδοχέων του γλουταμινικού οξέος και του γ-αμινοβουτυρικού οξέος (GABA) στα βασικά γάγγλια και τον φλοιό των εγκεφαλικών ημισφαιρίων του μυός weaver. Παράλληλα, μελετήθηκε η έκφραση των νευροπεπτιδίων εγκεφαλίνης και δυνορφίνης στα βασικά γάγγλια του μυός weaver. O μυς weaver χαρακτηρίζεται από προοδευτική, γενετικά επαγόμενη εκφύλιση των ντοπαμινεργικών κυττάρων του μεσεγκεφάλου, κυρίως αυτών οι οποίοι καταλήγουν στο ραβδωτό σώμα. Για αυτόν τον λόγο, θεωρείται ένα καλό μοντέλο της νόσου Parkinson και η μελέτη των νευροχημικών μεταβολών που συμβαίνουν στον εγκέφαλο του παραπάνω μυός, αποτελεί πολύτιμο εργαλείο για τη διερεύνηση των παθογενετικών μηχανισμών της νόσου. Mε την τεχνική του υβριδισμού in situ, προσδιορίστηκαν τα επίπεδα mRNA των υπομονάδων z1, ε1 και ε2 του υποδοχέα NMDA, των υπομονάδων KA2 και GluR6 του υποδοχέα καϊνικού οξέος, των υπομονάδων α1, α2, α4, β2 και β3 του υποδοχέα GABAA, καθώς και των πρόδρομων πολυπεπτιδίων προ-προεγκεφαλίνη και προδυνορφίνη. Η μελέτη πραγματοποιήθηκε σε φυσιολογικούς μύες (+/+) και μύες weaver (wv/wv), στις ηλικίες των 26 ημερών, 3 μηνών και 6 μηνών μετά τη γέννηση. Όσον αφορά στους υποδοχείς του γλουταμινικού οξέος, τα αποτελέσματά μας υπέδειξαν αύξηση στην έκφραση των υπομονάδων z1, ε2, ΚΑ2 και GluR6 στο ραβδωτό σώμα των μυών weaver, σε σχέση με τους φυσιολογικούς. Η αύξηση στο mRNA των υπομονάδων z1, ε2 και GluR6 παρατηρήθηκε μόνο στην ηλικία των 6 μηνών, ενώ το mRNA της υπομονάδας KA2, παρουσίασε αύξηση και στις τρεις ηλικίες που μελετήθηκαν. Οι αυξήσεις της έκφρασης των υπομονάδων z1, ε2, ΚΑ2 και GluR6 συμφωνούν και πιθανόν εξηγούν τις αυξήσεις στα επίπεδα των θέσεων δέσμευσης για τους υποδοχείς NMDA και μη-NMDA, οι οποίες έχουν βρεθεί από παλαιότερες μελέτες του εργαστηρίου μας στο ραβδωτό σώμα των μυών weaver ηλικίας 6 μηνών. Με βάση βιβλιογραφικά δεδομένα, υποστηρίζουμε ότι η καθυστερημένη αύξηση στην έκφραση των υπομονάδων z1, ε2 και GluR6 κατά πάσα πιθανότητα συντελείται μέσω επαγωγής του μεταγραφικού παράγοντα ΔfosB, σε απόκριση προς τη μείωση της ντοπαμίνης. Στον σωματοαισθητικό φλοιό των μυών weaver ηλικίας 26 ημερών, παρατηρήθηκε αύξηση στην έκφραση των υπομονάδων z1, ε1, ε2 και KA2, η οποία θα μπορούσε να οφείλεται στη μειωμένη θαλαμοφλοιϊκή γλουταμινεργική είσοδο. Όσον αφορά στους υποδοχείς GABAA, παρατηρήθηκε αύξηση στα επίπεδα mRNA των υπομονάδων α4 και β3, στο ραβδωτό σώμα των μυών weaver ηλικίας 6 μηνών, η οποία συμφωνεί και