Les antipsychotiques sont utilisés en clinique depuis plus de 50 ans pour pallier aux symptômes de la schizophrénie. Malgré une recherche intensive, les mécanismes cellulaires et moléculaires responsables de l’effet clinique de cette médication demeurent encore nébuleux. Ces drogues sont reconnues comme des antagonistes des récepteurs D2 de la dopamine et peuvent moduler la transcription génique dans le striatum. Au cours des recherches qui ont mené à l'écriture de cette thèse, nous avons exploré l’expression de Nur77, un facteur de transcription de la famille des récepteurs nucléaires, afin de caractériser le rôle de la dopamine, la sérotonine, l’adénosine et le glutamate dans la régulation génique contrôlée par les antagonistes D2. En premier lieu, nous avons examiné l’impact de la co-administration d’agents sérotonergiques et adrénergiques sur l’expression de l’ARNm de Nur77 induite par l’halopéridol, un antipsychotique de première génération. Nous avons observé que le 8-OH-DPAT et le MDL11939 préviennent partiellement l’induction de Nur77 dans le striatum. Au contraire, l’idazoxan potentialise l’effet de l’halopéridol sur l’expression de Nur77 alors que le prazosin reste sans effet. Ces résultats démontrent que l’expression striatale de Nur77 induite par l’halopéridol peut être modulée à la baisse avec un agoniste 5-HT1A ou un antagoniste 5-HT2A. Par la suite, nous avons évalué dans divers paradigmes expérimentaux l’effet de l’éticlopride, un antagoniste spécifique D2, afin d’explorer davantage le mécanisme de l’effet transcriptionnel des antagonistes D2. Étonnamment, la suppression de l’isoforme D2L chez la souris D2L KO ne réduit pas la réponse de l’éticlopride dans le striatum. Par contre, une lésion corticale avec l’acide iboténique bloque l’effet de l’éticlopride sur la transcription de Nur77, suggérant un rôle du glutamate. La combinaison d’un antagoniste des récepteurs métabotropes du glutamate de types 5 (mGluR5) et d’un antagoniste des récepteurs de l’adénosine A2A abolit complètement l’augmentation de la transcription de Nur77 induit par l’éticlopride dans le striatum. La modulation directe de l’expression striatale de Nur77 par les récepteurs mGluR5 et A2A a été confirmée dans un modèle de cultures organotypiques de tranches cérébrales. Ces résultats démontrent clairement que la modulation de l’expression génique dans le striatum, à la suite d’un traitement avec un antagoniste D2 pourrait être indépendante d’une interaction directe avec les récepteurs D2 post-synaptiques, et reposerait plutôt sur son interaction avec les récepteurs D2 hétérosynaptiques des afférences corticostriées et l’activation subséquente des récepteurs post-synaptiques du glutamate et de l’adénosine. En résumé, nos résultats suggèrent que l’interaction des antipsychotiques atypiques avec les récepteurs 5-HT2A et 5-HT1A pourrait expliquer la différence dans le patron d’expression génique induit par ces drogues en comparaison avec les antipsychotiques typiques. De plus, nos résultats révèlent un nouveau mécanisme d’action des antagonistes D2 et supportent un rôle primordial du glutamate et de l’adénosine dans les effets des antipsychotiques de première génération. / Antipsychotic drugs have been used to alleviate schizophrenia symptoms for more than 50 years. Despite extensive research, little is known about the molecular and cellular mechanism responsible for their clinical outcome. These drugs are usually recognized as dopamine D2 antagonists and are known to modulate gene expression in the striatum. In the present thesis, we used the expression of Nur77, a transcription factor of the orphan nuclear receptor family, to explore the role of dopamine, serotonin, glutamate and adenosine receptors in the effect of a dopamine D2 antagonist in the striatum. First, we examined the abilities of serotoninergic and adrenergic receptor drugs to modify the pattern of Nur77 mRNA expression induced by haloperidol, a first generation antipsychotic drug. We observed that 8-OH-DPAT and MDL11939 partially prevent haloperidol-induced Nur77 upregulation. On the contrary, idazoxan consistently potentiated haloperidol-induced Nur77 mRNA levels in the striatum whereas prazosin remained without effect. Taken together, these results show the ability of a 5-HT1A agonist or a 5-HT2A antagonist to reduce haloperidol-induced Nur77 striatal expression. Subsequently, we evaluated in different experimental designs the effect of eticlopride, a specific D2 antagonist, to provide additional information on the mechanism by which D2 antagonist controls transcriptional activity in the striatum. Surprisingly, deletion of the D2L receptor isoform did not reduce eticlopride-induced upregulation of Nur77 mRNA levels in the striatum. However, cortical lesions with ibotenic acid strongly reduced eticlopride-induced upregulation of Nur77 mRNA, suggesting a role for glutamate neurotransmission. A combination of a metabotropic glutamate type 5 (mGluR5) antagonist with an antagonist of its synergistic partner adenosine A2A receptor abolished eticlopride-induced upregulation of Nur77 mRNA levels in the striatum. Direct modulation of striatal Nur77 expression by glutamate and adenosine receptors was confirmed using corticostriatal organotypic cultures. Taken together, these results indicate that modulation of gene expression in the striatum, following a D2 antagonist, might not involve a direct interaction of the drug at postsynaptic D2 receptors, but rather relies on its interaction with corticostriatal presynaptic D2 receptors and subsequent activation of postsynaptic glutamate and adenosine receptors in the striatum. In summary, our results suggest that interaction of atypical antipsychotic drugs with 5-HT2A and 5-HT1A receptors participate in the differential pattern of gene expression induced by these drugs when compared with typical antipsychotic drugs. Moreover, our results uncover a new mechanism of action of D2 antagonists and support a prominent role of glutamate and adenosine in the effect of classic antipsychotic drugs.

