The neurotransmitters serotonin (5-HT) and dopamine (DA) play roles in eating disorders, mood disorders, such as depression and anxiety, and in the regulation of locomotion. The 5-HT2C receptor is one of fourteen 5-HT receptor subtypes that is expressed in regions of the brain including the hippocampus, amygdala, dorsal striatum, nucleus accumbens (NA) and substantia nigra, and is therefore implicated in behaviours and disorders associated with these regions. 5-HT has been shown to exert both a tonic and phasic inhibitory control, through the 5-HT2C receptor, on the firing rate and bursting activity of DA-containing neurones in the ventral tegmental area which enhances DA release in the NA and prefrontal cortex. In addition, the 5-HT2C receptor is under the control of a monophasic diurnal rhythm and is in a position to alter circadian regulation and behaviour due to its expression in the suprachiasmatic nucleus (the light entrainable oscillator (LEO)). It was hypothesised that elevating expression of the 5-HT2C receptor would have a detrimental effect on mood and cause hypolocomotion while reducing 5-HT2C receptor expression would improve mood, cause hyperphagia, obesity and hyperlocomotion. In order to investigate these hypotheses mouse models that either over- or under-expressed the 5-HT2C receptor were implemented. The 5-HT2C receptor expression pattern and levels were confirmed in all mouse lines. A behavioural phenotype of hypolocomotion and increased anxiety in the 5-HT2C receptor over-expressing mice and hyperphagia, obesity and hyperlocomotion in the 5-HT2C receptor under-expressing mice were found the latter is conistent with current literature. During backcrossing of the mouse lines onto the C57Bl/6 genetic background the abnormal behavioural phenotypes were lost suggesting that 5-HT2C receptor function is particulary sensitive to the genetic background on which it is being expressed. In response to altered expression levels of 5-HT2C receptor, compensatory alterations were found in the 5-HT system, with an inverse relationship existing between both the 5-HT1A receptor mRNA expression levels and 5-HT release in the hippocampus with the expression levels of the 5-HT2C receptor. Over-expression of 5-HT2C receptor appears to inhibit DA release in the cortex. The circadian experiments showed that under-expressing the 5-HT2C receptor did not alter the regulation of the food entrainable oscillator and there was a suggestion that the regulation of the LEO was affected. In summary, these results demonstrate that altered expression of 5-HT2C receptors results in abnormal behaviours consistent with its role in psychiatric disorders, but that the outcome is dependent on the genetic background.

Chronic neuropathic pain, as a result of nerve injury or dysfunction in the nervous system, is poorly understood and is usually inadequately treated with analgesics such as morphine. It is characterised by hyperalgesia (an increased sensitivity to a mild noxious stimulus), allodynia (interpretation of a normally innocuous stimulus as noxious) and spontaneous pain (with no precipitating stimulus). Central sensitisation may be due to changes within the central nervous system and particularly the dorsal horn of the spinal cord, which may help to explain the abnormal pain responses observed in humans and animals. Descending inputs from the brainstem can modulate nociception at the level of the spinal dorsal horn and serotonin (5-HT) is one of the neurotransmitters that may be involved. A long-circuited spinal-brainstem-spinal loop can act through 5-HT3 receptors on presynaptic terminals to facilitate afferent nociceptive inputs, especially in models of neuropathic pain. We show that 5-HT2C receptors may also act on dorsal horn neurones involved in nociceptive processing to facilitate their responsiveness. There are conflicting reports of the influence of 5-HT2 receptors in nociception, however there is evidence that activation of spinal 5-HT2A/2C receptors enhances the central transmission of nociceptive signals. Evidence that 5-HT2C receptor mRNA is more abundant in the dorsal horn of the spinal cord than that of any other 5HT2 receptor subtype points to this as a receptor worthy of investigation. We have shown that 5-HT2C and 5-HT2A receptor antagonists attenuate behavioural reflex sensitisation induced by nerve injury or inflammation, with a more marked influence on sensitivity to thermal rather than mechanical stimulation. We also observed delayed development of sensitisation following inflammation or nerve injury in transgenic mice with reduced expression of the 5-HT2C receptor compared to wild-type mice. We have also shown that the expression of the 5-HT2C receptor is increased in the dorsal horn of the spinal cord ipsilateral to nerve injury by immunohistochemical means and we have identified potential binding partners interacting with the carboxy terminal tail of the 5-HT2C receptor in spinal cord extracts. These findings indicate that the spinal 5-HT2C receptor activated by pathways originating in the brainstem may be of importance in contributing to the increased sensitivity to noxious stimuli following nerve injury or inflammatory damage.