μπορεί να εξηγήσει την αύξηση στα επίπεδα των θέσεων δέσμευσης για τους υποδοχείς GABAA, η οποία έχει βρεθεί σε προηγούμενη μελέτη του εργαστηρίου μας, στο ραβδωτό σώμα των μυών weaver ηλικίας 6 μηνών. Σκοπεύουμε να ελέγξουμε την πιθανότητα, η αύξηση της έκφρασης της υπομονάδας α4, να υποδεικνύει μία αύξηση του αριθμού των εξωσυναπτικών υποδοχέων GABAA στους νευρώνες προβολής του ραβδωτού σώματος. Στην ωχρά σφαίρα των μυών weaver ηλικίας 6 μηνών, παρατηρήθηκε μείωση των επιπέδων mRNA των υπομονάδων α1 και β2, υποδεικνύοντας μία μείωση του αριθμού των υποδοχέων GABAA, η οποία ήταν αναμενόμενη, λόγω της αυξημένης GABAεργικής εισόδου στην εν λόγω εγκεφαλική περιοχή του μυός weaver. Στον σωματοαισθητικό φλοιό, παρατηρήθηκε μείωση στην έκφραση των υπομονάδων α2 και β2 και ταυτόχρονα αύξηση στην έκφραση των υπομονάδων α4 και β3. Με βάση βιβλιογραφικά δεδομένα, προτείνουμε ότι οι μεταβολές αυτές μπορεί να αντανακλούν μείωση στον αριθμό των συναπτικών και αύξηση στον αριθμό των εξωσυναπτικών υποδοχέων GABAA, σε απόκριση προς τη μειωμένη GABAεργική είσοδο προς τους νευρώνες του σωματοαισθητικού φλοιού του μυός weaver. Όσον αφορά στην έκφραση των πολυπεπτιδίων, το mRNA της προ-προεγκεφαλίνης, παρουσίασε αύξηση στο ραβδωτό σώμα των μυών weaver, μόνο στην ηλικία των 6 μηνών, ενώ το mRNA της προδυνορφίνης, παρουσίασε μείωση στην παραπάνω περιοχή, στην ηλικία των 26 ημερών και αύξηση στις μεγαλύτερες ηλικίες. Σύμφωνα με τα βιβλιογραφικά δεδομένα υποστηρίζουμε ότι: α) η καθυστερημένη αύξηση της έκφρασης της προ-προεγκεφαλίνης στο ραβδωτό σώμα του μυός weaver, οφείλεται στη μείωση της τονικής ανασταλτικής ρυθμιστικής δράσης της ντοπαμίνης στην έκφραση του εν λόγω γονιδίου και πιθανώς συντελείται μέσω του μεταγραφικού παράγοντα ΔfosB, β) ο παραπάνω μεταγραφικός παράγοντας είναι κατά πάσα πιθανότητα υπεύθυνος και για την καθυστερημένη επαγωγή της έκφρασης της προδυνορφίνης στο ραβδωτό σώμα των μυών weaver και γ) η μείωση του παραπάνω mRNA στην ηλικία των 26 ημερών οφείλεται στη μείωση της τονικής διεγερτικής δράσης της ντοπαμίνης στην έκφραση του εν λόγω γονιδίου. Τέλος, το γεγονός ότι οι μεταβολές των mRNA των διαφόρων υπομονάδων και νευροπεπτιδίων δεν ήταν οι ίδιες στις διάφορες ηλικίες που μελετήθηκαν υποδεικνύει ότι κατά την πρόοδο της ντοπαμινεργικής εκφύλισης των ντοπαμινεργικών νευρώνων του μεσεγκεφάλου διαφορετικοί μηχανισμοί ευθύνονται για την πρόκληση των αλλαγών στην έκφραση των υπό μελέτη γονιδίων.