Major depression is a serious mood disorder affecting more than 120 million people worldwide, irrespective of their race or socio-economic status. This psychiatric disorder is predicted to become the second leading cause of disability by the year 2020, second only to heart diseases in the global population, without distinguishing differences in the incidence within defined age groups. Depression is known to affect people across all age groups, including children, adolescents, adults and geriatrics, although older age is associated with an increased susceptibility to major depression and other psychiatric conditions. Until the 1970‘s depression during childhood and adolescence was thought to be uncommon or non-existent. Recent epidemiological studies have demonstrated that there is a persistent escalation in the prevalence of depression in children and adolescents. Accordingly, the number of prescriptions for drugs to treat this disorder in juveniles has escalated significantly. With our current limited understanding of the safety and long-term effects of treatment with antidepressants, the clinician is left making decisions without sound evidence of safety. In addition, psychotropic drugs may affect neurodevelopment during childhood and adolescence and may consequently modulate susceptibility to psychiatric disorders later in life. The objective of the current study was to investigate the effects of early-life (pre-natal and postnatal) chronic treatment with venlafaxine, a dual action serotonin-noradrenalin reuptake inhibitor, during the developmental phase of the serotonin and norepinephrine pathways in stress-sensitive rats on measures of cognition, anxiety-like and depressive-like behaviour later in life. The study also investigated which age shows optimal behavioural changes later in life, following the above mentioned administration of venlafaxine. In addition we also determined the effects that the administration of venlafaxine has on the levels of monoamines l-norepinephrine (l-NE) and serotonin (5-HT) in the prefrontal cortex and the hippocampus. A number of translational animal models of psychiatric disorders have been described and validated, and is suitable for such investigations. For the current study we used stress-sensitive Flinders Sensitive Line (FSL) rats and their controls, Flinders Resistant Line (FRL) rats. Pregnant dams were injected subcutaneously for 14 days with 10 mg/kg venlafaxine or saline from pre-natal day 15 (ND-15) to ND-01. New-born pups were then injected subcutaneously with 3 mg/kg venlafaxine or saline for 14 days from postnatal day 3 (ND+03) to ND+17. These doses were determined from previous studies reported in literature. Four rat treatment groups of both FSL and FRL rats received injections during pre-natal + postnatal ages as follows: saline + saline, venlafaxine + saline, saline + venlafaxine and venlafaxine + venlafaxine. Following the drug treatments, all rat groups were housed under normal conditions until the indicated time to be subjected to a battery of behavioural tests, including the novel object recognition test (nORT), locomotor activity test (Digiscan®), elevated plus maze (EPM) and forced-swim test (FST), scheduled on either ND+35, ND+60 or ND+90. Separate treatment groups were used for each age group. After the behavioural tests animals were decapitated, the brains removed and the prefrontal cortex and hippocampus dissected out. These were analysed at a later stage using an HPLC with electrochemical detection to determine the levels of the monoamines l-NE and 5-HT. All animal procedures were approved by the Ethics Committee of the North-West University (approval number: NWU-00045-10-S5), and are in accordance with the recommendations of the National Institutes of Health guide for the care and use of laboratory animals. The data from the current study suggest that in general FRL rats were not influenced by the early-life treatment with venlafaxine, as observed in the nORT, EPM or FST on ND+35, ND+60 or ND+90. There was minimal changes seen in the immobile behaviour in the FST of FRL rats that received prenatal venlafaxine. As expected, depressive-like behaviour in the FST was significantly enhanced in FSL rats relative to corresponding FRL rat groups as observed at ND+35 and ND+60, but not ND+90. Importantly, depressive-like behaviour was reversed following pre- and postnatal treatment with venlafaxine in FSL rats at ND+60, relative to the corresponding FRL rat groups. Reversal of depressive-like behaviour in FSL rats were not observed at ND+35 or ND+90, suggesting a delayed response that is reversed later in adulthood. The data from the nORT, Digiscan® or EPM did not reveal any significant differences between the various FSL treatment groups, including at ND+60. The current study therefore demonstrated that the treatment regimen employed had a transient effect on depressive-like behaviour later in life and suggested that genetic susceptibility plays an important role in the treatment of depression. This was suggested by the venlafaxine-induced decrease in immobile behaviour exhibited by FSL rats at ND+60 in the FST, and the subsequent increase in immobile behaviour at ND+90. In general, the most significant venlafaxine-induced effects were seen in FSL rats, suggesting genetic susceptibility plays an important role. / MSc (Pharmacology), North-West University, Potchefstroom Campus, 2014