Previous research indicates that serotonin (5-HT) enhances the acquisition of stress-induced changes in behavior; although it is unclear which serotonin receptors mediate this enhancement. 5-HT2 receptors are potential candidates because activation at these receptors is associated with increased fear and anxiety. In this study we investigated whether pharmacological treatments targeting 5-HT2A and 5-HT2C receptors modulated the acquisition and expression of conditioned defeat. Conditioned defeat is a social defeat model in Syrian hamsters (Mesocricetus auratus) that is characterized by increased submissive and defensive behavior and a loss of territorial aggression following social defeat. In experiment 1, we injected the 5-HT2C receptor agonist mCPP (0.3, 1.0, or 3.0 mg/kg) or vehicle prior to social defeat and tested subjects for conditioned defeat behavior in a social interaction test 24 hours later. In experiment 2, subjects received a social defeat, and 24 hours later we injected mCPP (0.3, 1.0, or 3.0 mg/kg) or vehicle prior to a social interaction test. We found that injection of mCPP increased the expression, but not acquisition, of conditioned defeat. In experiment 3, we injected the 5-HT2A receptor antagonist MDL 11,939 (0.5 or 2.0 mg/kg) or vehicle prior to a social defeat and tested subjects for conditioned defeat behavior. In experiment 4, subjects received a social defeat, and 24 hours later we injected MDL 11,939 (0.5 or 2.0 mg/kg) or vehicle prior to a social interaction test. We found that injection of MDL 11,939 significantly decreased the acquisition, but not expression, of conditioned defeat. These data suggest that pharmacological activation of 5-HT2C receptors enhances the expression of conditioned defeat, while pharmacological blockade of 5-HT2A receptors impairs the acquisition of conditioned defeat. These data extend other studies indicating that 5-HT signaling at 5-HT2A receptors facilitate memories for aversive events and 5-HT signaling at 5-HT2C receptors enhance stress-induced anxiety.

Thesis (Ph.D. in Human Medical Genetics) -- University of Colorado, 2006. / Typescript. Includes bibliographical references (leaves 118-127). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Le système sérotoninergique central a longtemps été associé au contrôle du comportement alimentaire et à la modulation des effets des drogues psychostimulantes sur le comportement.Le récepteur 5-HT2C est présent dans les centres hypothalamiques et particulièrement dans le noyau arqué dans l’hypothalamus (ARC) qui contrôle la régulation homéostatique de la prise alimentaire. On le retrouve aussi dans l’aire tegmentaire ventrale (VTA), une région importante dans la motivation de plusieurs comportements notamment la consommation alimentaire.Dans cette étude, l’hypothèse testée est, d’une part, que le récepteur 5-HT2C exprimé dans le VTA jouerait un rôle dans le contrôle de l’activité locomotrice induite par l’amphétamine et d’autre part qu’il exercerait son effet hypophagique a travers l’ARC.Des micro-injections localisées dans le VTA et dans l’ARC ont été réalisées afin d’évaluer les effets d’un agoniste sélectif du récepteur 5-HT2C, AR231630, sur l’hyperlocomotion stimulée par l’amphétamine ainsi que sur la prise alimentaire chez le rat.Dans les tests sur l’activité locomotrice, AR231630, lorsque qu’injecté dans la VTA, et non pas dans l’ARC, réduit de façon dose-dépendante l’ambulation provoquée par l’amphétamine.Dans les tests sur le comportement alimentaire, l’injection du composé AR231630 dans l’ARC n’induit pas de réduction de la prise alimentaire, contrairement au VTA.Une expérience suivante montre que les effets inhibants de l’administration périphérique d’AR231630 (5mg/kg) sur l’alimentation sont spécifiquement inversés par un prétraitement dans le VTA avec un antagoniste sélectif du récepteur 5-HT2C, SB242084.Ces résultats suggèrent que les récepteurs 5-HT2C dans le VTA participent à la fois au comportement alimentaire et à la fonction de la récompense, et ce, potentiellement par le même mécanisme. / Central serotonin systems have long been associated with the control of ingestive behavior, and the modulation of behavioral effects of psychostimulants.The 5-HT2C receptor is present in hypothalamic centers particularly the arcuate nucleus (ARC) controlling homeostatic regulation of food intake as well as in the ventral tegmental area (VTA), a region important for motivational aspects of multiple behaviors, including feeding.In the present study, the hypothesis was tested that the 5-HT2CR in the VTA may control amphetamine-evoked locomotor activity and 5-HT2CR in the ARC may regulate food consumption. Localized microinjections into the VTA or into the ARC were used to assess the effects of a highly selective 5-HT2C agonist, AR231630, on the locomotor stimulant effect of amphetamine as well as food intake. In the tests for locomotor activity, AR231630 into the VTA, but not into the ARC, dose-dependently reduced locomotor activity elicited by amphetamine. Unexpectedly, in tests for food intake, intra-ARC injection of AR231630 did not reduce food intake even at doses of 10ug, whereas intra-VTA injection of 10ug AR231630 did. A subsequent experiment determined that the suppressant effect of peripheral administration of AR231630 (5 mg/kg) on feeding was partially reversed by pretreatment with the selective 5-HT2CR antagonist SB242084 into the VTA (5ug). These findings suggest that 5-HT2CR in the VTA participates in both food intake and brain reward function, and possibly through the same pathway.

