A atribuição da percepção da dor pelos peixes é um assunto controverso no meio científico. Alguns autores associam a percepção da dor a estruturas neocorticais que estão ausentes em peixes. Entretanto, estudos recentes têm demonstrado que os peixes são capazes de perceber e responder a estímulos nocivos de maneira semelhante ao que é observado em mamíferos, sendo estas respostas sensíveis à administração de morfina. Além disso, estudos pioneiros de nosso laboratório demonstraram a existência de um sistema analgésico endógeno em peixes. O objetivo deste estudo foi avaliar se este sistema analgésico endógeno pode ser ativado pelo estresse. A natureza neuroquímica deste sistema e a participação de uma região telencefálica, o telencéfalo dorsomedial (Dm), na modulação da antinocicepção também foram investigados. Nossos dados demonstram que o estresse de restrição de 3 e 5 minutos de duração inibe a resposta comportamental à injeção subcutânea de formalina a 3% na região da nadadeira adiposa no peixe Leporinus macrocephalus, sugerindo que este procedimento é capaz de ativar um sistema antinociceptivo endógeno. Além disso, a antinocicepção induzida pelo estresse de restrição de 3 e 5 min é de curta duração, sendo observada apenas por 5 min após o término da restrição. A análise da natureza neuroquímica da antinocicepção induzida pelo estresse de restrição revelou participação do sistema opióde e canabinoide na modulação desta resposta. O tratamento prévio com injeção intraperitoneal de naloxona (30 mg.kg-1), um antagonista opioide não seletivo, bloqueou a antinocicepção induzida pela restrição de 3 min de duração, mas não foi capaz de inibir a antinocicepção induzida pela restrição de 5 min de duração. Já o tratamento prévio com injeção intraperitoneal de AM251 (3 mg.kg-1), um antagonista de receptores canabinoides tipo 1, bloqueou a antinocicepção induzida pelo estresse de restrição de 3 e 5 min de duração, sugerindo que o sistema canabinoide desempenha um papel fundamental na antinocicepção induzida por esta modalidade de estresse na espécie estudada. Nosso estudo também demonstrou que a região do telencéfalo dorsomedial está envolvida na modulação da antinocicepção induzida pelo estresse de restrição no peixe L. macrocephalus. A microinjeção de midazolan (40 e 80 nmol), um agonista de receptores benzodiazepínicos, no telencéfalo Dm bloqueou a antinocicepção induzida pela restrição de 3 e 5 min de duração. Além disso, o tratamento prévio com flumazenil (80 e 160 nmol), um antagonista específico de receptores benzodiazepínicos, inibiu os efeitos do tratamento com midazolan, demonstrando que o bloqueio da antinocicepção promovido pelo midazolan ocorre pela ativação específica dos receptores benzodiazepínicos. Juntos estes resultados trazem novas perspectivas acerca do entendimento sobre a percepção nociceptiva em peixes. Este é o primeiro trabalho que traz evidências acerca da existência de um sistema de modulação da dor ativado pelo estresse e demonstra a participação de uma região encefálica específica na modulação desta antinocicepção. Estes resultados indicam que as vias analgésicas endógenas em peixes são ativadas de maneira semelhante aos mamíferos, sugerindo que estes animais possuem um processamento complexo da informação nociceptiva. / The assignment of pain perception by fish is controversial among scientists. Some authors associate the pain perception to neocortical structures that are absent in fish. However, recent studies have shown that fish are able to perceive and respond to noxious stimuli, similar to observed in mammals, and this responses are sensitive to morphine administration. Furthermore, pioneering studies from our laboratory have demonstrated the existence of an endogenous analgesic system in fish. This study aimed to evaluate if this endogenous analgesic system can be activated by stress, the neurochemical nature of this system and involvement of a telencephalic region, the dorsomedial (Dm) telencephalon, in the antinociception modulation. Our data demonstrate that 3 and 5 min of restraint stress inhibits the behavioral response to subcutaneous injection of formalin 3 % in the adipose fin in the fish Leporinus macrocephalus, suggesting that this procedure can activate an endogenous antinociceptive system. Furthermore, stress-induced antinociception induced by 3 and 5 min of restraint is short, with the antinociceptive effects being observed only for 5 min after the restriction. The