In the present study we investigated the expression of the subunits of glutamate and γ-aminobutyric acid (GABA) receptors in basal ganglia and cerebral cortex of the weaver mouse. We also studied the expression of striatal neuropeptides, which are important neuromodulators of the synaptic transmission in the basal ganglia circuitry. The weaver mouse is characterized by a progressive, genetically induced degeneration of the mesencephalic dopaminergic neurons, especially those that project to the striatum. For this reason, the weaver mouse is a useful model for clarifying the pathogenetic mechanisms that underly Parkinson’s disease. Using the in situ hybridization method, the mRNA levels of the ΝΜDA subunits z1, ε1 and ε2, the kainate subunits KΑ2 and GluR6, the GABAA subunits α1, α2, α4, β2 and β3, as well as the mRNA levels of the precursor polypeptides pre-proenkephalin and prodynorphin, were estimated. The study was performed using wild-type (+/+) and weaver mice (wv/wv) of the following ages: 26 days, 3 months and 6 months. Concerning the glutamate receptors, an increase in the mRNA levels of z1, ε2, KA2 and GluR6 subunits was indicated in the weaver striatum, compared to the wild type. The z1, ε2 and GluR6 mRNA increases were observed only at the age of 6 months, whereas the KA2 mRNA increase was observed at all three ages studied. The increases in z1, ε2, ΚΑ2 and GluR6 mRNA expression are in agreement and probably explain the increased levels of ΝΜDA- and non-NMDA-sensitive binding sites that we had previously found in the 6 months old weaver striatum. Based on bibliographic data, we suggest that the delayed increases in z1, ε2 and GluR6 mRNA levels, are probably mediated by the delayed induction of the ΔfosB transcription factor, in response to the reduction of striatal dopamine levels. In the somatosensory cortex of 26 day old weaver mice, an increase in the levels of z1, ε1, ε2 and ΚΑ2 mRNAs was observed. The above increases can be attributed to the decreased thalamocortical glutamatergic imput. Concerning the GABAA receptors, the observed increases of the α4 and β3 mRNA levels in the 6 months old weaver striatum are in agreement and probably explain the increased levels of GABAA binding sites that we had previously found in the 6 months old weaver striatum. We are going to test the hypothesis, that the α4 mRNA increase might indicate an increase in the number of extrasynaptic GABAA receptors in striatal projection neurons. In the 6 months old weaver globus pallidus, the observed decrease in α1 and β2 mRNA levels was expected, since the GABAergic transmission is increased in the above region of the weaver brain. In the weaver somatosensory cortex, a decrease in the α2 and β2 mRNA levels and an increase in the α4 and β3 mRNA levels were observed. Based on bibliographic data, we suggest that the above alterations probably indicate a differential regulation of the synaptic versus extrasynaptic cortical GABAA receptors, in response to the decreased GABAergic presynaptic input to the weaver cortical neurons. Concerning the expression of the striatal neuropeptides, the pre-proenkephalin mRNA was increased in the weaver striatum, only at the age of 6 months. In contrast, prodynorphin mRNA was decreased in the 26 day old weaver striatum, whereas it was increased in the 3 and 6 months old weaver striatum. Based on bibliographic data, we suggest that: a) the delayed increase in the expression of pre-proenkephalin could be caused by the reduction of the tonic dopaminergic inhibitory control on the expression of the above gene in the dopamine-depleted weaver striatum and is probably mediated by the ΔfosB transcription factor; b) the above transcription factor could be responsible for the delayed induction of the prodynorphin expression in the weaver striatum as well, and c) the decrease of prodynorphin mRNA in the 26 day old weaver striatum could be attributed to the reduction of the dopaminergic stimulatory control on the expression of the above gene. Finally, the different pattern of expression alterations among the three ages studied indicates that distinct mechanisms are responsible for the observed changes, during the progress of the dopaminergic degeneration of the weaver brain.