Evidências na literatura apontam o núcleo basolateral do complexo amigdalóide como uma estrutura importante na elaboração e controle das respostas defensivas. O sistema serotonérgico é um dos tantos sistemas de neurotransmissão que estão envolvidos nessas respostas. Dentre os diferentes subtipos de receptores serotonérgicos existentes, os do tipo 5-HT2C é um dos mais estudados com relação à mediação dos processos de ansiedade. A estimulação de receptores 5-HT2C tem efeito do tipo ansiogênico em diferentes modelos animais de ansiedade. Porém, não se conhece ainda a real participação desses receptores localizados no núcleo basolateral do complexo amigdalóide sobre as respostas de esquiva e fuga geradas no labirinto em T elevado relacionas, respectivamente, com ansiedade generalizada e pânico. Assim, o presente trabalho teve por objetivo verificar o papel dos receptores serotonérgicos do tipo 5-HT2C do núcleo basolateral do complexo amigdalóide na modulação das respostas defensivas associadas à ansiedade generalizada e ao pânico. Foram realizadas injeções bilaterais intra-núcleo basolateral do agonista endógeno serotonina, do agonista preferencial de receptores 5-HT2C MK-212 ou do antagonista seletivo desses receptores SB242084 em ratos submetidos ao teste do labirinto em T elevado e teste de transição claro-escuro. Os resultados mostram que a administração do agonista endógeno serotonina e do agonista de receptores do tipo 5-HT2C MK-212 promove uma facilitação na aquisição da esquiva inibitória em ratos testados no labirinto em T elevado. A administração dos agonistas também diminui o tempo gasto pelos animais no compartimento claro da caixa claro-escuro, também sugerindo um efeito do tipo ansiogênico. Por outro lado, a administração local do antagonista dos receptores 5-HT2C SB-242084 prejudica tal resposta. Esse mesmo tratamento não altera a resposta no teste de transição claro-escuro. A administração prévia de SB-242084 é capaz de bloquear o efeito ansiogênico promovido pela serotonina. Nem a ativação nem o bloqueio desses receptores exerceu efeito sobre a resposta de fuga. Em suma, nossos resultados sugerem que os receptores 5-HT2C do núcleo basolateral do complexo amigdalóide têm papel regulatório sobre a resposta de esquiva inibitória, mas não sobre a resposta de fuga gerada no labirinto em T elevado. / A wealth of evidence highlights the importance of serotonergic mechanisms within the basolateral nucleus of the amygdala for the regulation of defensive responses. However, conflicting evidence exists on the role of played by different 5-HT receptor subtypes such as 5-HT1A, 5-HT2A and 5-HT2C receptors in this process. For instance, previous studies show that whereas activation of the former two subtypes causes anxiolytic-like effect in the elevated T-maze, stimulation of 5-HT2C receptors has anxiogenic consequences in different animal models of anxiety. In this study we further explored the role played by 5-HT2C receptors of the basolateral nucleus of the amygdala in the regulation of defensive behaviors that have been associated with generalized anxiety and panic. The results showed that bilateral injection of the endogenous agonist serotonin or the 5-HT2C receptor agonist MK-212 facilitates inhibitory avoidance acquisition in rats tested in the elevated T-maze. Both drugs decreased the time spent by the animals in the light compartment of a light-dark box, also suggesting an anxiogenic-like effect. On the other hand, local administration of the 5-HT2C receptor antagonist SB-242084 impaired inhibitory avoidance acquisition in the elevated T-maze, but was without effect upon the behaviors measured in the light-dark transition test. Previous administration of SB-242084 blocked the anxiogenic effect of serotonin on inhibitory avoidance acquisition in the T-maze. Neither the blockade nor the activation of 5-HT2C receptors of the basolateral nucleus of the amygdala affected escape performance in the elevated T-maze. In summary, the results obtained in the present study are indicative that 5-HT2C receptors in the basolateral nucleus of the amygdala are selectively involved in the regulation of defensive behaviors that are associated with generalized anxiety but not panic disorder.