analysis of the neurocheamical nature of antinociception induced by restraint stress revealed the involvement of opioid and cannabinoid systems in the modulation of this response. The pre-treatment with intraperitoneal injection of naloxone (30 mg.kg-1), a non-selective opioid receptors antagonist, blocked the antinociception induced by 3 min of restraint, but was not able to inhibit the antinociception induced by 5 min of restraint. The pre-treatment with intraperitoneal injection of AM251 ( 3 mg.kg-1), a type 1 cannabinoid receptors antagonist, blocked the stress-induced antinociception promoted by 3 and 5 min of restraint, suggesting that the cannabinoid system plays a critical role in this type of stress-induced antinociception in the studied species. Our study also showed that the dorsomedial telencephalon is involved in the modulation of stress-induced antinociception in fish L. macrocephalus. The microinjection of midazolan (40 and 80 nmol), a benzodiazepine receptors agonist, in the Dm blocked the stress-induced antinociception promoted by 3 and 5 min of restraint. Furthermore, pre-treatment with flumazenil (80 and 160 nmol), a benzodiazepine receptors selective antagonist, inhibited the effects of the midazolan treatment, demonstrating that the antinociception blockade by midazolan is promoted by specific activation of benzodiazepine receptors. Together these results provide new insights on the understanding of nociceptive perception in fish. This is the first study that demonstrates evidence for the existence of a pain modulation system activated by stress in fish and demonstrates the involvement of a specific brain region in the modulation of this antinociception. These results indicate that the endogenous analgesic pathways in fish are activated in a similar manner to mammals, suggesting that these animals have a complex processing of nociceptive information.

The neuropeptide, corticotropin-releasing factor (CRF), plays a critical role in the central regulation of various stress-related behaviours, including those unique to subjects with prior cocaine experience. The three series of experiments presented in this dissertation explored the role of two neurochemical systems, the teneurin C-terminal associated peptides (TCAP) and the endocannabinoids (eCBs), in several cocaine- and anxiety-related behaviours induced or mediated by CRF. The first series of experiments examined the effects of TCAP-1 on the reinstatement of cocaine seeking and expression of cocaine-induced behavioural sensitization. Repeated (5-day), but not acute, TCAP-1 treatment blocked the reinstatement of cocaine seeking induced by central injections of CRF. TCAP-1 was, however, without effect on footshock- or cocaine-induced reinstatement. Repeated TCAP-1 further interfered with the expression of behavioural sensitization to a CRF, but not a cocaine, challenge. These findings suggest that TCAP-1 normalizes CRF signaling dysregulated by cocaine exposure to interfere in the subsequent effects of CRF on cocaine-related behaviours. A parallel series of experiments investigated the role of eCB signaling at CB1 receptors in the reinstatement of cocaine seeking and cocaine-sensitized locomotion. Pretreatment with the CB1 receptor antagonist, AM251, selectively interfered with CRF-, but not footshock- or cocaine-induced reinstatement. AM251 further blocked the expression of behavioural sensitization induced by challenge injections of both CRF and cocaine. These findings reveal a mediating role for CB1 receptor transmission in the effects of CRF on cocaine-related behaviours. A final series of experiments examined the role of CB1 receptor transmission in the behavioural anxiety induced by central injections of CRF, and by withdrawal from chronic cocaine exposure. AM251, although itself anxiogenic, reversed anxiety induced by CRF and cocaine withdrawal. Furthermore, AM251 elevated plasma corticosterone levels, indicative of increased HPA axis activity, irrespective of CRF treatment or cocaine withdrawal. These findings suggest that CRF- and cocaine withdrawal-induced anxiety are mediated, at least in part, by CB1 receptor transmission, independent of HPA axis regulation. The collective findings are discussed within a framework of CRF-TCAP-eCB interactions, wherein TCAP-1 and AM251 are proposed to act in parallel to modulate amygdalar CRF transmission, and thus regulate the expression of cocaine- and anxiety-related behaviours.

