The influence of central dopaminergic fibers on drug-induced oral activity in rats has not been well studied. Rats were treated 3 days after birth with bilateral intracerebroventricular 6-hydroxydopamine (6-OHDA; 134 ωg total, base form) to destroy dopaminergic fibers in the brain. Control rats received vehicle by the same route. At about 10 weeks of age, a challenge dose of the dopamine D2 receptor antagonist, spiperone (40 ωg/kg, IP), produced an 8-fold increase in the number of oral movements during a 60-minute observation period, vs. the control group. SKF 38393 (3.0 mg/kg, IP), a D1 agonist, produced the same number of oral movements as spiperone in the 6-OHDA group, representing a 2.4-fold increase over the controls. The Bmax and Kd for both D1 and D2 receptors was not changed in rat striatum by neonatal 6-OHDA treatment, even though dopamine content was reduced by 96%. These findings demonstrate that oral activity in rats can be greatly altered, even when there is no change in absolute numbers of D1 and D2 receptors and no change in the ratio of D1:D2 receptors.

Receptor priming is a recently discovered phenomenon by which receptor agonists produce abrupt and long-lived supersensitization of receptors. Induction of dopamine (DA) D2 receptor supersensitivity by the agonist quinpirole was discovered approximately 15 years ago, and was found to occur consistently if rats were treated repeatedly at daily or weekly or monthly intervals with low or high doses of quinpirole. In this review we summarize and discuss some of the major studies that underlie DA D2 receptor supersen-sitivity, describe behavioral processes that are known to be altered by DA D2 receptor supersensitivity, and discuss the importance of DA innervation on expression of enhanced behaviors. DA D2 receptor supersen-sitivity represents one of the neural mechanisms implicated in psychiatric disorders. Also, DA D2 receptor supersensitivity and increased DA D3 receptor expression are associated with motor dyskinesias, as in L-DOPA-treated Parkinson's disease patients. An understanding of receptor priming, a knowledge of the types of behavioral expression associated with DA D2 receptor supersensitivity, and an understanding of mechanisms associated with receptor supersen-sitization, can lead to improvements in the treatments of psychiatric and neurological disorders.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23571号 / 医博第4785号 / 新制||医||1054(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 齊藤 博英, 教授 大森 孝一, 教授 大鶴 繁 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Tardive dyskinesia (TD) is a serious side effect of certain antipsychotic medications that are used to treat schizophrenia (SCZ) and other mental illnesses. The methylation status of the insulin receptor substrate 1 (IRS1) gene is reportedly associated with SCZ; however, no study, to the best of the authors' knowledge, has focused on the quantitative DNA methylation levels of the IRS1 gene using pyrosequencing in SCZ with or without TD. The present study aimed to quantify DNA methylation levels of 4 CpG sites in the IRS1 gene using a Chinese sample including SCZ patients with TD and without TD (NTD) and healthy controls (HCs). The general linear model (GLM) was used to detect DNA methylation levels among the 3 proposed groups (TD vs. NTD vs. HC). Mean DNA methylation levels of 4 CpG sites demonstrated normal distribution. Pearson's correlation analysis did not reveal any significant correlations between the DNA methylation levels of the 4 CpG sites and the severity of SCZ. GLM revealed significant differences between the 3 groups for CpG site 1 and the average of the 4 CpG sites (P=0.0001 and P=0.0126, respectively). Furthermore, the TD, NTD and TD + NTD groups demonstrated lower methylation levels in CpG site 1 (P=0.0003, P<0.0001 and P<0.0001, respectively) and the average of 4 CpG sites (P=0.0176, P=0.0063 and P=0.003, respectively) compared with the HC group. The results revealed that both NTD and TD patients had significantly decreased DNA methylation levels compared with healthy controls, which indicated a significant association between the DNA methylation levels of the IRS1 gene with SCZ and TD.