Uma série de evidências indica que o núcleo dorsal da rafe (NDR) é uma estrutura heterogênea, composta por populações de neurônios serotoninérgicos, anatômica e funcionalmente distintas, que foram implicadas de maneira diferente na fisiopatologia da ansiedade. Evidências do nosso grupo mostraram que a facilitação da neurotransmissão serotoninérgica do subnúcleo dorsomedial (DRD) do NDR favorece a aquisição da esquiva inibitória, sugerindo um efeito ansiogênico, e inibe a expressão da resposta de fuga, efeito do tipo panicolítico, no labirinto em T elevado (LTE). Por outro lado, a ativação das asas laterais do NDR apenas altera o desempenho na tarefa de fuga, efeito do tipo panicolítico, no LTE. Essas descobertas sustentam que diferentes vias serotoninérgicas provenientes do NDR modulam comportamentos associados à ansiedade e ao pânico. O presente estudo teve como objetivo investigar se as asas laterais enviam projeções serotoninérgicas para a substância cinzenta periaquedutal dorsal (SCPd), estruturas chave na modulação de comportamentos relacionados ao pânico, através da técnica de neurotraçamento anterógrado. Em seguida, avaliamos, através de manipulações farmacológicas, o papel desta via na modulação de respostas defensivas avaliadas no LTE. Estendemos esta investigação farmacológicas para o hipotálamo dorsomedial (dmHIP), outra estrutura chave na evocação de comportamentos relacionados ao pânico. Além disso, investigamos o papel da via serotoninérgica que parte do DRD para o núcleo basolateral de amígdala (BLA) na evocação de comportamentos defensivos no ETM e no teste de transição claro-escuro. Com base em estudos prévios do nosso grupo que mostram que a tarefa de esquiva inibitória no LTE ativa o subnúcleo caudal (DRC), realizamos as primeiras observações das consequências comportamentais no LTE após manipulações farmacológicas deste subnúcleo. Primeiramente, verificamos se as asas laterais enviam projeções serotoninérgicas para a SCPd através do neurotraçador anterógrado amina dextrano biotinilado não fluorescente e da imunomarcação para a enzima triptofano hidroxilase. Em seguida, investigamos o efeito da microinjeção do antagonista seletivo de receptores 5-HT1A WAY-100635 nas asas laterais de ratos que receberam administração prévia da mesma droga na SCPd. Dados anteriores do nosso grupo mostraram que a administração de WAY-100635 no NDR, sem diferenciar os subnúcleos, promove efeito do tipo panicolítico no LTE, e este efeito era bloqueado pelo antagonismo dos receptores 5-HT1A na SCPd. Nossa hipótese é que o antagonismo dos receptores 5-HT1A nas asas laterais resulte em um aumento na liberação de serotonina (5-HT) na SCPd, promovendo o efeito do tipo panicolítico. Portanto, o WAY-100635 administrado anteriormente na SCPd bloquearia o efeito panicolítico observado na tarefa de fuga no LTE. Estendemos essa investigação farmacológica, também utilizando o WAY-100635, para o dmHIP. Em passo seguinte, com base em resultados prévios de imuno-histoquímica do nosso grupo que mostraram que a tarefa de esquiva inibitória, mas não a resposta de fuga, ativa o DRC, avaliamos o comportamentos de ratos submetidos ao LTE após a manipulação farmacológica dos receptores do tipo 5-HT1A neste subnúcleo. Esperávamos que a microinjeção de WAY-100635 no DRC facilitasse a aquisição da esquiva inibitória, enquanto que a administração do agonista seletivo de receptores 5-HT1A, 8-OH-DPAT, prejudicasse esta tarefa no LTE. Em uma etapa posterior, investigamos se o efeito ansiogênico observado após a administração de WAY- 100635 no DRD era dependente da ativação de receptores 5-HT2C no BLA. Nosso grupo mostrou que a administração de 5-HT no BLA promove efeito ansiogênico no LTE, e este efeito é bloqueado pelo antagonista de receptores 5-HT2C, SB-242084. Para responder esta pergunta, injetamos WAY-100635 no DRD de ratos que receberam a administração prévia de SB-242084 no BLA. Assim, se o antagonismo de receptores 5-HT1A no DRD resulta em um aumento da liberação de 5-HT no BLA, esperávamos que o SB-242084 administrado no BLA bloquearia o efeito ansiogênico observado no LTE. Nossos resultados mostraram que as asas laterais envia projeções serotoninérgicas para a SCPd. Os resultados farmacológicos mostraram que o tratamento prévio com WAY-100635 na SCPd bloqueia o efeito de tipo panicolítico observado após a administração de WAY-100635 nas asas laterais, sem interferir na tarefa de esquiva inibitória no LTE. Por outro lado, o tratamento prévio com SB-242084 no BLA bloqueou o efeito ansiogênico observado após a administração de WAY-100635 no DRD, sem interferir na resposta de fuga no LTE. Este resultado também foi confirmado em outro teste relacionado à ansiedade, o teste de transição claro-escuro. As manipulações farmacológicas no DRC apontaram para o não envolvimento deste subnúcleo na modulação de respostas associadas à ansiedade e ao pânico. Nossos resultados sugerem ainda que as vias asas laterais- SCPd e asas laterais-dmHIP modulam comportamentos de defesa associados ao pânico, enquanto a via DRD-BLA está