The neuropeptide, corticotropin-releasing factor (CRF), plays a critical role in the central regulation of various stress-related behaviours, including those unique to subjects with prior cocaine experience. The three series of experiments presented in this dissertation explored the role of two neurochemical systems, the teneurin C-terminal associated peptides (TCAP) and the endocannabinoids (eCBs), in several cocaine- and anxiety-related behaviours induced or mediated by CRF. The first series of experiments examined the effects of TCAP-1 on the reinstatement of cocaine seeking and expression of cocaine-induced behavioural sensitization. Repeated (5-day), but not acute, TCAP-1 treatment blocked the reinstatement of cocaine seeking induced by central injections of CRF. TCAP-1 was, however, without effect on footshock- or cocaine-induced reinstatement. Repeated TCAP-1 further interfered with the expression of behavioural sensitization to a CRF, but not a cocaine, challenge. These findings suggest that TCAP-1 normalizes CRF signaling dysregulated by cocaine exposure to interfere in the subsequent effects of CRF on cocaine-related behaviours. A parallel series of experiments investigated the role of eCB signaling at CB1 receptors in the reinstatement of cocaine seeking and cocaine-sensitized locomotion. Pretreatment with the CB1 receptor antagonist, AM251, selectively interfered with CRF-, but not footshock- or cocaine-induced reinstatement. AM251 further blocked the expression of behavioural sensitization induced by challenge injections of both CRF and cocaine. These findings reveal a mediating role for CB1 receptor transmission in the effects of CRF on cocaine-related behaviours. A final series of experiments examined the role of CB1 receptor transmission in the behavioural anxiety induced by central injections of CRF, and by withdrawal from chronic cocaine exposure. AM251, although itself anxiogenic, reversed anxiety induced by CRF and cocaine withdrawal. Furthermore, AM251 elevated plasma corticosterone levels, indicative of increased HPA axis activity, irrespective of CRF treatment or cocaine withdrawal. These findings suggest that CRF- and cocaine withdrawal-induced anxiety are mediated, at least in part, by CB1 receptor transmission, independent of HPA axis regulation. The collective findings are discussed within a framework of CRF-TCAP-eCB interactions, wherein TCAP-1 and AM251 are proposed to act in parallel to modulate amygdalar CRF transmission, and thus regulate the expression of cocaine- and anxiety-related behaviours.

A atribuição da percepção da dor pelos peixes é um assunto controverso no meio científico. Alguns autores associam a percepção da dor a estruturas neocorticais que estão ausentes em peixes. Entretanto, estudos recentes têm demonstrado que os peixes são capazes de perceber e responder a estímulos nocivos de maneira semelhante ao que é observado em mamíferos, sendo estas respostas sensíveis à administração de morfina. Além disso, estudos pioneiros de nosso laboratório demonstraram a existência de um sistema analgésico endógeno em peixes. O objetivo deste estudo foi avaliar se este sistema analgésico endógeno pode ser ativado pelo estresse. A natureza neuroquímica deste sistema e a participação de uma região telencefálica, o telencéfalo dorsomedial (Dm), na modulação da antinocicepção também foram investigados. Nossos dados demonstram que o estresse de restrição de 3 e 5 minutos de duração inibe a resposta comportamental à injeção subcutânea de formalina a 3% na região da nadadeira adiposa no peixe Leporinus macrocephalus, sugerindo que este procedimento é capaz de ativar um sistema antinociceptivo endógeno. Além disso, a antinocicepção induzida pelo estresse de restrição de 3 e 5 min é de curta duração, sendo observada apenas por 5 min após o término da restrição. A análise da natureza neuroquímica da antinocicepção induzida pelo estresse de restrição revelou participação do sistema opióde e canabinoide na modulação desta resposta. O tratamento prévio com injeção intraperitoneal de naloxona (30 mg.kg-1), um antagonista opioide não seletivo, bloqueou a antinocicepção induzida pela restrição de 3 min de duração, mas não foi capaz de inibir a antinocicepção induzida pela restrição de 5 min de duração. Já o tratamento prévio com injeção intraperitoneal de AM251 (3 mg.kg-1), um antagonista de receptores canabinoides tipo 1, bloqueou a antinocicepção induzida pelo estresse de restrição de 3 e 5 min de duração, sugerindo que o sistema canabinoide desempenha um papel fundamental na antinocicepção induzida por esta modalidade de estresse na espécie estudada. Nosso estudo também demonstrou que a região do telencéfalo dorsomedial está envolvida na modulação da antinocicepção induzida pelo estresse de restrição no peixe L. macrocephalus. A microinjeção de midazolan (40 e 80 nmol), um agonista de receptores benzodiazepínicos, no telencéfalo Dm bloqueou a antinocicepção induzida pela restrição de 3 e 5 min de duração. Além disso, o tratamento prévio com flumazenil (80 e 160 nmol), um antagonista específico de receptores