Η παρούσα εργασία αφορά στη μελέτη της ανταγωνιστικής αλληλεπίδρασης των Α1/D1 υποδοχέων στο επίπεδο έκφρασης του πρώιμου γονιδίου zif/268 (δείκτης νευρωνικής δραστηριότητας) και της in vivo μεταγωγής σήματος των Α1 και Α2Α υποδοχέων αδενοσίνης κάτω από τη ντοπαμινεργική απονεύρωση στο μυ weaver. Ο μυς weaver αποτελεί ένα γενετικό μοντέλο ντοπαμινεργικής απονεύρωσης, η οποία συμβαίνει σταδιακά, έτσι ώστε το μοντέλο αυτό να προσομοιάζει τη Νόσο Πάρκινσον (ΝΠ) στον άνθρωπο. Στο πρώτο στάδιο της μελέτης προέκυψε το ενδιαφέρον αποτέλεσμα ότι με την ταυτόχρονη ενεργοποίηση των Α1 και D1 υποδοχέων παρατηρήθηκε η αναμενόμενη ανταγωνιστική αλληλεπίδραση (ενδεχομένως μέσω σχηματισμού του ετεροδιμερούς), ενώ με την ενεργοποίηση μόνο των Α1 υποδοχέων στους weaver μύες παρατηρήθηκε αυξημένη ενεργοποίηση των νευρώνων του ραβδωτού σώματος και συγκεκριμένων περιοχών του εγκεφαλικού φλοιού. Η ενεργοποίηση αυτή ήταν μη αναμενόμενη, δεδομένου ότι οι Α1 υποδοχείς (A1Rs) είναι συζευγμένοι με Gi πρωτεΐνες και καταστέλλουν τη μεταγωγή σήματος που οδηγεί στην επαγωγή του zif/268 μέσω του D1R/Gs/cAMP/PKA/pDARPP-32/pCREB μονοπατιού. Η ακόλουθη διερεύνηση του μηχανισμού έδειξε ότι η Α1R-επαγόμενη ενεργοποίηση του zif/268 καταστέλλεται από τον ειδικό ανταγωνιστή των Α2Α υποδοχέων αδενοσίνης (A2ARs) ZM241385 και από τον ειδικό αγωνιστή των D2 υποδοχέων ντοπαμίνης Quinpirole, υποδεικνύοντας την ενεργοποίηση των Α2ΑRs και άρα ενεργοποίηση της έμμεσης οδού. Το αποτέλεσμα αυτό επιβεβαιώθηκε, δεδομένου ότι η διέγερση των Α1Rs προκάλεσε αύξηση της έκφρασης του mRNA της εγκεφαλίνης, αλλά όχι της δυνορφίνης, που αποτελούν δείκτες ενεργοποίησης της έμμεσης και της άμεσης οδού, αντίστοιχα. Το γεγονός ότι ο αγωνιστής των Α1Rs δεν προκαλεί στα φυσιολογικά ζώα ενεργοποίηση του zif/268 mRNA υποδεικνύει την υπερευαίσθητη απόκριση των Α2ARs. Η μελέτη της υπερευαίσθητης αυτής απόκρισης στο μυ weaver έγινε με τη διερεύνηση της μεταγωγής σήματος μετά από in vivo ενεργοποίηση των Α2ΑRs: α) του καθιερωμένου μονοπατιού Α2ΑRs/Gs/AC/cAMP/PKA/pDARPP-32/pCREB, το οποίο οδηγεί στη επαγωγή του zif/268 και β) του