relacionada à modulação das respostas relacionadas à ansiedade. / A wealth of evidence indicates that the dorsal raphe nucleus (DR) is a heterogeneous structure, composed of anatomically and functionally distinct populations of serotonergic neurons, which have been differently implicated in the pathophysiology of anxiety. Evidences from our group showed that serotonergic neurotransmission activation via dorsomedial subnucleus (DRD) of the DR facilitated inhibitory avoidance acquisition, suggesting an anxiogenic effect, and inhibited escape expression, a panicolytic-like effect in the elevated T maze (ETM). On the other hand, in the ETM, activation of the lateral wings (lwDR) of the DR only altered escape response, a panicolytic-like effect. These findings support that different DR serotonergic pathways regulate anxiety and panic-like behaviors. The present study aimed to investigate whether the lwDR send serotonergic projections to the dorsal periaqueductal gray (dPAG), a key structure in the modulation of panic-related behaviors, through the anterograde neurotrace technique. Next, we evaluated, through pharmacological manipulations, the role of this pathway in the modulation of defensive responses evaluated in the ETM. We extend this pharmacological investigation to the dorsomedial hypothalamus (dmHIP), another key structure in the evocation of panic-related behaviors. In addition, we investigated the role of the serotonergic pathway from the DRD to the basolateral nucleus of amygdala (BLA) in the elicitation of defensive behaviors in the ETM and in the light-dark transition test. Based on previous studies from our group that show that inhibitory avoidance task in the ETM activates the caudal subnucleus (DRC) of the DR, we performed the first behavioral observations in the ETM after the pharmacological manipulations of this DR subnucleus. First, we labeled the lwDR to dPAG pathway using the anterograde tracer non-fluorescent biotinylated dextran amine and performed an immunostaining for the enzyme tryptophan hydroxylase. Next, we investigated the effect of the microinjection of the selective 5-HT1A antagonist WAY-100635 in the lwDR of rats that have received previous administration of the same drug in the dPAG. Previous data from our group showed that the administration of WAY-100635 in the DR, without differentiating subnuclei, promotes panicolytic effect in the ETM, and this effect was blocked by the 5-HT1A receptors antagonism in the dPAG. Our hypothesis is that the antagonism of 5-HT1A receptors in the lwDR results in an increase in the release of serotonin (5-HT) in the dPAG, promoting the panicolytic effect. Therefore, WAY- 100635 previously administered in dPAG would block the panicolytic effect observed in the escape task in the ETM. We extended this pharmacological investigation, also using WAY-100635, to dmHIP. In the next step, based on previous immunohistochemistry results from our group that show that the inhibitory avoidance task, but not the escape response, activates the DRC, we investigated the behavior of rats submitted to the ETM after the pharmacological manipulation of the 5-HT1A receptors in this subnucleus. We expected that the microinjection of WAY-100635 in the DRC would facilitate the inhibitory avoidance acquisition, whereas the administration of the selective 5-HT1A agonist 8-OH-DPAT would hinder this task in the ETM. At a later stage, we investigated whether the anxiogenic effect observed after administration of WAY-100635 in the DRD is dependent on activation of 5-HT2C receptors in the BLA. Our group has shown that the administration of 5-HT in the BLA promotes an anxiogenic effect in the ETM, and this effect was blocked by the 5- HT2C receptor antagonist, SB-242084. To answer these questions, we microinjected WAY-100635 in the DRD of rats that received prior administration of SB-242084 in the BLA. Thus, if 5-HT1A receptor antagonism in the DRD results in an increase of 5- HT released in the BLA, we expected that SB-242084 administered in the BLA blocks the anxiogenic effect in the LTE. Our results showed that the lwDR send serotonergic projections to the dPAG. The pharmacological results showed that previous treatment with WAY-100635 in the dPAG or dmHIP blocked the panicolytic-like effect observed after WAY-100635 administration in the lwDR, without interfering in the inhibitory avoidance response in the ETM. On the other hand, previous treatment with SB-242084 in the BLA blocked the anxiogenic effect observed after WAY- 100635 administration in the DRD, without interfering in the escape response in the ETM. The later result was also confirmed in another anxiety-related test, the light/dark transition test. The results of the pharmacological manipulations of the DRC do not support an involvement of this subnucleus in the modulation of defensive responses associated with anxiety and panic. Our results suggest that lwDR-PAG and lwDR-dmHIP pathways modulate panic-like defense behaviors while DRD-BLA pathway is implicated in the modulation of anxiety responses.