Biogene Amine sind kleine organische Verbindungen, die sowohl bei Wirbeltieren als auch bei Wirbellosen als Neurotransmitter, Neuromodulatoren und/oder Neurohormone wirken können. Sie bilden eine bedeutende Gruppe von Botenstoffen und entfalten ihre Wirkungen über die Bindung an eine bestimmte Klasse von Rezeptorproteinen, die als G-Protein-gekoppelte Rezeptoren bezeichnet werden. Bei Insekten gehören zur Substanzklasse der biogenen Amine die Botenstoffe Dopamin, Tyramin, Octopamin, Serotonin und Histamin. Neben vielen anderen Wirkung ist z.B. gezeigt worden, daß einige dieser biogenen Amine bei der Honigbiene (Apis mellifera) die Geschmacksempfindlichkeit für Zuckerwasser-Reize modulieren können. Ich habe verschiedene Aspekte der aminergen Signaltransduktion an den „Modellorganismen“ Honigbiene und Amerikanische Großschabe (Periplaneta americana) untersucht. Aus der Honigbiene, einem „Modellorganismus“ für das Studium von Lern- und Gedächtnisvorgängen, wurden zwei Dopamin-Rezeptoren, ein Tyramin-Rezeptor, ein Octopamin-Rezeptor und ein Serotonin-Rezeptor charakterisiert. Die Rezeptoren wurden in kultivierten Säugerzellen exprimiert, um ihre pharmakologischen und funktionellen Eigenschaften (Kopplung an intrazelluläre Botenstoffwege) zu analysieren. Weiterhin wurde mit Hilfe verschiedener Techniken (RT-PCR, Northern-Blotting, in situ-Hybridisierung) untersucht, wo und wann während der Entwicklung die entsprechenden Rezeptor-mRNAs im Gehirn der Honigbiene exprimiert werden. Als Modellobjekt zur Untersuchung der zellulären Wirkungen biogener Amine wurden die Speicheldrüsen der Amerikanischen Großschabe genutzt. An isolierten Speicheldrüsen läßt sich sowohl mit Dopamin als auch mit Serotonin Speichelproduktion auslösen, wobei Speichelarten unterschiedlicher Zusammensetzung gebildet werden. Dopamin induziert die Bildung eines völlig proteinfreien, wäßrigen Speichels. Serotonin bewirkt die Sekretion eines proteinhaltigen Speichels. Die Serotonin-induzierte Proteinsekretion wird durch eine Erhöhung der Konzentration des intrazellulären Botenstoffs cAMP vermittelt. Es wurden die pharmakologischen Eigenschaften der Dopamin-Rezeptoren der Schaben-Speicheldrüsen untersucht sowie mit der molekularen Charakterisierung putativer aminerger Rezeptoren der Schabe begonnen. Weiterhin habe ich das ebony-Gen der Schabe charakterisiert. Dieses Gen kodiert für ein Enzym, das wahrscheinlich bei der Schabe (wie bei anderen Insekten) an der Inaktivierung biogener Amine beteiligt ist und im Gehirn und in den Speicheldrüsen der Schabe exprimiert wird. / Biogenic amines are small organic compounds that act as neurotransmitters, neuromodulators and/or neurohormones in vertebrates and in invertebrates. They form an important group of messenger substances and mediate their diverse effects by binding to membrane receptors that primarily belong to the large gene-family of G protein-coupled receptors. In insects, the group of biogenic amine messengers consists of five members: dopamine, tyramine, octopamine, serotonin, and histamine. Besides many other effects, some of these biogenic amines were shown, for example, to modulate gustatory sensitivity to sucrose stimuli in the honeybee (Apis mellifera). I have investigated various aspects of the aminergic signal transduction in the “model organisms” honeybee and American cockroach (Periplaneta americana). So far, I have characterized two dopamine receptors, a tyramine receptor, an octopamine receptor and a serotonin receptor of the honeybee, which is well-known for its learning and memory capacities. The receptors where expressed in cultivated mammalian cells in order to analyze their pharmacological and functional (i.e., second messenger coupling) properties. The spatiotemporal expression patterns of the respective receptor mRNA were investigated in the honeybee brain by using different techniques (RT PCR, Northern blotting, in situ-hybridization). The salivary glands of the American cockroach were used as a model object in order to investigate the cellular effects of biogenic amines. Both dopamine and serotonin trigger salivary secretion in isolated salivary glands. The quality of the secreted saliva is, however, different. Stimulation of the glands by serotonin results in the production of a protein-rich saliva, whereas stimulation by dopamine results in saliva that is protein-free. Serotonin-induced protein secretion is mediated by an increase in the intracellular concentration of cAMP. The pharmacological properties of dopamine receptors associated with cockroach salivary glands were investigated and the molecular characterization of putative aminergic receptors of the cockroach was initiated. Furthermore, I have characterized the ebony gene of the cockroach. This gene encodes an enzyme that is probably involved in the inactivation of biogenic amines in the cockroach (as in other insects). The ebony gene is expressed in the brain and in the salivary glands of the cockroach.