Dystonia is defined as a neurological syndrome characterized by involuntary sustained or intermittent muscle contractions causing twisting, often repetitive movements, and postures. Paroxysmal dyskinesias are episodic movement disorders encompassing dystonia, chorea, athetosis, and ballism in conscious individuals. Several decades of research have enhanced the understanding of the etiology of human dystonia and dyskinesias that are associated with dystonia, but the pathophysiology remains largely unknown. The spontaneous occurrence of hereditary dystonia and paroxysmal dyskinesia is well documented in rodents used as animal models in basic dystonia research. Several hyperkinetic movement disorders, described in dogs, horses and cattle, show similarities to these human movement disorders. Although dystonia is regarded as the third most common movement disorder in humans, it is often misdiagnosed because of the heterogeneity of etiology and clinical presentation. Since these conditions are poorly known in veterinary practice, their prevalence may be underestimated in veterinary medicine. In order to attract attention to these movement disorders, i.e., dystonia and paroxysmal dyskinesias associated with dystonia, and to enhance interest in translational research, this review gives a brief overview of the current literature regarding dystonia/paroxysmal dyskinesia in humans and summarizes similar hereditary movement disorders reported in domestic animals.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Haloperidol-induced orofacial dyskinesia (OD) is a putative animal model of tardive dyskinesia (TD) whose pathophysiology has been related to an increase in dopamine turnover, reduction in gabaergic neurotransmission and oxidative stress. Schizophrenic patients have been reported to eat a diet higher in fat than the general population and dietary fat intake can lead to an increase in oxidative stress in animal models. Thus, the first objective of this study was to determine if the association of high fat diet ingestion with prolonged haloperidol treatment could alter the OD and lead to oxidative stress in the rat brain (article 1). Haloperidol decanoate administration (38 mg/kg, which is equivalent 1mg/kg/day) monthly for a period of 6 months to rats fed previously with a HF diet caused an increase in OD. Furthermore, haloperidol caused an increase in the levels of lipid peroxidation in striatum and substantia nigra only in rats fed with the HF diet. Another objective of this work was to evaluate the effects of V. officinalis (1% administered in the drink water) in the animal model of OD induced by long-term treatment with haloperidol (article 2). V. officinalis has been widely used to treat insomnia since it posses GABAmimetic and antioxidant properties. Concomitant treatment with V. officinalis did not modify the intensity or prevalence of OD. We did not find any statistical differences among the groups when oxidative stress parameters were evaluated. On the other hand, haloperidol treatment significantly decreased [3H]-dopamine uptake in slices from striatum of rats, an effect unaltered by V. officinalis. The treatment with V. officinalis reduced the locomotor activity in the open field and increased the time spent on open arm in the plus maze test, confirming the anxiolitic effect of V. fficinalis. Taken together, these results indicate that a high fat diet caused a transitory increase in haloperidol-induced OD in rats and this, at least in part, can be related to the haloperidol-induced oxidative stress in brain structures. Our data also suggest that the reduction in dopamine transport can be a possible mechanism related to the maintenance of chronic OD in rats feeding NF diet. Finally, V. officinalis seems not to be efficacious in the reduction of OD in rats. / A discinesia orofacial (DO) induzida por haloperidol consiste num modelo de discinesia tardia (DT) cuja patofisiologia tem sido relacionada a um aumento na renovação de dopamina, à redução na neurotransmissão gabaérgica e ao estresse oxidativo. Existem relatos de que pacientes esquizofrênicos ingerem uma maior quantidade de gordura na dieta do que a população em geral e o aumento da ingestão de gordura pode levar a um aumento do estresse oxidativo em modelos animais. Assim, um primeiro objetivo deste estudo foi determinar se a associação de ingestão de dieta rica em gordura concomitante ao tratamento prolongado com haloperidol poderia alterar a DO e levar ao estresse oxidativo em cérebro de ratos (artigo 1). A administração de decanoato de haloperidol (38 mg/kg, equivalente a 1mg/kg/dia) mensalmente, por um período de 6 meses, a ratos previamente alimentados com dietas normolipídica (NL) e hiperlipídica (HL) causou um aumento na DO. Além disso, o haloperidol causou um aumento nos níveis de peroxidação lipídica em estriado e substantia nigra somente em ratos ingerindo dieta HL. Um outro objetivo deste trabalho foi avaliar os efeitos da V. officinalis (administrada na água de beber na proporção de 1mL de tintura de raíz para 100mL de água) no modelo animal de DO induzido por tratamento prolongado com haloperidol (artigo 2). A V. officinalis tem sido largamente utilizada para o tratamento de insônia possuindo propriedades GABA-miméticas e antioxidantes. O tratamento concomitante com V. officinalis não modificou a intensidade ou a prevalência da DO. Além disto, não encontramos nenhuma diferença estatística entre os grupos quando os parâmetros de estresse oxidativo foram avaliados. Por outro lado, o tratamento com haloperidol reduziu significativamente a captação de [3H]-dopamina em fatias de estriado de ratos, um efeito inalterado pela V. officinalis. O tratamento com V. officinalis reduziu a atividade locomotora no campo aberto e aumentou o tempo de permanência no braço aberto do labirinto em cruz elevado, o que confirma o efeito ansiolítico da V. officinalis. Em conjunto, estes resultados indicam que a dieta rica em gordura causou um aumento transitório da DO induzida por haloperidol em ratos o que pode, em parte, ser relacionado ao estresse oxidativo induzido por haloperidol em estruturas do cérebro envolvidas com a DO. Nossos dados também sugerem que a redução no transporte de dopamina pode ser um possível mecanismo relacionado à manutenção da DO crônica em ratos ingerindo dieta NL. Além disso, a V. officinalis parece não ser eficaz na redução da DO em ratos.

