Οι θυρεοειδικές ορμόνες (Τ3 και Τ4) είναι απαραίτητες στην ανάπτυξη, τη διαφοροποίηση και την ωρίμαση του νευρικού συστήματος. H δράση τους έχει συσχετιστεί με αλλαγές στη συμπεριφορά, ψυχικές και κινητικές δυσλειτουργίες.Η ντοπαμινεργική νεύρωση των βασικών γαγγλίων και κυρίως του ραβδωτού, είναι γνωστό ότι, παίζει κεντρικό ρόλο σ’ ένα ευρύ φάσμα κινητικών, γνωστικών και συναισθηματικών λειτουργιών.Ο υποθυρεοειδισμός προκαλεί μορφολογικές διαταραχές σε εγκέφαλο ενήλικα επίμυ, ακριβέστερα μείωση, αξονικής και δενδριτικής ανάπτυξης των νευρώνων του ραβδωτού, και τροποποιεί την ικανότητα δέσμευσης ανταγωνιστών της ντοπαμίνης. Όμως το κυτταρικό και μοριακό υπόβαθρο των ανωτέρω επιδράσεων παραμένει κατά ένα μεγάλο μέρος αδιευκρίνιστο. Κατά την διατριβή αυτή μελετήθηκε η επίδραση του υποθυρεοειδισμού στο ραβδωτό σώμα αρσενικών ενήλικων μυών στην κατάσταση του υποθυρεοειδισμού, προκειμένου να διευκρινισθεί περαιτέρω το υπόβαθρο των δράσεων των θυρεοειδικών ορμονών στο ντοπαμινεργικό σύστημα, στον ενήλικα εγκέφαλο.Με αυτοραδιογραφική μελέτη, δείχθηκε μειωμένη ποσοτική κατανομή των D2, όχι όμως και των D1,υποδοχέων ντοπαμίνης στα βασικά γάγγλια εγκεφάλου αρσενικών ενηλίκων υποθυρεοειδικών μυών συγκριτικά με τους ευθυρεοειδικούς. Με μελέτη κορεσμού και ανάλυση κατά Scatchard παρατηρήθηκε μη στατιστικά σημαντική διαφορά στη χημική συγγένεια του ανταγωνιστή των D2 υποδοχέων ντοπαμίνης [3H]-raclopride με τους υποδοχεις αυτούς. Παρατηρήθηκε επίσης στατιστικά σημαντική μείωση, 37%, στο ποσό δέσμευσης (Bmax) του ραδιενεργού προσδέτη στους D2 ντοπαμινεργικούς υποδοχείς των αρσενικών υποθυρεοειδικών μυών σε σχέση με τους ευθυρεοειδικούς. Διερευνώντας τα χαρακτηριστικά δέσμευσης των D2 ντοπαμινεργικών υποδοχέων, βρέθηκε στατιστικά σημαντικά αυξημένη η χημική συγγένεια της ντοπαμίνης σε σχέση με αυτή του ανταγωνιστή των D2 υποδοχέων ντοπαμίνης [3H]-raclopride για τη θέση υψηλής χημικής συγγένειας και μειωμένη η ποσοστιαία αναλογία των θέσεων δέσμευσης που αφορούν τη θέση υψηλής χημικής συγγένειας στο ραβδωτό σώμα υποθυρεοειδικών μυών συγκριτικά με τους ευθυρεοειδικούς. Αντίθετα στους υποθυρεοειδικούς μύες βρέθηκε μειωμένη η χημική συγγένεια στη θέση χαμηλής χημικής συγγένειας. Η ‘in vitro’ προσθήκη T3 σε ομογενοποίημα ραβδωτού σώματος εγκεφάλου αρσενικών ευθυρεοειδικών μυών προκάλεσε στατιστικά σημαντική μείωση της χημικής συγγένειας στη θέση υψηλής χημικής συγγένειας, συγκριτικά με το αντίστοιχο δείγμα ευθυρεοειδικών μυών. Επίσης, με ενεργοποίηση των Α2Α υποδοχέων αδενοσίνης μέσω του αγωνιστή των Α2Α υποδοχέων αδενοσίνης CGS 21680, παρατηρήθηκε πολλαπλάσια μείωση της χημικής συγγένειας στη θέση υψηλής χημικής συγγένειας των υποθυρεοειδικών δειγμάτων σε σχέση με τα αντίστοιχα των ευθυρεοειδικών, που σημαίνει ενισχυμένη αλληλεπίδραση των Α2Α /D2 υποδοχέων. Τέλος, τα αποτελέσματά μας έδειξαν ότι η επαγόμενη σύνθεση του cΑΜΡ από το SKF38393 (αγωνιστής των D1 υποδοχέων ντοπαμίνης) είναι στατιστικά σημαντικά μεγαλύτερη στο ραβδωτό των υποθυρεοειδικών μυών και ότι το σύστημα αλληλεπίδρασης D1/D2 υποδοχέων ντοπαμίνης στο ραβδωτό διαφέρει μεταξύ ευθυρεοειδικών και υποθυρεοειδικών μυών. Συγκεκριμένα η από το SKF38393 επαγόμενη σύνθεση του cΑΜΡ όταν αναστέλλεται υπό την παράλληλη δράση της κουινπιρόλης (αγωνιστής των D2 υποδοχέων ντοπαμίνης), παρουσιάζει στο ραβδωτό των ευθυρεοειδικών μυών μια δοσοεξαρτώμενη και αναλογική αναστολή της επαγόμενης σύνθεσης του cΑΜΡ και στο ραβδωτό των υποθυρεοειδικών μυών μια δοσοεξαρτώμενη αλλά όχι αναλογική αναστολή της επαγόμενης