Affective disorders represent one of the greatest global burdens of disease. Work in patients with affective disorders demonstrates that serotonin (5-HT) signaling within the prefrontal cortex, particularly at the level of the 5-HT receptors, plays an integral role in both the pathology and treatment of these diseases. Surprisingly, the characterization of the prefrontal 5-HT receptors under both healthy and pathological conditions remains incomplete. The technique of whole cell electrophysiological recording provides an unparalleled tool for investigating the functional effects of these 5-HT receptors on neurons in acute prefrontal cortical slices. The objectives of my thesis were to delve deeper into the 5-HT receptor subtypes that modulate the prefrontal cortex in the healthy control rodents and to examine how this modulation was disrupted in a rodent model of affective disorders. In work from healthy control rodents, I examined two prefrontal 5-HT receptor-mediated currents. I show for the first time the presence of the 5-HT1A receptor during the early postnatal period, a critical developmental window during which this receptor programs adult anxiety behaviors. In adulthood, I characterized an inhibitory current mediated by the 5-ht5A receptor; findings that will permit the classification of this receptor within the 5-HT receptor family. Collectively, this investigation of functional early 5-HT1A receptors and adult 5-ht5A receptors offers a novel conceptual framework for understanding 5-HT receptor modulation of the healthy prefrontal cortex. To model vulnerability to affective disorder in the rodent, I used the early stress of maternal separation. In early stress rodents, I observed a marked increase in 5-HT1A receptor currents during the early postnatal period, the critical time window for the programming of anxiety. By comparison, in adulthood I found that rodents exposed to early stress displayed increased 5-HT2A receptor currents. These findings provide novel insight into the developmental and long-lasting pathology underlying early stress, indicating that the early prefrontal 5-HT1A receptor and adult prefrontal 5-HT2A receptors as a potential therapeutic target in treatment of affective disorders At a fundamental level, the findings provided herein offer critical insight into the cellular mechanisms underlying affective disorders, one of the most debilitating and costly diseases worldwide.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 5 uppsatser.

O gênero Psychotria destaca-se na família RUBIACEAE pela produção de alcalóides bioativos e por sua taxonomia complexa, sendo muitas vezes relacionado aos gêneros Palicourea, Cephaelis, Calycodendron e Calycosia. A divisão de Psychotria nos subgêneros Psychotria, Tetramerae e Heteropsychotria foi proposta com o objetivo de auxiliar a classificação quimiotaxonômica do gênero. Estudos demonstram que o subgênero Psychotria (espécies pantropicais) produz alcalóides poliméricos, formados por duas ou mais unidades de triptamina; enquanto o subgênero Heteropsychotria parece estar envolvido com a produção de alcalóides indol monoterpênicos, de acordo com pesquisas realizadas com diferentes espécies coletadas no Sul do Brasil. O isolamento e purificação dos alcalóides estrictosamida, ácido estrictosidínico e miriantosina, a partir de Psychotria myriantha, corroboram com esta hipótese, permitindo a inclusão da espécie no subgênero Heteropsychotria. A literatura descreve várias atividades para extratos e alcalóides isolados de espécies de Psychotria, como antimicrobiana e analgésica, por exemplo. Neste trabalho, o extrato n-butanólico de alcalóides de P. myriantha, além de seus alcalóides isolados, apresentaram atividade inibidora da migração de leucócitos, sugerindo um efeito antiinflamatório, e capacidade de inibir a ação da enzima acetilcolinesterase. Extratos e alcalóides isolados da espécie foram avaliados quanto à atividade antioxidante em CCD, frente ao DPPH, apresentando resultado negativo. O extrato EBA e o alcalóide ácido estrictosidínico aumentaram o tempo de latência no teste da retirada da cauda frente ao estímulo térmico, indicando uma atividade analgésica do tipo opióide. A influência do ácido estrictosidínico, alcalóide isolado em maior quantidade em massa, sobre os níveis de DA, DOPAC, 3-MT, HVA, 5-HT e 5-HIAA em estruturas cerebrais de ratos foi verificada. Hipocampos de animais que receberam injeção intra-hipocampal bilateral de ácido estrictosidínico (20 μg/μL) apresentaram redução de 83,4 % nos níveis de serotonina, em comparação ao grupo controle, enquanto os córtices desses animais apresentaram redução nos níveis de DOPAC (35,9%), 3-MT (24,7%) e 5-HIAA (9%). Hipocampos e estriados de ratos tratados com injeção i.p. de ácido estrictosidínico (10 mg/kg) demonstraram diminuição de 63,4 e 28,7% nos níveis de 5-HT, respectivamente. As alterações nos níveis de aminas biogênicas nas estruturas avaliadas, além das atividades analgésica e inibidora da acetilcolinesterase, indicam que P. myriantha e espécies do subgênero Heteropsychotria constituem uma potencial fonte de substâncias bioativas no tratamento de distúrbios do sistema nervoso central. / Psychotria genus is an important in RUBIACEAE due to bioactive alkaloids production and complex taxonomy, being related to Palicourea, Cephaelis, Calycodendron and Calycosia genera. The division of Psychotria in Psychotria, Tetramerae and Heteropsychotria