μονοπατιού των ΜΑΡΚ. Τα αποτελέσματα έδειξαν αυξημένα βασικά επίπεδα φωσφορυλίωσης της DARPP-32 στη θέση Thr-34. Τα αυξημένα επίπεδα της φωσφορυλιωμένης DARPP-32 πολλαπλασιάζουν τη δράση της ΡΚΑ και άρα διευκολύνουν τη μεταγωγή σήματος μέσω Α2ΑRs/Gs/AC/cAMP/PKA/pDARPP-32/pCREB μονοπατιού. Επομένως, η υπερευαίσθητη απόκριση των Α2ΑRs κάτω από την έλλειψη ντοπαμίνης στο μυ weaver φαίνεται να οφείλεται στα αυξημένα ενδογενή επίπεδα της φωσφορυλιωμένης DARPP-32. Ενδιαφέρον παρουσιάζει το γεγονός ότι τα βασικά επίπεδα φωσφορυλίωσης των πρωτεϊνών ERK1/2(MAPK44/42) είναι αυξημένα στον μυ weaver, αλλά μειώνονται σημαντικά μετά από την ενεργοποίηση των Α2ΑRs. Δεν γνωρίζουμε το μηχανισμό μέσω του οποίου αυξάνονται τα ενδογενή επίπεδα των ERK1/2 και πρέπει να διερευνηθεί περαιτέρω. Το συμπέρασμα όμως που εξάγεται είναι ότι κάτω από τη ντοπαμινεργική απονεύρωση η μεταβίβαση σήματος μέσω των Α2ΑRs δεν ενεργοποιεί την οδό των MAP κινασών. Στην παρούσα in vivo μελέτη αναδεικνύεται ο ρόλος των Α1 και Α2Α υποδοχέων στην λειτουργία των βασικών γαγγλίων κάτω από τη ντοπαμινεργική απονεύρωση. Τα αποτελέσματα αυτά έχουν ιδιαίτερη σημασία δεδομένου ότι εμφανίζονται σε ένα γενετικό μοντέλο παρκινσονισμού, στο οποίο η εκφύλιση των ντοπαμινεργικών νευρώνων είναι σταδιακή και προσομοιάζει τη ΝΠ, και όχι οξεία, όπως σε άλλα τοξικά μοντέλα. Επιπλέον, τα αποτελέσματα αυτά παρουσιάζουν ενδεχομένως κλινικό ενδιαφέρον, δεδομένου ότι η ενεργοποίηση της έμμεσης οδού μέσω Α1Rs από την ενδογενή αδενοσίνη θα επιδείνωνε περαιτέρω τις κινητικές δυσλειτουργίες της ΝΠ. Η πληροφορία αυτή, καθώς και η γνώση για την ενισχυμένη μεταγωγή σήματος μέσω των Α2Α υποδοχέων ενισχύουν την πρόταση για χρήση των Α2Α ανταγωνιστών ως αντιπαρκινσονικά φάρμακα. Δεδομένου ότι σήμερα το ενδιαφέρον είναι στραμμένο στη δημιουργία διμερών προσδεμάτων (bivalent ligands) που μπορούν να δρουν ταυτόχρονα σε δύο υποδοχείς, η συγκεκριμένη πληροφορία θα μπορούσε να χρησιμοποιηθεί για μελλοντική δημιουργία φαρμακευτικού σχήματος που να δρα ταυτόχρονα ως αγωνιστής των D2 υποδοχέων ντοπαμίνης και ως ανταγωνιστής των Α2Α υποδοχέων αδενοσίνης.