σύνθεσης του cΑΜΡ. Ενώ η επαγόμενη από το CGS21680 (αγωνιστής των Α2Α υποδοχέων δείχνει επίσης μεγαλύτερη επαγωγή σύνθεσης cAMP στα υποθυρεοειδικά ζώα αλλά και ενισχυμένη αλληλεπίδραση των Α2Α /D2 υποδοχέων στούς υποθυρεοειδικούς μύες, με στατιστικά σημαντικά μειωμένη σύνθεση του cΑΜΡ μόνο στους υποθυρεοειδικούς μύες. Τα αποτελέσματα της επαγόμενης σύνθεσης του cΑΜΡ πιθανόν παραπέμπουν σε τροποποιημένη δράση των G-πρωτεϊνών στους υποθυρεοειδικούς μύες. Τα αποτελέσματά μας δείχνουν επίδραση των θυρεοειδικών ορμονών στο ραβδωτό σώμα τόσο στον αριθμό των D2 ντοπαμινεργικών υποδοχέων όσο και στην μεταβίβαση σήματος μέσω των ντοπαμινεργικών υποδοχέων. Επίσης υποστηρίζουν την εμπλοκή των θυρεοειδικών ορμονών στην αλληλεπίδραση των ντοπαμινεργικών υποδοχέων με τους υποδοχείς αδενοσίνης σε μεμβρανικό επίπεδο καθώς και σε επίπεδο δευτερογενών μηνυμάτων, δείχνοντας ενισχυμένη αλληλεπίδραση των Α2Α /D2 υποδοχέων στους υποθυρεοειδικούς μύες. / Thyroid hormones (T3 and T4) are necessary in differentiation and development but also in the maturation of the central neural system. Their action has been connected with alterations in behavior, as well as with psychological and movement disorders. It is known that the dopaminergic (DA) innervation of the basal ganglia, and in particular of the striatum, plays a central role in most movement, cognitive and emotional functions. Hypothyroidism induces morphological alterations, and more precisely, reductions in the axonal and dendritic sprouting of striatal neurons of adult rat brain and also modifications in the ability of dopamine antagonists’ ligand-binding. However, the underling mechanisms involved at the cellular and molecular level remain unidentified. Aim: In the current dissertation, we studied the effect of hypothyroidism in the striatum of adult, male Balb-c mice, in order to get a better understanding on the underling mechanism(s) of the effect of thyroid hormones on the DAergic system in adult brain. Results: Autoradiography showed a quantitative reduction in the level of D2 receptors in the basal ganglia of adult, male, hypothyroid mice versus that in eythyroid mice. Saturation studies revealed a non- statistically significant alteration in the chemical affinity of D2 receptor antagonist [3H]-raclopride for these receptors. However, a statistically significant reduction (37 %) in Bmax of the radiolabeled ligand was observed in D2 dopaminergic receptors of the hypothyroid mice versus the euthyroid. Similar studies on the pharmacological profile of D2 DAergic receptor binding, showed a statistically significant increase in the binding affinity of dopamine versus [3H]-raclopride, at the high affinity site, and a reduction in the percentage of the binding sites in this site, in the striatum of male, adult hypothyroid mice versus eythyroid. The opposite effect was observed in the low affinity site, where we showed a reduction in the binding affinity. Furthermore, the “in vitro” addition of T3 in striatal homogenates of male, adult euthyroid brain resulted in a statistically significant reduction in the binding affinity, of the high affinity site, compared to euthyroid samples. Also, by activation of A2A adenosine receptors through application of the CGS21680 agonist, a statistically significant reduction was observed in the binding affinity, when at the high affinity site, of the hypothyroid compared to eythyroid samples. Finally the effect of hypothyroidism in the induction of cAMP synthesis was