subgenera was proposed with the aim of aiding the genus chemotaxonomic classification. Studies demonstrate that Psychotria subgenus produce polyindoline alkaloids formed by two or more triptamine units; whereas Heteropsychotria subgenus seems to be involved with indole monoterpene alkaloids production, according to researches with different species collected in Southern Brazil. Isolation and purification of strictosamide, strictosidinic acid and miriantosine from Psychotria myriantha corroborated this hypothesis, allowing its inclusion into Heteropsychotria subgenus. The scientific literature describes several activities to Psychotria extracts and alkaloids, such as antimicrobial and analgesic, for example. In this work, P. myriantha n-butanolic alkaloid extract and isolated compounds inhibited the leukocyte migration, suggesting an antiinflammatory activity, and a weak ability to inhibit the action of acetylcholinesterase enzyme. Alkaloids and extracts from P. myriantha were evaluated as regards their antioxidant activity using DPPH, with no positive results. EBA and strictosidinic acid increased the latency in the tail flick model, indicating an opioid analgesic activity. The influence of strictosidinic acid on the levels of DA, DOPAC, 3-MT, HVA, 5-HT and 5-HIAA in brain structures of rats was verified. Hippocampus with intra-hippocampal injection of strictosidinic acid (20 μg/μL) displayed a decrease of 83.4% in serotonin levels, in comparison with control group; whereas the cortex showed a decrease in the levels of DOPAC (35.9%), 3-MT (24.7%) and 5-HIAA (9%). Hippocampus and striatum that received intraperitoneal injection of strictosidinic acid (10 mg/kg) showed 5-HT levels reduction of 63.4 e 28.7%, respectively. The biogenic amine levels alterations in the studied structures, associated with the analgesic and acetylcholinesterase inhibitor activities, suggest that P. myriantha and species from the Heteropsychotria subgenus constitute a font of bioactive compounds in the treatment of central nervous system disturbs.

Connectomics – the study of neural connectivity – is primarily concerned with the mapping and characterisation of wired synaptic links; however, it is well established that long-distance chemical signalling via extrasynaptic volume transmission is also critical to brain function. As these interactions are not visible in the physical structure of the nervous system, current approaches to connectomics are unable to capture them. This work addresses the problem of missing extrasynaptic interactions by demonstrating for the first time that whole-animal volume transmission networks can be mapped from gene expression and ligand-receptor interaction data, and analysed as part of the connectome. Complete networks are presented for the monoamine systems of Caenorhabditis elegans, along with a representative sample of selected neuropeptide systems. A network analysis of the synaptic (wired) and extrasynaptic (wireless) connectomes is presented which reveals complex topological properties, including extrasynaptic rich-club organisation with interconnected hubs distinct from those in the synaptic and gap junction networks, and highly significant multilink motifs pinpointing locations in the network where aminergic and neuropeptide signalling is likely to modulate synaptic activity. Thus, the neuronal connectome can be modelled as a multiplex network with synaptic, gap junction, and neuromodulatory layers representing inter-neuronal interactions with different dynamics and polarity. This represents a prototype for understanding how extrasynaptic signalling can be integrated into connectomics research, and provides a novel dataset for the development of multilayer network algorithms.

O gênero Psychotria destaca-se na família RUBIACEAE pela produção de alcalóides bioativos e por sua taxonomia complexa, sendo muitas vezes relacionado aos gêneros Palicourea, Cephaelis, Calycodendron e Calycosia. A divisão de Psychotria nos subgêneros Psychotria, Tetramerae e Heteropsychotria foi proposta com o objetivo de auxiliar a classificação quimiotaxonômica do gênero. Estudos demonstram que o subgênero Psychotria (espécies pantropicais) produz alcalóides poliméricos, formados por duas ou mais unidades de triptamina; enquanto o subgênero Heteropsychotria parece estar envolvido com a produção de alcalóides indol monoterpênicos, de acordo com pesquisas realizadas com diferentes espécies coletadas no Sul do Brasil. O isolamento e purificação dos alcalóides estrictosamida, ácido estrictosidínico e miriantosina, a partir de Psychotria myriantha, corroboram com esta hipótese, permitindo a inclusão da espécie no subgênero Heteropsychotria. A literatura descreve várias atividades para extratos e alcalóides isolados de espécies de Psychotria, como antimicrobiana e analgésica, por exemplo. Neste trabalho, o extrato n-butanólico de alcalóides de P. myriantha, além de seus alcalóides isolados, apresentaram atividade inibidora da migração de leucócitos, sugerindo um efeito antiinflamatório, e capacidade de inibir a ação da enzima acetilcolinesterase. Extratos e alcalóides isolados da espécie foram avaliados quanto à atividade antioxidante em CCD, frente ao DPPH, apresentando resultado negativo. O extrato EBA e o alcalóide ácido estrictosidínico aumentaram o tempo de latência no teste da retirada da cauda frente ao estímulo térmico, indicando uma atividade analgésica do tipo opióide. A