Uma série de evidências indica que o núcleo dorsal da rafe (NDR) é uma estrutura heterogênea, composta por populações de neurônios serotoninérgicos, anatômica e funcionalmente distintas, que foram implicadas de maneira diferente na fisiopatologia da ansiedade. Evidências do nosso grupo mostraram que a facilitação da neurotransmissão serotoninérgica do subnúcleo dorsomedial (DRD) do NDR favorece a aquisição da esquiva inibitória, sugerindo um efeito ansiogênico, e inibe a expressão da resposta de fuga, efeito do tipo panicolítico, no labirinto em T elevado (LTE). Por outro lado, a ativação das asas laterais do NDR apenas altera o desempenho na tarefa de fuga, efeito do tipo panicolítico, no LTE. Essas descobertas sustentam que diferentes vias serotoninérgicas provenientes do NDR modulam comportamentos associados à ansiedade e ao pânico. O presente estudo teve como objetivo investigar se as asas laterais enviam projeções serotoninérgicas para a substância cinzenta periaquedutal dorsal (SCPd), estruturas chave na modulação de comportamentos relacionados ao pânico, através da técnica de neurotraçamento anterógrado. Em seguida, avaliamos, através de manipulações farmacológicas, o papel desta via na modulação de respostas defensivas avaliadas no LTE. Estendemos esta investigação farmacológicas para o hipotálamo dorsomedial (dmHIP), outra estrutura chave na evocação de comportamentos relacionados ao pânico. Além disso, investigamos o papel da via serotoninérgica que parte do DRD para o núcleo basolateral de amígdala (BLA) na evocação de comportamentos defensivos no ETM e no teste de transição claro-escuro. Com base em estudos prévios do nosso grupo que mostram que a tarefa de esquiva inibitória no LTE ativa o subnúcleo caudal (DRC), realizamos as primeiras observações das consequências comportamentais no LTE após manipulações farmacológicas deste subnúcleo. Primeiramente, verificamos se as asas laterais enviam projeções serotoninérgicas para a SCPd através do neurotraçador anterógrado amina dextrano biotinilado não fluorescente e da imunomarcação para a enzima triptofano hidroxilase. Em seguida, investigamos o efeito da microinjeção do antagonista seletivo de receptores 5-HT1A WAY-100635 nas asas laterais de ratos que receberam administração prévia da mesma droga na SCPd. Dados anteriores do nosso grupo mostraram que a administração de WAY-100635 no NDR, sem diferenciar os subnúcleos, promove efeito do tipo panicolítico no LTE, e este efeito era bloqueado pelo antagonismo dos receptores 5-HT1A na SCPd. Nossa hipótese é que o antagonismo dos receptores 5-HT1A nas asas laterais resulte em um aumento na liberação de serotonina (5-HT) na SCPd, promovendo o efeito do tipo panicolítico. Portanto, o WAY-100635 administrado anteriormente na SCPd bloquearia o efeito panicolítico observado na tarefa de fuga no LTE. Estendemos essa investigação farmacológica, também utilizando o WAY-100635, para o dmHIP. Em passo seguinte, com base em resultados prévios de imuno-histoquímica do nosso grupo que mostraram que a tarefa de esquiva inibitória, mas não a resposta de fuga, ativa o DRC, avaliamos o comportamentos de ratos submetidos ao LTE após a manipulação farmacológica dos receptores do tipo 5-HT1A neste subnúcleo. Esperávamos que a microinjeção de WAY-100635 no DRC facilitasse a aquisição da esquiva inibitória, enquanto que a administração do agonista seletivo de receptores 5-HT1A, 8-OH-DPAT, prejudicasse esta tarefa no LTE. Em uma etapa posterior, investigamos se o efeito ansiogênico observado após a administração de WAY- 100635 no DRD era dependente da ativação de receptores 5-HT2C no BLA. Nosso grupo mostrou que a administração de 5-HT no BLA promove efeito ansiogênico no LTE, e este efeito é bloqueado pelo antagonista de receptores 5-HT2C, SB-242084. Para responder esta pergunta, injetamos WAY-100635 no DRD de ratos que receberam a administração prévia de SB-242084 no BLA. Assim, se o antagonismo de receptores 5-HT1A no DRD resulta em um aumento da liberação de 5-HT no BLA, esperávamos que o SB-242084 administrado no BLA bloquearia o efeito ansiogênico observado no LTE. Nossos resultados mostraram que as