also examined. Our data showed that the SKF38393 (a D1 dopamine receptor agonist)-driven cAMP synthesis is statistically significant in the striatum of adult, male hypothyroid mice and that the D1/D2 interaction system of dopamine receptors is different between euthyroid and hypothyroid mice. This means that when SKF38393-driven cAMP synthesis is suppressed by quinpirole (a D2 dopamine receptor agonist), a dose dependent inhibition is observed in the striatum of euthyroid mice, which is similar, but not proportional to the inhibition observed in the striatum of hypothyroid mice. While the CGS21680 (a Α2Α adenosine receptror agonist)- driven cAMP synthesis is statistically significant in the striatum of adult, male hypothyroid mice versus to euthyroid and that the A2A/D2 interaction system of adenosine/dopamine receptors is different between euthyroid and hypothyroid mice. Only in hypothyroid mice the A2A-diven cAMP synthesis is statistically suppressed in a dose dependent manner, by costimulation of D2.receptors. Conclusions: Our results show the effect of thyroid hormones on the number of DAergic receptors level and on their signal transduction levels, after dopamine receptors activation in the striatum of adult, male mice. These observations mark the involvement of thyroid hormones in the interactions of the DAergic receptors with the adenosinergic receptors, both at the receptor-receptor and the signal transduction level.

Dopamine receptors belong to the G protein-coupled receptor (GPCR) superfamily and are classified into two families: D1-like (D1R and D5R) and D2-like (D2R, D3R and D4R), based on their ability to stimulate or inhibit adenylyl cyclase (AC). Classically, GPCRs (including D2R and D3R) are desensitized by the activation of the serine/threonine protein kinase C (PKC) upon phorbol-12-myristate-13-acetate (PMA) treatment. Previous studies demonstrate that while human D5R (hD5R) is also strongly desensitized upon PMA treatment, the human D1R (hD1R) undergo a robust PMA-induced sensitization. The aim of this PhD thesis was to explore how the canonical PKC- or phorbol ester-linked pathway can control the responsiveness of two similar GPCRs like hD1R and hD5R in an opposite fashion. Our data indicate that hD1R sensitization and hD5R desensitization are not mediated by a direct modulation of AC activity by PKC. Using a chimeric approach, we identified the third intracellular loop (IL3) as the key structural determinant controlling in an opposite manner the PMA-mediated regulation of hD1R and hD5R. To delineate the potential PKC phosphorylation sites, a series of mutation of serine (Ser) and threonine (Thr) located into IL3 of hD1R and hD5R were used. No hD1R mutation decreased the PMA-mediated sensitization. This suggests that hD1R phosphorylation is not required for PMA-induced sensitization. In contrast, our results indicate that PMA-mediated hD5R desensitization occurs through a hierarchical phosphorylation of Ser260, Ser261, Ser271 and Ser274. Notably, these hD5R mutants exhibited a PMA-induced sensitization, reminiscent of the PMA-induced hD1R sensitization. Additionally, using short hairpin RNAs (shRNAs), we showed that PKCε is the potentiating PKC while the desensitizing isoform is δ. Overall, our work suggests the presence or absence of specific Ser residues on IL3 of hD1-like receptors dictate if phosphorylation-dependent desensitization (through PKCδ) or phosphorylation-independent potentiation (via PKCε) will occur.