influência do ácido estrictosidínico, alcalóide isolado em maior quantidade em massa, sobre os níveis de DA, DOPAC, 3-MT, HVA, 5-HT e 5-HIAA em estruturas cerebrais de ratos foi verificada. Hipocampos de animais que receberam injeção intra-hipocampal bilateral de ácido estrictosidínico (20 μg/μL) apresentaram redução de 83,4 % nos níveis de serotonina, em comparação ao grupo controle, enquanto os córtices desses animais apresentaram redução nos níveis de DOPAC (35,9%), 3-MT (24,7%) e 5-HIAA (9%). Hipocampos e estriados de ratos tratados com injeção i.p. de ácido estrictosidínico (10 mg/kg) demonstraram diminuição de 63,4 e 28,7% nos níveis de 5-HT, respectivamente. As alterações nos níveis de aminas biogênicas nas estruturas avaliadas, além das atividades analgésica e inibidora da acetilcolinesterase, indicam que P. myriantha e espécies do subgênero Heteropsychotria constituem uma potencial fonte de substâncias bioativas no tratamento de distúrbios do sistema nervoso central. / Psychotria genus is an important in RUBIACEAE due to bioactive alkaloids production and complex taxonomy, being related to Palicourea, Cephaelis, Calycodendron and Calycosia genera. The division of Psychotria in Psychotria, Tetramerae and Heteropsychotria subgenera was proposed with the aim of aiding the genus chemotaxonomic classification. Studies demonstrate that Psychotria subgenus produce polyindoline alkaloids formed by two or more triptamine units; whereas Heteropsychotria subgenus seems to be involved with indole monoterpene alkaloids production, according to researches with different species collected in Southern Brazil. Isolation and purification of strictosamide, strictosidinic acid and miriantosine from Psychotria myriantha corroborated this hypothesis, allowing its inclusion into Heteropsychotria subgenus. The scientific literature describes several activities to Psychotria extracts and alkaloids, such as antimicrobial and analgesic, for example. In this work, P. myriantha n-butanolic alkaloid extract and isolated compounds inhibited the leukocyte migration, suggesting an antiinflammatory activity, and a weak ability to inhibit the action of acetylcholinesterase enzyme. Alkaloids and extracts from P. myriantha were evaluated as regards their antioxidant activity using DPPH, with no positive results. EBA and strictosidinic acid increased the latency in the tail flick model, indicating an opioid analgesic activity. The influence of strictosidinic acid on the levels of DA, DOPAC, 3-MT, HVA, 5-HT and 5-HIAA in brain structures of rats was verified. Hippocampus with intra-hippocampal injection of strictosidinic acid (20 μg/μL) displayed a decrease of 83.4% in serotonin levels, in comparison with control group; whereas the cortex showed a decrease in the levels of DOPAC (35.9%), 3-MT (24.7%) and 5-HIAA (9%). Hippocampus and striatum that received intraperitoneal injection of strictosidinic acid (10 mg/kg) showed 5-HT levels reduction of 63.4 e 28.7%, respectively. The biogenic amine levels alterations in the studied structures, associated with the analgesic and acetylcholinesterase inhibitor activities, suggest that P. myriantha and species from the Heteropsychotria subgenus constitute a font of bioactive compounds in the treatment of central nervous system disturbs.

Este trabalho apresenta a continuidade da avaliação farmacológica das substâncias LASSBio-579, LASSBio-580 e LASSBio-581, através de ensaios in vitro e in vivo, em busca de um novo protótipo para o desenvolvimento de novos fármacos antipsicóticos de segunda geração. LASSBio-581 se liga a receptores D2-like (Ki=0,95 μM), 5-HT1A (Ki=1,2 μM) e 5-HT2A (Ki=11 μM) com afinidades moderadas. Esta substância é capaz de reduzir a temperatura corporal de roedores, um efeito provavelmente mediado pela ativação de receptores 5-HT1A, e inibir o desenvolvimento de head-twiches e ear-scratches induzidos pela administração de um antagonista de receptores 5-HT2A. Estes efeitos demonstram a capacidade de LASSBio-581 em modular o sistema serotonérgico in vivo e in vitro. Porém, quanto avaliado em modelos animais preditivos de ação antipsicótica, LASSBio-581 foi inativo. LASSBio-580 também é capaz de se ligar a receptores D2-like (Ki=0,73 μM), 5-HT1A (Ki=0,48 μM) e 5-HT2A (Ki=5,7 μM) com afinidades moderadas. Esta substância não foi capaz de inibir o desenvolvimento do comportamento de escalada nem a redução da temperatura corporal de roedores induzidos por apomorfina, não apresentando potencial atividade antipsicótica nos ensaios realizados. Já LASSBio-579 é capaz de modular três diferentes sistemas neurotransmissores envolvidos na patofisiologia da esquizofrenia: a neurotransmissão dopaminérgica, serotonérgica e glutamatérgica. Esta substância se liga a receptores D2-like (Ki=0,11 μM), 5-HT1A (Ki=0,09 μM) e 5-HT2A (Ki=2,2 μM) com afinidades adequadas para uma molécula protótipo que se liga a múltiplos alvos. Apresenta ação antidopaminérgica in vivo, demonstrada em três modelos animais preditivos de atividade antipsicótica (sintomas positivos): inibição da estereotipia anfetamínica (NEVES et al., 2003), bloqueio do comportamento de escalada induzido por apomorfina e hipotermia apomorfínica. A ação agonista 5-HT1A de LASSBio-579 in vivo foi claramente demonstrada através de ensaios de aferição da temperatura corporal, onde o efeito hipotérmico induzido por esta substância é completamente bloqueado pela pré-administração de WAY 100635. Porém, a