The present work studied the antagonistic interaction of A1/D1 receptors at the level of mRNA expression of the immediate early gene zif/268 (used as a marker of neuronal function). In parallel we studied the in vivo signal transduction of A1 and A2A adenosine receptors under dopamine deficiency in weaver mutant. The weaver mutant represents the only genetic animal model of gradual nigrostriatal neuron degeneration, which can be characterized as a pathophysiological phenocopy of Parkinson’s Disease. In the first part of the study, the co-activation of A1 and D1 receptors revealed the well-known antagonistic interaction of these receptors (possibly through the formation of A1/D1 heterodimer) in weaver mutant. An interesting result was that the activation of A1 receptors alone did induce zif/268 mRNA expression in stiatal and specific cortical neurons in weaver mutant. This induction was not expected, since A1 receptors are Gi-coupled and suppress the signal transduction pathway that leads to zif/268 induction through AC/PKA/p-DARPP-32/pCREB cascade. Further study, revealed that the A1 receptor-induced zif/268 mRNA expression is counteracted by the A2A receptor selective antagonist ZM241385 and by the D2 receptor selective agonist Quinpirole, suggesting the activation of A2A receptors and thus the activation of the “indirect pathway”. Moreover, A1 receptors activation induced the expression of enkephalin mRNA, but not of dynorphin, which are considered as marker of neuronal activation of the “indirect” and the “direct” pathway, respectively. The fact that the A1 receptor agonist did not induced zif/268 mRNA expression in +/+ animals indicates that under dopamine deficiency the A2A receptors react with a supersensitive response. This response was analyzed in weaver mouse after in vivo A2A receptor activation: a) by examining the classical signal transduction pathway of A2A receptors/AC/PKA/p-DARPP-32/pCREB, which leads to zif/268 expression and b) by studying the MAPK cascade. Results showed increased basal phosphorylation levels of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, MW 32kDa) of Thr-34 in weaver compared to control mice. Increased phosphoThr34-DARPP-32 would amplify the effects of the PKA and thus facilitating the signal transduction through A2A receptors/AC/PKA/p-DARPP-32/pCREB. Therefore, the A2A receptors supersensitive response under dopamine deficiency in weaver mutant seems to be due to elevated endogenous phosphorylation levels of DARPP-32. Interestingly, while the basal phosphorylation levels of ERK1/2 (MAPK44/42) are elevated in weaver mutant, they are significantly reduced after A2A receptor activation. Although we do not know the mechanism through which the endogenous ERK1/2 levels are elevated, the conclusion is that, under dopamine deficiency, A2A receptors do not activate MAPK cascade. The present in vivo study demonstrates the role of A1 and A2A adenosine receptors in the function of basal ganglia under dopamine deficiency. Our results are significant since the expreriments were performed in a genetic parkinsonian model, in which the dopaminergic neurons are gradually degenerated and thus simulate the human PD, and not in an acute toxic model. Moreover, these results could be of possible clinical relevance, since the activation of A1 receptors by endogenous adenosine would exaggerate the motor dysfunctions of PD. Furthermore, the enhanced signal transduction pathway through A2A receptors supports the suggestion that the A2A receptor antagonists as antiparkinsonian agents. Given the well-known A2A/D2 antagonistic interaction, new therapeutical prospectives would involve the development of pharmacological bivalent ligands, which can interact with the A2A/D2 receptors and act simultaneously as A2A receptor antagonists and as D2 receptor agonists.

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder. The earliest and most severe neuropathological change in HD occurs within the striatum. Exogenous excitotoxic lesioning of the rodent and non-human primate (NHP) striatum is used to model HD. Apart from NHPs, no other excitotoxic large animal model of HD has been established. Sheep have the potential to be an important species for modelling neurodegenerative disease, primarily because of neuroanatomical similarities between the sheep and human brain. This thesis describes the development of an excitotoxic sheep model of HD using the excitotoxin, quinolinic acid (QA). QA is an N-methyl-D-aspartate (NMDA) glutamate receptor agonist that produces pathological changes within the striatum that resemble those seen in HD. Sixteen castrated-male, 18 month old, Merino-Border Leicester cross sheep underwent two surgical procedures, four weeks apart, to infuse 75 μl of 180 mM QA (experimental group) or 75 μl of saline (control group) into the left (first surgery) and then the right (second surgery) caudate nucleus of the striatum. Longitudinal magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS) and diffusion tensor imaging (DTI) of the brains of the sheep was performed on a 3-Tesla scanner pre-surgically, one week after the first surgery, five weeks after the first surgery and sixteen weeks after the first surgery to investigate the neuropathological changes that occur in