Dopamine receptors belong to the G protein-coupled receptor (GPCR) superfamily and are classified into two families: D1-like (D1R and D5R) and D2-like (D2R, D3R and D4R), based on their ability to stimulate or inhibit adenylyl cyclase (AC). Classically, GPCRs (including D2R and D3R) are desensitized by the activation of the serine/threonine protein kinase C (PKC) upon phorbol-12-myristate-13-acetate (PMA) treatment. Previous studies demonstrate that while human D5R (hD5R) is also strongly desensitized upon PMA treatment, the human D1R (hD1R) undergo a robust PMA-induced sensitization. The aim of this PhD thesis was to explore how the canonical PKC- or phorbol ester-linked pathway can control the responsiveness of two similar GPCRs like hD1R and hD5R in an opposite fashion. Our data indicate that hD1R sensitization and hD5R desensitization are not mediated by a direct modulation of AC activity by PKC. Using a chimeric approach, we identified the third intracellular loop (IL3) as the key structural determinant controlling in an opposite manner the PMA-mediated regulation of hD1R and hD5R. To delineate the potential PKC phosphorylation sites, a series of mutation of serine (Ser) and threonine (Thr) located into IL3 of hD1R and hD5R were used. No hD1R mutation decreased the PMA-mediated sensitization. This suggests that hD1R phosphorylation is not required for PMA-induced sensitization. In contrast, our results indicate that PMA-mediated hD5R desensitization occurs through a hierarchical phosphorylation of Ser260, Ser261, Ser271 and Ser274. Notably, these hD5R mutants exhibited a PMA-induced sensitization, reminiscent of the PMA-induced hD1R sensitization. Additionally, using short hairpin RNAs (shRNAs), we showed that PKCε is the potentiating PKC while the desensitizing isoform is δ. Overall, our work suggests the presence or absence of specific Ser residues on IL3 of hD1-like receptors dictate if phosphorylation-dependent desensitization (through PKCδ) or phosphorylation-independent potentiation (via PKCε) will occur.

Male and Female newborn rats were primed with either quinpirole 0.05 mg/kg IP or SKF-38393 0.1 mg/kg IP on days 1-11, 12-22 and 23-33 of their lives. When the rats reached the age of 13 weeks, they were placed on metal rods in an activity avoidance chamber, and light and electric current of 30V/0.8 mA were used on them as conditioning stimuli. Avoidance of the electric shock was considered a positive conditioned reaction. Training and memorizing the conditioned avoidance reflex consisted of a series of ten trials, 60 seconds apart, once a week for ten weeks. The mean number of positive responses after quinpirole was more profound in all priming intervals tested, as compared to SKF-38393, and was higher as the priming started later in life. Significantly higher scores were obtained by the female rats primed with quinpirole, as compared to the male rats primed with the same drug. These differences were much weaker with SKF-38393. These findings confirm that the central D2 receptor system is involved in learn ing and memorizing of Conditioned Avoidance Reflex much more than the D1 receptors do, and that female rats are more sensitive and retentive to this reflex.