habilidade de LASSBio-579 em modular a atividade de receptores 5-HT2A in vivo permanece incerta. Ensaios eletrofisiológicos preliminares demonstraram um aumento da liberação de glutamato induzido por LASSBio-579 que parece ser mediado pela ativação de receptores 5-HT2A, porém comportamentos ou efeitos relacionados a ativação deste sub-tipo de receptor serotonérgico não foram identificados em roedores tratados com LASSBio-579. Além disso, a administração de LASSBio-579 não induziu efeitos catatônicos em camundongos em doses até 12 vezes superiores àquela ativa no modelo do bloqueio do comportamento de escalada induzido por apomorfina. Estes resultados demonstram que a estratégia de planejamento de fármacos baseado na estrutura do ligante empregada neste trabalho se mostrou bem sucedida. LASSBio-579 pode ser considerado um novo protótipo de fármaco antipsicótico de segunda geração, ativo em modelos animais de sintomas positivos da esquizofrenia e com baixo potencial de indução de efeitos motores. Porém, algumas limitações em seu perfil farmacológico pode ser identificadas. A afinidade desta substância por receptores dopaminérgicos e serotonérgicos é considerada moderada e inferior a de antipsicóticos atualmente no mercado. Ainda, LASSBio-579 induziu um prejuízo na coordenação motora em roedores e apresentou um perfil farmacocinético pouco adequado a utilização clinica (CONRADO et al., 2008). Estes dados encorajam a busca de substâncias com um perfil farmacológico superior ao de LASSBio-579. Neste sentido, uma triagem farmacológica de 18 derivados N-fenilpiperazínicos análogos a LASSBio-579 foi realizada. Os resultados obtidos nos ensaios de radioligação a receptores D2-like, 5-HT1A e 5-HT2A foram utilizados na proposição de relações qualitativas entre estrutura química das substâncias e a afinidade apresentada pelos diferentes receptores. A partir dos resultados obtidos in vitro, cinco outras substâncias foram selecionadas para avaliação da potencial atividade frente a sintomas positivos da esquizofrenia no modelo de bloqueio do comportamento de escalada induzido por apomorfina. Neste ensaio, apenas LASSBio-664 apresentou atividade, sem induzir catatonia nos animais. Porém, esta substância também induz um importante prejuízo motor nos animais. Ensaios adicionais são necessários a fim de diferenciar o perfil farmacológico de LASSBio-664 e LASSBio-579. Outro objetivo deste trabalho foi iniciar o desenvolvimento de um modelo animal de sintomas da esquizofrenia. Os resultados obtidos até o momento apontam para a possibilidade do desenvolvimento de um modelo relacionado a sintomas negativos/cognitivos da esquizofrenia baseados na esposição à natação forçada repetida. Foi demonstrado que apenas clozapina e não imipramina é capaz de reverter o aumento de imobilidade ao longo dos dias acarretado pela exposição repetida à natação forçada em roedores. Este dado demonstra uma potencial validade preditiva, o primeiro grau de validação necessário para um novo modelo animal. O efeito de LASSBio-579 também foi avaliado neste procolo experimental. / This study strengthened the pharmacological evaluation of the heterocyclic Nphenylpiperazine derivatives LASSBio-579, LASSBio-580 and LASSBio-581 by means of in vitro and in vivo pharmacological assays searching a new second generations antipsychotic lead compound. LASSBio-581 presented moderate affinitties for D2-like (Ki=0.95 μM), 5-HT1A (Ki=1.2 μM) e 5-HT2A (Ki=11 μM). This compound induced an hypothermic effect in rodents probably mediated by 5-HT1A receptor activation. LASSBio-581 administration inhibited the induction of head-twiches and ear-scratches by a 5-HT2A agonist. These results shown that LASSBio-581 modulates serotonergic neurotransmission in vivo and in vitro. However, it was inactive on animal models predictive of antipsychotic activity. LASSBio-580 presented moderate affinitties for D2-like (Ki=0.73 μM), 5-HT1A (Ki=0.48 μM) e 5-HT2A (Ki=5.7 μM). This compound did not inhibited apomophine-induced climbing nor apomorphine-induced hypothermia. Thus, among the three compounds initially evaluated, LASSBio-579 was the only one that exhibited promising results. This derivative was able to modulate three neurotransmitter systems involved in schizophrenia’s pathophysiology: dopaminergic, serotonergic and glutamateric ones. As a multi-target lead compound, LASSBio-579 presented adequated affinities for D2-like, 5-HT1A and 5-HT2A receptors (Ki D2-like = 0.11 μM, 5-HT1A = 0.093 μM and 5-HT2A = 2.2 μM). Its antidopaminergic in vivo effect was demonstrated in three animal models of positive symptons of schizophrenia: amphetamineinduced stereotypy (NEVES et al., 2003), apomorphine-induced climbing behavior and apomorphine-induced hypothermia. Regarding the serotonergic system, LASSBio-579 was considered a 5-HT1A receptor agonist, since the hypothermia induced by this compound was blocked by WAY 100,635 pre-administration. The ability of LASSBio-579 to modulate 5-HT2A receptors was not fully characterized. Electrophysiological assays demonstrated an increase on synaptic glutamate release induced by LASSBio-579 that may be related to 5-HT2A receptor activation, however this compound did not induce any behavior related to 5-HT2A activation in rodents. In addition, LASSBio-579 did not induce catalepsy in mice at doses 12 folds higher than those active at apomorphine-induced climbing test. Thus, LASSBio-579 represents a new antipsychotic lead