Certains neuropeptides (enképhaline et neurotensine) sont des modulateurs du système dopaminergique. Chez les rongeurs, le traitement avec l’antipsychotique typique halopéridol (antagoniste des récepteurs D2), augmente fortement leurs niveaux d’ARNm dans le striatum, une structure centrale du système dopaminergique qui contrôle l’activité locomotrice. Comme l’halopéridol est associé avec de nombreux effets secondaires moteurs, on peut penser que la modulation des neuropeptides est possiblement un mécanisme d’adaptation visant à rétablir l’homéostasie du système dopaminergique après le blocage des récepteurs D2. Cependant, le mécanisme moléculaire de cette régulation transcriptionnelle n’est pas bien compris. Nur77 est un facteur de transcription de la famille des récepteurs nucléaires orphelins qui agit en tant que gène d’induction précoce. Le niveau de son ARNm est aussi fortement augmenté dans le striatum suivant un traitement avec halopéridol. Plusieurs évidences nous suggèrent que Nur77 est impliqué dans la modulation transcriptionnelle des neuropeptides. Nur77 peut former des hétérodimères fonctionnels avec le récepteur rétinoïde X (RXR). En accord avec une activité transcriptionnelle d’un complexe Nur77/RXR, l’agoniste RXR (DHA) réduit tandis que l’antagoniste RXR (HX531) augmente les troubles moteurs induits par un traitement chronique à l’halopéridol chez les souris sauvages tandis que ces ligands pour RXR n’ont aucun effet chez les souris Nur77 nulles. Nos travaux ont révélé que l’antagoniste RXR (HX531) réduit l’augmentation des niveaux d’enképhaline suivant un traitement chronique avec l’halopéridol. Nous avons ensuite démontré la liaison in vitro de Nur77 sur un élément de réponse présent dans le promoteur proximal de la proenképhaline, le peptide précurseur de l’enképhaline. Ces résultats supportent l’hypothèse que Nur77, en combinaison avec RXR, pourrait participer à la régulation transcriptionnelle des neuropeptides dans le striatum et donc contribuer à la neuroadaptation du système dopaminergique suivant un traitement aux antipsychotiques typiques. / Neuropeptides (enkephalin and neurotensin) are modulators of dopaminergic system. In the rodent’s striatum, antipsychotic drugs strongly modulate their mRNA levels. For exemple, haloperidol (D2 receptor antagonist) increases their mRNA levels in the striatum, a central structure of dopaminergic system that control locomotor activity. Since haloperidol is associated with many motor side effects, it is likely that neuropeptides modulation is a compensatory mechanism to restore the dopaminergic system homeostasis after D2 receptor blockade. However, molecular mechanism of this transcriptional regulation is not well understood. Nur77 is a transcription factor of the orphan nuclear receptor family that acts as an immediate early gene. In the striatum, its mRNA level is strongly increased following haloperidol treatment. Several evidences suggest that Nur77 is involved in neuropeptides transcriptional modulation. Nur77 can form functional heterodimers with retinoid X receptor (RXR). In agreement with a transcriptional activity of Nur77/RXR complex, RXR agonist (DHA) decreases while the RXR antagonist (HX531) exacerbates acute and chronic motor side effects of haloperidol treatment in wild type mice but remain without effect in Nur77 knockout mice. Our work shows that RXR antagonist (HX531) significantly reduced the increased levels of enkephalin following chronic treatment with haloperidol. We have set up the EMSA (electro-mobility shift assay) to demonstrate the in vitro binding of Nur77 on a responsive element from proenkephalin’s proximal promotor. These results support the hypothesis that Nur77 could be involve in transcriptional modulation of neuropeptides in the striatum and thus, contribute to neuroadaptation of dopaminergic system after treatment with typical antipsychotic drugs.

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<p>Dyskinesias or abnormal involuntary movements are a debilitating complication of long-term levodopa treatment of Parkinson’s disease (PD) that is widely experienced and may compromise the efficacy of the drug therapy. Tardive dyskinesia is another important adverse effect seen with antipsychotic drug treatment. The neural mechanisms underlying levodopa- and neuroleptic-induced dyskinesia are not clear and involvement of the endogenous opioid neuropeptide system has been implicated. In this thesis, the role of the opioid system is investigated in models of dyskinesia and PD using behavioral, neurochemical and advanced analytical chemistry techniques. In addition, the motor effects of a new partial dopamine agonist with normalizing properties on both reduced and elevated dopamine transmission are studied and a new model for tardive dyskinesia is presented.</p><p>Using microdialysis in combination with micro-electrospray mass spectrometry, the <i>in vivo</i> processing of the opioid neuropeptide dynorphin A(1-17) was studied and 32 metabolites were detected in the striatum. Altered <i>in vivo</i> metabolism of the peptide was found in a model of PD with more metabolites formed in the dopamine-depleted striatum. Moreover, dynorphin A(1-17) was differently processed in levodopa-, bromocriptine and saline-treated animals. </p><p>Levodopa treatment caused an increase in the mRNA expression of the precursor of dynorphin, preproenkephalin-B as well as the precursor of enkephalin, preproenkephalin-A, in all sub-regions of the dopamine-depleted striatum. A non-selective opioid receptor antagonist, naloxone, was found to reduce levodopa-induced dyskinesia with maintained antiparkinsonian response and a normalization of hyperkinesia. Moreover, the new drug GMC1111 showed dopamine stabilizing properties in models of levodopa-induced dyskinesia and PD. This might prove useful in the treatment of PD.</p><p>Altogether, these results suggest that the endogenous opioid system is involved in the pathophysiology of levodopa-induced dyskinesia.</p>