É reconhecido que as experiências que geram reações de medo são praticamente indeléveis do encéfalo dos organismos e que condicionamentos aversivos suscitam inúmeras respostas defensivas, como o congelamento, sendo esta resposta um indicador de medo em roedores. Vários trabalhos têm apontado para a relação entre alterações na transmissão dopaminérgica e os estados aversivos. Entretanto, observam-se resultados conflitantes com a utilização de drogas dopaminérgicas em diferentes modelos animais de ansiedade. Assim, investigações devem ainda ser realizadas objetivando avaliar a funcionalidade da modulação dopaminérgica nos estados emocionais aversivos. O objetivo do presente estudo foi avaliar o envolvimento de mecanismos dopaminérgicos na expressão do medo condicionado ao contexto. Inicialmente foram avaliados os efeitos de agonistas (SKF 38393 e quimpirole) e antagonistas (SCH 23390 e sulpirida) de receptores D1 e D2 administrados sistemicamente sobre a expressão do medo condicionado contextual, sendo mensurado o tempo de congelamento dos animais. A atividade motora foi avaliada com o teste do campo aberto. Os resultados indicam que os receptores da família D2, e não D1, estão envolvidos na expressão do medo condicionado contextual, uma vez que a administração de quimpirole e sulpirida, mas não de SCH 23390 e SKF 38393, levou a uma diminuição do congelamento condicionado ao contexto. Não houve alterações na atividade motora dos animais. Com base nestes resultados foi levantada a hipótese de que a capacidade da sulpirida e do quimpirole em diminuir o medo condicionado poderia ocorrer devido a uma ação em receptores pós-sinápticos de estruturas do sistema mesocorticolímbico e em autoreceptores da área tegmental ventral (ATV), respectivamente, levando ao efeito comum de diminuição da atividade dopaminérgica. A fim de testar esta hipótese, foram realizadas microinjeções de quimpirole na ATV. Os resultados obtidos mostram uma diminuição da expressão do congelamento condicionado e que os efeitos obtidos com a administração sistêmica desse agonista de receptores D2 provavelmente devem-se a sua ação na ATV. Portanto, a ATV parece atuar na modulação das respostas de medo condicionado e a ativação desta estrutura deve ser importante para a recuperação da aprendizagem aversiva ocorrida no dia do condicionamento. / It is well established that experiences that generate fear reactions are practically unforgettable and that aversive conditioning raises several defensive responses such as freezing, which is an index of fear in rodents. Several studies have pointed to the existence of a relationship between changes in dopaminergic neurotransmission and aversive states. However, there are conflicting results in the literature with the use of dopaminergic drugs in different animal models of anxiety. Thus, further investigations should be conducted to evaluate the importance of dopaminergic modulation of aversive states. The aim of the present study was to evaluate the involvement of dopaminergic neurotransmission in the expression of contextual conditioned fear in rats. Initially, we evaluated the effects of intraperitoneal injections of D1 and D2 receptors agonists (SKF 38393 and quinpirole) and antagonists (SCH 23390 and sulpiride) in the expression of contextual conditioned fear by measuring the time of freezing response of the animals. The motor activity was evaluated in the open field test. The results indicate that the D2 receptors, but not D1 receptors, are involved in the expression of contextual conditioned fear, since administration of quinpirole and sulpiride, but not SCH 23390 and SKF 38393, decreased conditioned freezing to the context. There were no changes in motor activity of animals. Based on these results it was hypothesized that quinpirole and sulpiride probably acted on presynaptic and postsynaptic D2 receptors, respectively, leading to a decrease of dopaminergic neurotransmission in both cases. To test this hypothesis, microinjections of quinpirole were performed into the ventral tegmental area (VTA). The results show a decrease in the expression of conditioned freezing, indicating that the effects obtained with the intraperitoneal administration of the dopamine D2 receptor agonist is probably due to its action in the VTA. Therefore, dopaminergic mechanisms in the VTA seem to be important in the modulation of conditioned fear responses and activation of this structure appears to take place during the fear memory following the context aversive conditioning.