vivo after QA lesioning of the sheep striatum. The phenotypic consequences of lesioning the sheep striatum with QA were investigated using a veterinary neurological examination, dopamine agonist induced rotation and a two-choice discrimination task. The author / investigator was blind to the treatment group. MRI revealed QA-lesion hyperintensity and dilation of the lateral ventricles, consistent with atrophy of the caudate nucleus. MRS and DTI revealed a significant decrease in the neuronal marker N-acetylaspartate (NAA), and in fractional anisotropy (FA) in the acutely-lesioned (one week after surgery) striatae of the QA-lesioned sheep, followed by recovery in NAA and a significant increase in FA in the chronic (five to sixteen weeks) QA-lesioned striatae. NAA and FA changes are consistent with neuronal loss and structural disruption in the acute lesion, followed by recovery of reversibly impaired neurons, structural reorganisation and gliosis in the chronic lesion. Heterogeneous neuronal loss and damage and gliosis were visible on histological analysis of the QA-lesioned sheep striatae, supporting the in vivo MRS and DTI detected changes. Neurological examination of the sheep revealed evidence of laterality and mild hind limb motor paresis in seven out of eight of the QA-lesioned sheep, however the examination was not informative of lesion characteristics. A directional bias was evident in the QA-lesioned sheep during rotation studies. However, the direction and magnitude of bias in individual sheep at any one timepoint varied markedly, making identification of QA-lesioned individuals difficult. There was no difference between the QA-lesioned and saline-treated sheep in performance of the acquisition and reversal phases of the two-choice discrimination task. The behavioural studies described in this thesis were not suitable for comprehensive identification and characterisation of QA lesions in the striatum of sheep. This is the first description of the development of an acute excitotoxic sheep model of HD. The experiments demonstrate that longitudinal analysis of the neuropathological changes in the QA-lesioned sheep striatum is possible using advanced magnetic resonance modalities performed on a clinically relevant 3-Tesla scanner and that neuropathological changes are consistent with HD-like pathology in other species. Furthermore, phenotypic investigation of the QA-lesioned sheep is possible, however more refined methods than those described need to be utilised. The excitotoxic sheep model of HD is clinically relevant HD model with potential for use in disease mechanism and therapy investigations.
Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2020

Indiana University-Purdue University Indianapolis (IUPUI)
Magnetic resonance (MR) spectroscopy is a neuroimaging technique. It is widely used to quantify the concentration of important metabolites in a brain tissue. Imbalance in concentration of brain metabolites has been found to be associated with development of neurological impairment. There has been increasing trend of using MR spectroscopy as a diagnosis tool for neurological disorders. We established statistical methodology to analyze data obtained from the MR spectroscopy in the context of the HIV associated neurological disorder. First, we have developed novel methodology to study the association of marker of neurological disorder with MR spectrum from brain and how this association evolves with time. The entire problem fits into the framework of scalar-on-function regression model with individual spectrum being the functional predictor. We have extended one of the existing cross-sectional scalar-on-function regression techniques to longitudinal set-up. Advantage of proposed method includes: 1) ability to model flexible time-varying association between response and functional predictor and (2) ability to incorporate prior information. Second part of research attempts to study the influence of the clinical and demographic factors on the progression of brain metabolites over time. In order to understand the influence of these factors in fully non-parametric way, we proposed LongCART algorithm to construct regression tree with longitudinal data. Such a regression tree helps to identify smaller subpopulations (characterized by baseline factors) with differential longitudinal profile and hence helps us to identify influence of baseline factors. Advantage of LongCART algorithm includes: (1) it maintains of type-I error in determining best split, (2) substantially reduces computation time and (2) applicable even observations are taken at subject-specific time-points. Finally, we carried out an in-depth analysis of longitudinal changes in the brain metabolite concentrations in three brain regions, namely, white matter, gray matter and basal ganglia in chronically infected HIV patients enrolled in HIV Neuroimaging Consortium study. We studied the influence of important baseline factors (clinical and demographic) on these longitudinal profiles of brain metabolites using LongCART algorithm in order to identify subgroup of patients at higher risk of neurological impairment.
Partial research support was provided by the National Institutes of Health grants U01-MH083545, R01-CA126205 and U01-CA086368