benzodiazepínicos, inibiu os efeitos do tratamento com midazolan, demonstrando que o bloqueio da antinocicepção promovido pelo midazolan ocorre pela ativação específica dos receptores benzodiazepínicos. Juntos estes resultados trazem novas perspectivas acerca do entendimento sobre a percepção nociceptiva em peixes. Este é o primeiro trabalho que traz evidências acerca da existência de um sistema de modulação da dor ativado pelo estresse e demonstra a participação de uma região encefálica específica na modulação desta antinocicepção. Estes resultados indicam que as vias analgésicas endógenas em peixes são ativadas de maneira semelhante aos mamíferos, sugerindo que estes animais possuem um processamento complexo da informação nociceptiva. / The assignment of pain perception by fish is controversial among scientists. Some authors associate the pain perception to neocortical structures that are absent in fish. However, recent studies have shown that fish are able to perceive and respond to noxious stimuli, similar to observed in mammals, and this responses are sensitive to morphine administration. Furthermore, pioneering studies from our laboratory have demonstrated the existence of an endogenous analgesic system in fish. This study aimed to evaluate if this endogenous analgesic system can be activated by stress, the neurochemical nature of this system and involvement of a telencephalic region, the dorsomedial (Dm) telencephalon, in the antinociception modulation. Our data demonstrate that 3 and 5 min of restraint stress inhibits the behavioral response to subcutaneous injection of formalin 3 % in the adipose fin in the fish Leporinus macrocephalus, suggesting that this procedure can activate an endogenous antinociceptive system. Furthermore, stress-induced antinociception induced by 3 and 5 min of restraint is short, with the antinociceptive effects being observed only for 5 min after the restriction. The analysis of the neurocheamical nature of antinociception induced by restraint stress revealed the involvement of opioid and cannabinoid systems in the modulation of this response. The pre-treatment with intraperitoneal injection of naloxone (30 mg.kg-1), a non-selective opioid receptors antagonist, blocked the antinociception induced by 3 min of restraint, but was not able to inhibit the antinociception induced by 5 min of restraint. The pre-treatment with intraperitoneal injection of AM251 ( 3 mg.kg-1), a type 1 cannabinoid receptors antagonist, blocked the stress-induced antinociception promoted by 3 and 5 min of restraint, suggesting that the cannabinoid system plays a critical role in this type of stress-induced antinociception in the studied species. Our study also showed that the dorsomedial telencephalon is involved in the modulation of stress-induced antinociception in fish L. macrocephalus. The microinjection of midazolan (40 and 80 nmol), a benzodiazepine receptors agonist, in the Dm blocked the stress-induced antinociception promoted by 3 and 5 min of restraint. Furthermore, pre-treatment with flumazenil (80 and 160 nmol), a benzodiazepine receptors selective antagonist, inhibited the effects of the midazolan treatment, demonstrating that the antinociception blockade by midazolan is promoted by specific activation of benzodiazepine receptors. Together these results provide new insights on the understanding of nociceptive perception in fish. This is the first study that demonstrates evidence for the existence of a pain modulation system activated by stress in fish and demonstrates the involvement of a specific brain region in the modulation of this antinociception. These results indicate that the endogenous analgesic pathways in fish are activated in a similar manner to mammals, suggesting that these animals have a complex processing of nociceptive information.

Marihuana (Cannabis sativa) é uma planta amplamente usada pelo ser humano há séculos e suas várias aplicações têm benefícios importantes. A planta Cannabis sativa tem sido usada pelo homem como comida, em práticas medicinais e rituais religiosos. Seus efeitos incluem analgesia, alteração na percepção, cognição, memória e atividade psicomotora. Os compostos canabinoides têm sido usados na quimioterapia do câncer e AIDS. No entanto, o uso da marijuana é um problema devido aos seus efeitos indesejados, nesse caso, a atividade psicotrópica apresentada pelos compostos canabinoides. Devido ao grande interesse nos efeitos causados pelos compostos extraídos da Cannabis, vários estudos têm sido realizados com o objetivo de melhor entender a relação entre a estrutura química e a atividade biológica de compostos canabinoides, bem como as suas interações com os receptores canabinoides, CB1 e CB2. Ambos são receptores de sete transmembranas (TM) que pertencem à família classe A, como a da rodopsina bovina, dos receptores acoplados à proteína-G (GPCRs). Esta Tese representa um estudo da modelagem molecular do receptor CB1 baseado na estrutura da rodopsina bovina já publicada, uma vez que a maioria dos efeitos terapêuticos dos canabinoides tem sido mostrado serem mediados pelo receptor canabinoide