Myopia is a refractive defect of the eye in which collimated light produces images focused in front of the retina. Myopia can be artificially induced in animal models by form deprivation (form deprivation myopia, FDM) or by application of negative lenses (lens induced myopia, LIM). In this study myopia was induced using diffusers. The project had two main aims: 1. To determine if there is an interaction between the GABAergic system and dopaminergic system in the retina in terms of myopia? 2. To determine if there is an interaction between the GABAergic system and cholinergic system in the retina in terms of myopia? Firstly, an experiment focusing on the interaction between dopaminergic receptors antagonists and GABAC receptor antagonist was developed. Comparison of the different drug treated eye with the control was found and the effects of combination injections were compared to individual drug injections. Use of different blockers for various subtype of receptors simplified the understandings the underlying pharmacological interventions for GABAC receptor antagonist TPMPA. The D1 subtype of receptors was found to be involved in transmission of signals from GABAC receptors. Our results showed that D1 receptor antagonist SCH-23390 antagonizes the actions of TPMPA. In addition to this it was also found that possibly 5HT receptor may also play an important role in modulation of signaling from GABA receptor to dopaminergic receptors in the retina. These results were consistent with the drug combination effects for agonists. GABA A/C receptor agonist muscimol negativate the efficacy of D1 receptor agonist SKF-38393 but the activity of D2/4 receptor agonist quinpirole was not affected by muscimol. Although dopaminergic receptors are found to interact with GABAergic signaling, but an alternative interaction with anticholinergic (most widely studied antimyopic agents) could not be ruled out. This problem led to a follow-up experiment, in which GABA receptors intervention in anticholinergic agents was studied. The GABAergic receptor agonist muscimol when injected with anticholinergics (atropine and pirenzepine) showed a moderate interaction. As muscimol interacted with atropine to a lesser extent a more specific M1/5 receptor antagonist pirenzepine (earlier found to inhibit myopia) was used under these circumstances. The second aim to study the interaction between muscimol and pirenzepine showed more interaction with GABAA/C receptor agonist. There were data suggesting that there is a muscarinic and GABAergic interaction in retina, such that each modulation of each receptor had an effect on FDM. However, a drug combination treatment helped in understanding the underlying mechanism. Several previous studies have indicated that there exist a strong interaction between excitatory neurotransmitter acetylcholine and inhibitory transmitter GABA in retina. The results of this study indicate a similar finding. Thus results of this study may be summarized as: 1. D1 antagonists and not D2 antagonists blocks the antimyopic effects of GABAC antagonist TPMPA 2. GABA A/C agonist muscimol partially blocks the antimyopic activity of anticholinergics (e.g. atropine and pirenzepine).

É reconhecido que as experiências que geram reações de medo são praticamente indeléveis do encéfalo dos organismos e que condicionamentos aversivos suscitam inúmeras respostas defensivas, como o congelamento, sendo esta resposta um indicador de medo em roedores. Vários trabalhos têm apontado para a relação entre alterações na transmissão dopaminérgica e os estados aversivos. Entretanto, observam-se resultados conflitantes com a utilização de drogas dopaminérgicas em diferentes modelos animais de ansiedade. Assim, investigações devem ainda ser realizadas objetivando avaliar a funcionalidade da modulação dopaminérgica nos estados emocionais aversivos. O objetivo do presente estudo foi avaliar o envolvimento de mecanismos dopaminérgicos na expressão do medo condicionado ao contexto. Inicialmente foram avaliados os efeitos de agonistas (SKF 38393 e quimpirole) e antagonistas (SCH 23390 e sulpirida) de receptores D1 e D2 administrados sistemicamente sobre a expressão do medo condicionado contextual, sendo mensurado o tempo de congelamento dos animais. A atividade motora foi avaliada com o teste do campo aberto. Os resultados indicam que os receptores da família D2, e não D1, estão envolvidos na expressão do medo condicionado contextual, uma vez que a administração de quimpirole e sulpirida, mas não de SCH 23390 e SKF 38393, levou a uma