asas laterais envia projeções serotoninérgicas para a SCPd. Os resultados farmacológicos mostraram que o tratamento prévio com WAY-100635 na SCPd bloqueia o efeito de tipo panicolítico observado após a administração de WAY-100635 nas asas laterais, sem interferir na tarefa de esquiva inibitória no LTE. Por outro lado, o tratamento prévio com SB-242084 no BLA bloqueou o efeito ansiogênico observado após a administração de WAY-100635 no DRD, sem interferir na resposta de fuga no LTE. Este resultado também foi confirmado em outro teste relacionado à ansiedade, o teste de transição claro-escuro. As manipulações farmacológicas no DRC apontaram para o não envolvimento deste subnúcleo na modulação de respostas associadas à ansiedade e ao pânico. Nossos resultados sugerem ainda que as vias asas laterais- SCPd e asas laterais-dmHIP modulam comportamentos de defesa associados ao pânico, enquanto a via DRD-BLA está relacionada à modulação das respostas relacionadas à ansiedade. / A wealth of evidence indicates that the dorsal raphe nucleus (DR) is a heterogeneous structure, composed of anatomically and functionally distinct populations of serotonergic neurons, which have been differently implicated in the pathophysiology of anxiety. Evidences from our group showed that serotonergic neurotransmission activation via dorsomedial subnucleus (DRD) of the DR facilitated inhibitory avoidance acquisition, suggesting an anxiogenic effect, and inhibited escape expression, a panicolytic-like effect in the elevated T maze (ETM). On the other hand, in the ETM, activation of the lateral wings (lwDR) of the DR only altered escape response, a panicolytic-like effect. These findings support that different DR serotonergic pathways regulate anxiety and panic-like behaviors. The present study aimed to investigate whether the lwDR send serotonergic projections to the dorsal periaqueductal gray (dPAG), a key structure in the modulation of panic-related behaviors, through the anterograde neurotrace technique. Next, we evaluated, through pharmacological manipulations, the role of this pathway in the modulation of defensive responses evaluated in the ETM. We extend this pharmacological investigation to the dorsomedial hypothalamus (dmHIP), another key structure in the evocation of panic-related behaviors. In addition, we investigated the role of the serotonergic pathway from the DRD to the basolateral nucleus of amygdala (BLA) in the elicitation of defensive behaviors in the ETM and in the light-dark transition test. Based on previous studies from our group that show that inhibitory avoidance task in the ETM activates the caudal subnucleus (DRC) of the DR, we performed the first behavioral observations in the ETM after the pharmacological manipulations of this DR subnucleus. First, we labeled the lwDR to dPAG pathway using the anterograde tracer non-fluorescent biotinylated dextran amine and performed an immunostaining for the enzyme tryptophan hydroxylase. Next, we investigated the effect of the microinjection of the selective 5-HT1A antagonist WAY-100635 in the lwDR of rats that have received previous administration of the same drug in the dPAG. Previous data from our group showed that the administration of WAY-100635 in the DR, without differentiating subnuclei, promotes panicolytic effect in the ETM, and this effect was blocked by the 5-HT1A receptors antagonism in the dPAG. Our hypothesis is that the antagonism of 5-HT1A receptors in the lwDR results in an increase in the release of serotonin (5-HT) in the dPAG, promoting the panicolytic effect. Therefore, WAY- 100635 previously administered in dPAG would block the panicolytic effect observed in the escape task in the ETM. We extended this pharmacological investigation, also using WAY-100635, to dmHIP. In the next step, based on previous immunohistochemistry results from our group that show that the inhibitory avoidance task, but not the escape response, activates the DRC, we investigated the behavior of rats submitted to the ETM after the pharmacological manipulation of the 5-HT1A receptors in this subnucleus. We expected that the microinjection of WAY-100635 in the