compound active in animal models of positive symptoms of schizophrenia and with a mild propensity to induce motor side effects. However, some limitations on LASSBio-579’s pharmacological profile could be identified. The affinity of LASSBio-579 for dopamine and serotonin receptors is moderate and lower than those presented by second generation antipsychotics. Furthermore, this compound induced a mild decrease on locomotion and exploratory behavior, a meaningful motor coordination impairment and presents a limited oral bioavailability and a low brain penetration (CONRADO et al., 2008). Considering this, a pharmacologycal screening of 18 N-phenylpiperazine derivatives were done in order to achieve an optimized analogue of LASSBio-579. Structural features of this molecular scaffold were discussed regarding binding affinity and selectivity for D2-like, 5-HT1A and 5-HT2A receptors. Among the compounds prepared, LASSBio-664 exhibited an adequate binding profile and a potential for schizophrenia positive symptoms treatment without cataleptogenic effects. However, the motor coordination impairment remained. Additional pharmacological characterization of LASSBio-664 is still needed to find differences from LASSBio-579’s profile. Another aim of this study was to start the development of an animal model of schizophrenia symptons. It was shown that clozapine but not imipramine presented an anti-immobility effect in animals repeated exposed to forced swimming. This result points to the usefullness of repeated forced swimming protocol for developing new animal models predictive of antipsychotic action. The effect of LASSBio-579 in this protocol was also evaluated.

O sistema serotoninérgico há muito tempo vem sendo relacionado ao comportamento agressivo e particularmente o subtipo de receptor 5-HT1A está envolvido na modulação da agressividade. Estudos prévios utilizando agonistas que atuam seletivamente sobre estes receptores demonstraram tanto redução como aumento da agressão. Sabe-se que estes receptores são encontrados em diversas regiões encefálicas onde atuam como auto e heteroreceptores, apresentando diferentes funções, dependendo de sua localização. Heteroreceptores 5-HT1A, além de outras regiões, são encontrados no córtex pré-frontal, uma região encefálica particularmente importante no controle inibitório de comportamentos agressivos e impulsivos. O objetivo do presente estudo foi verificar os possíveis efeitos antiagressivos da microinjeção do agonista seletivo dos receptores 5-HT1A, F15599, na região ventro-orbital do córtex pré-frontal de camundongos machos da linhagem CF-1, previamente submetidos ao protocolo de provocação social, caracterizado por elevar os níveis de agressividade a padrões considerados violentos. Nossos resultados mostraram que a microinjeção das menores doses de F15599 (0,03 e 0,1 μg) reduziu significativamente a frequência de mordidas além de ocorrer uma tendência à redução da frequência de ataques laterais para o grupo de animais que recebeu a dose de 0,03 μg. Estes efeitos anti-agressivos não foram acompanhados por alterações nos demais elementos comportamentais relacionados à agressividade: perseguir o intruso, cheirar o intruso e sacudir a cauda. Também não foram observadas alterações na duração dos elementos comportamentais não agressivos caminhar e rearing. Ocorreu aumento apenas na duração do comportamento de grooming para o grupo de animais que recebeu a dose de 0,03 μg do agonista. Os resultados do presente estudo confirmaram o envolvimento da região VO do CPF e do sistema serotoninérgico, mais precisamente do subtipo de receptor 5-HT1A na modulação da agressividade exacerbada. / The serotonergic system has long been linked to aggressive behavior and particularly 5-HT1A receptor subtype is involved in modulation of aggressiveness. Previous studies using agonists that act selectively on these receptors showed both reduction and increased aggression. It is known that these receptors are found in several brain regions where they act as auto and heteroreceptors, with different functions, depending on their location. 5-HT1A heteroreceptors, as well as other regions, are found in the prefrontal cortex (PFC), a brain region particularly important in the inhibitory control of aggressive and impulsive behaviors. The aim of this study was to investigate the possible anti-aggressive effects of microinjections of F15599, a selective 5-HT1A agonist, in the ventral orbital prefrontal cortex (VO PFC) of CF-1 male mice, previously submitted to social instigation, characterized by increasing aggression to high levels. Our results showed that the microinjection of the lower doses of F15599 (0.03 and 0.1 μg) significantly reduced attack bite frequency. Furthermore, there was a tendency to reduce sideway threats for the group that received 0.03 μg agonist microinjection. These anti-aggressive effects were not accompanied by changes in other elements of the behavioral repertoire related to aggression: pursuit the intruder, sniff the intruder and tail rattle. There were also no changes observed in the duration of nonaggressive behavioral repertoire elements, walking and rearing. Only for grooming behavior was an increased duration observed for the group that received 0.03 μg agonist dose. The results of this study confirmed the involvement of VO PFC and serotonergic system, specifically de 5- HT1A receptor subtype, in the modulation of escalated aggressive behavior.