diminuição do congelamento condicionado ao contexto. Não houve alterações na atividade motora dos animais. Com base nestes resultados foi levantada a hipótese de que a capacidade da sulpirida e do quimpirole em diminuir o medo condicionado poderia ocorrer devido a uma ação em receptores pós-sinápticos de estruturas do sistema mesocorticolímbico e em autoreceptores da área tegmental ventral (ATV), respectivamente, levando ao efeito comum de diminuição da atividade dopaminérgica. A fim de testar esta hipótese, foram realizadas microinjeções de quimpirole na ATV. Os resultados obtidos mostram uma diminuição da expressão do congelamento condicionado e que os efeitos obtidos com a administração sistêmica desse agonista de receptores D2 provavelmente devem-se a sua ação na ATV. Portanto, a ATV parece atuar na modulação das respostas de medo condicionado e a ativação desta estrutura deve ser importante para a recuperação da aprendizagem aversiva ocorrida no dia do condicionamento. / It is well established that experiences that generate fear reactions are practically unforgettable and that aversive conditioning raises several defensive responses such as freezing, which is an index of fear in rodents. Several studies have pointed to the existence of a relationship between changes in dopaminergic neurotransmission and aversive states. However, there are conflicting results in the literature with the use of dopaminergic drugs in different animal models of anxiety. Thus, further investigations should be conducted to evaluate the importance of dopaminergic modulation of aversive states. The aim of the present study was to evaluate the involvement of dopaminergic neurotransmission in the expression of contextual conditioned fear in rats. Initially, we evaluated the effects of intraperitoneal injections of D1 and D2 receptors agonists (SKF 38393 and quinpirole) and antagonists (SCH 23390 and sulpiride) in the expression of contextual conditioned fear by measuring the time of freezing response of the animals. The motor activity was evaluated in the open field test. The results indicate that the D2 receptors, but not D1 receptors, are involved in the expression of contextual conditioned fear, since administration of quinpirole and sulpiride, but not SCH 23390 and SKF 38393, decreased conditioned freezing to the context. There were no changes in motor activity of animals. Based on these results it was hypothesized that quinpirole and sulpiride probably acted on presynaptic and postsynaptic D2 receptors, respectively, leading to a decrease of dopaminergic neurotransmission in both cases. To test this hypothesis, microinjections of quinpirole were performed into the ventral tegmental area (VTA). The results show a decrease in the expression of conditioned freezing, indicating that the effects obtained with the intraperitoneal administration of the dopamine D2 receptor agonist is probably due to its action in the VTA. Therefore, dopaminergic mechanisms in the VTA seem to be important in the modulation of conditioned fear responses and activation of this structure appears to take place during the fear memory following the context aversive conditioning.

Nitric acid has been implicated in a variety of physiological functions of the mammalian brain, and in a large number of its pathologies. Recently we have demonstrated that a nitric oxide donor (L-arginine) and a nitric-oxide-synthase-inhibitor (nitro-L-arginine-methyl-ester) modified the response of central al dopamine D 1 and D 3 receptors to some of their agonists. In the present study we demonstrate the modulatory effect of L-arginine, nitro-L-arginine-methyl-ester and molsidomine (another nitric oxide donor) on the reactivity of the central dopamine receptors to specific agonists and antagonists. The agonists tested were SKF-38393, 7-OH-DPAT and quinpirole, and the antagonists - SCH-23390 and haloperidol. They were evaluated in the rat by the following behavioral methods: locomotor activity, locomotor coordination, rearings and cataleptogenic activity (D 2 modulation); grooming time (D 1 activation); yawning (D 3 activation) and ethanol- and phenobarbital-sleeping-time parameters after SKF-38393 or quinpirole pretreatment. Our results suggest that nitro-L-arginine-methyl-ester is effective in modulating the reactivity of the central dopamine receptors D 2, D 1 and D 3, to their agonists and antagonists, and that it is much more effective than L-arginine in regulating the righting reflex after ethanol and phenobarbital, in both female and male mature rats.