CB1. Esse trabalho fornece, também, uma investigação da interação ligante-receptor e um estudo da ativação do receptor CB1. Ao final, foi feito um estudo de docking a fim de entender as principais interações que ocorrem entre o ∆9 -THC, a principal molécula psicoativa presente na Cannabis, e seu receptor CB1. / Marijuana (Cannabis sativa) is a widely used plant and its various applications have important benefits. The plant Cannabis sativa has been used by man for centuries for eating, medicinal practices and religious rituals. In human subjects, its effects include analgesia, alterations in perceptions, cognition, memory and psychomotor activity. The cannabinoid compounds have been used in the cancer chemotherapy and AIDS, but the use of marijuana is a problem due to its unwanted effects (the psychotropic activity presented by the cannabinoid compounds). Due to the great interest in the effects caused by the compounds extracted from the Cannabis, several studies have been carried out with the aim to better understand the relationship between the chemical structure and the biological activity of cannabinoid compounds, as well as their interaction with the cannabinoid receptors (CB1 and CB2). Both are seven-transmembrane (TM) receptors that belong to the rhodopsin-like family Class A of G protein coupled receptors (GPCRs). This work represents a study of molecular modeling of the CB1 receptor based upon the published bovine rhodopsin structure, once the most of the therapeutic effects of cannabinoids compounds have been shown to be mediated through the CB1 cannabinoid receptor. This work also provides an investigation of the CB1 receptor-ligand interaction and a study of the CB1 receptor activation. A docking study was also performed in order to understand the main interactions that occur between ∆9 -THC, the principal psychoactive molecule present in cannabis, and its receptor CB1.

Marihuana (Cannabis sativa) é uma planta amplamente usada pelo ser humano há séculos e suas várias aplicações têm benefícios importantes. A planta Cannabis sativa tem sido usada pelo homem como comida, em práticas medicinais e rituais religiosos. Seus efeitos incluem analgesia, alteração na percepção, cognição, memória e atividade psicomotora. Os compostos canabinoides têm sido usados na quimioterapia do câncer e AIDS. No entanto, o uso da marijuana é um problema devido aos seus efeitos indesejados, nesse caso, a atividade psicotrópica apresentada pelos compostos canabinoides. Devido ao grande interesse nos efeitos causados pelos compostos extraídos da Cannabis, vários estudos têm sido realizados com o objetivo de melhor entender a relação entre a estrutura química e a atividade biológica de compostos canabinoides, bem como as suas interações com os receptores canabinoides, CB1 e CB2. Ambos são receptores de sete transmembranas (TM) que pertencem à família classe A, como a da rodopsina bovina, dos receptores acoplados à proteína-G (GPCRs). Esta Tese representa um estudo da modelagem molecular do receptor CB1 baseado na estrutura da rodopsina bovina já publicada, uma vez que a maioria dos efeitos terapêuticos dos canabinoides tem sido mostrado serem mediados pelo receptor canabinoide CB1. Esse trabalho fornece, também, uma investigação da interação ligante-receptor e um estudo da ativação do receptor CB1. Ao final, foi feito um estudo de docking a fim de entender as principais interações que ocorrem entre o ∆9 -THC, a principal molécula psicoativa presente na Cannabis, e seu receptor CB1. / Marijuana (Cannabis sativa) is a widely used plant and its various applications have important benefits. The plant Cannabis sativa has been used by man for centuries for eating, medicinal practices and religious rituals. In human subjects, its effects include analgesia, alterations in perceptions, cognition, memory and psychomotor activity. The cannabinoid compounds have been used in the cancer chemotherapy and AIDS, but the use of marijuana is a problem due to its unwanted effects (the psychotropic activity presented by the cannabinoid compounds). Due to the great interest in the effects caused by the compounds extracted from the Cannabis, several studies have been carried out with the aim to better understand the relationship between the chemical structure and the biological activity of cannabinoid compounds, as well as their interaction with the cannabinoid receptors (CB1 and CB2). Both are seven-transmembrane (TM) receptors that belong to the rhodopsin-like family Class A of G protein coupled receptors (GPCRs). This work represents a study of molecular modeling of the CB1 receptor based upon the published bovine rhodopsin structure, once the most of the therapeutic effects of cannabinoids compounds have been shown to be mediated through the CB1 cannabinoid receptor. This work also provides an investigation of the CB1 receptor-ligand interaction and a study of the CB1 receptor activation. A docking study was also performed in order to understand the main interactions that occur between ∆9 -THC, the principal psychoactive molecule present in cannabis, and its receptor CB1.