A travers ce projet de recherche nous avons exploré différents aspects d’une dérégulation du système dopaminergique sur les troubles du repos, en prenant pour exemple deux maladies neurologiques : la maladie de Willis-Ekbom (MWE) et la maladie de Parkinson (MP). La MWE est une maladie neurologique sensorimotrice caractérisée par des douleurs dans les membres inférieurs, s’accompagnant d’un besoin irrépressible de bouger et ce, suivant un profil circadien. Ainsi, le premier volet de ces travaux s’est appliqué à reproduire chez le macaque, les principales altérations du métabolisme du fer et de celui de la dopamine reportées dans la MWE. Tout d’abord, nous avons établit les bases physiologiques des variations circadiennes des concentrations du fer et de ses biomarqueurs au niveau central et périphérique. Puis, nous avons développé un protocole simple, uniquement basé sur des prélèvements sanguins répétés, permettant d’induire efficacement une déplétion en fer sérique et de ses protéines associées. Finalement, ce protocole nous a permis d’explorer les liens entre l’altération de l’homéostasie du fer au niveau du système nerveux central, les perturbations neurochimiques dans différentes structures cérébrales ainsi que les modifications locomotrices qui en résultent. Le second volet de cette thèse a testé l’impact des agonistes des récepteurs dopaminergiques de type D1 (SKF38393) et D2 (quinpirole), sur les troubles du sommeil dans un modèle macaque de la MP, à l’aide d’enregistrements polysomnographiques. Pour cela, nous avons évalué les effets de ces agents pharmacologiques sur l’émergence de la somnolence diurne et sur l’altération du sommeil paradoxal, induits par une intoxication au MPTP. Nos résultats mettent en évidence que le quinpirole est inefficace pour restaurer les niveaux de base de ces deux paramètres. En revanche, le SKF38393 permet une diminution notable de la somnolence diurne ainsi qu’une restauration du sommeil paradoxal. Finalement, les perturbations monoaminergiques liées à la déplétion en fer ouvrent de multiples perspectives de recherche sur la physiopathologie de la MWE. De même, l’amélioration des troubles veille-sommeil par l’agoniste des récepteurs D1, offre de nouvelles pistes thérapeutiques quant à la prise en charge des troubles du repos dans la MP. L’ensemble de nos résultats apporte un niveau de compréhension supplémentaire quant au rôle de la dopamine dans les altérations du repos. / During this thesis project we explored several aspects of the impact of a dopaminergic system dysregulation on the rest alterations, through two neurological diseases: the Willis-Ekbom’s disease (WED) and Parkinson’s diseases (PD). The WED is a neurological sensorimotor disorder mainly characterized by pain in lower limbs. It preferentially appears in the evening and transiently and partially is alleviated by motor activity. Thus, the first part of this work aimed at reproducing the main dysfunctions of the iron and dopaminergic metabolisms observed in WED, in the macaque monkey. We first established the circadian variations of iron-indicator concentrations in serum and cerebrospinal fluid. Then we developed a rapid protocol based on repeated blood withdrawals, allowing to efficiently induce serum iron depletion. Finally, this protocol enabled us to investigate the relationship between iron metabolism dysfunctions, neurochemical alterations and the subsequent locomotor behavioural changes. In the second part, of this research project we examined the impact of selective D1 (SKF38393) and D2 (quinpirole) receptor agonists on the sleep impairments in a macaque model of PD using the polysomnographic recording technique. Thus we investigated the effects of these two pharmacological compounds on the daytime sleepiness and on the paradoxical sleep induced by MPTP intoxication. Our results demonstrated the inefficacy of quinpirole to restore these two altered sleep parameters. By contrast, SKF38393 significantly decreased daytime napping and substantially restored paradoxical sleep. Finally, the monoaminergic dysregulations, induced by iron depletion, may offer multiple perspectives to unravel the WED pathophysiology. In the same line, the beneficial effects exhibited by the D1 receptor agonist bring new therapeutic avenues to treat sleep-wake disorders in PD. Together, the global results bring new insights in the underlying mechanisms of sleep impairment involving dopamine.