DRC would facilitate the inhibitory avoidance acquisition, whereas the administration of the selective 5-HT1A agonist 8-OH-DPAT would hinder this task in the ETM. At a later stage, we investigated whether the anxiogenic effect observed after administration of WAY-100635 in the DRD is dependent on activation of 5-HT2C receptors in the BLA. Our group has shown that the administration of 5-HT in the BLA promotes an anxiogenic effect in the ETM, and this effect was blocked by the 5- HT2C receptor antagonist, SB-242084. To answer these questions, we microinjected WAY-100635 in the DRD of rats that received prior administration of SB-242084 in the BLA. Thus, if 5-HT1A receptor antagonism in the DRD results in an increase of 5- HT released in the BLA, we expected that SB-242084 administered in the BLA blocks the anxiogenic effect in the LTE. Our results showed that the lwDR send serotonergic projections to the dPAG. The pharmacological results showed that previous treatment with WAY-100635 in the dPAG or dmHIP blocked the panicolytic-like effect observed after WAY-100635 administration in the lwDR, without interfering in the inhibitory avoidance response in the ETM. On the other hand, previous treatment with SB-242084 in the BLA blocked the anxiogenic effect observed after WAY- 100635 administration in the DRD, without interfering in the escape response in the ETM. The later result was also confirmed in another anxiety-related test, the light/dark transition test. The results of the pharmacological manipulations of the DRC do not support an involvement of this subnucleus in the modulation of defensive responses associated with anxiety and panic. Our results suggest that lwDR-PAG and lwDR-dmHIP pathways modulate panic-like defense behaviors while DRD-BLA pathway is implicated in the modulation of anxiety responses.

Estudos com microinjeções de drogas vêm sendo realizados na tentativa de se compreender a participação da neurotransmissão serotonérgica do hipocampo na modulação de comportamentos defensivos relacionados à ansiedade. Nesse sentido, observou-se que a ativação dos receptores do tipo 5-HT1A do hipocampo dorsal (HD) promoveu efeito do tipo ansiogênico sobre a resposta de esquiva inibitória, sem alterar a resposta de fuga, em ratos submetidos ao labirinto em T elevado (LTE). Essa alteração seletiva na resposta de esquiva inibitória sustenta a hipótese da participação do hipocampo na fisiopatologia do transtorno de ansiedade generalizada, uma vez que, as respostas defensivas de esquiva inibitória e fuga, expressas no LTE, têm sido relacionadas respectivamente ao transtorno de ansiedade generalizada e ao transtorno do pânico. Além de receptores do tipo 5- HT1A, destaca-se no HD a presença de receptores do tipo 5-HT2C. Tem sido observado que a estimulação desses últimos em áreas límbicas associadas à ansiedade, como a amígdala, promove efeito do tipo ansiogênico em diferentes modelos animais de ansiedade. Porém, ainda é desconhecida a participação desses receptores presentes no HD sobre as resposta de esquiva inibitória e fuga, geradas no LTE. Assim, o presente estudo procurou avaliar a participação dos receptores serotonérgicos do tipo 5-HT2C na modulação de respostas defensivas relacionadas à ansiedade generalizada e ao transtorno do pânico. Os resultados mostram que injeções bilaterais intra-HD dos agonistas de receptores 5-HT2C MK-212 ou RO600175 prejudicaram a aquisição da resposta de esquiva inibitória, em ratos testados no LTE, indicando um efeito do tipo ansiolítico. Por outro lado, a administração do antagonista de receptores 5-HT2C SB-242084 promoveu efeito oposto sobre essa mesma resposta. Adicionalmente administração do agonista preferencial de receptores 5-HT2A DOI não foi capaz de promover efeito em nenhuma das doses utilizadas. Nenhum dos tratamentos empregados alterou a resposta de fuga no LTE. O efeito ansiolítico da ativação dos receptores 5-HT2C, bem como o efeito ansiogênico resultante do seu bloqueio, foram confirmados no teste do beber punido de Vogel. Em suma, nossos resultados sugerem que os receptores do tipo 5-HT2C do hipocampo dorsal estão envolvidos na modulação de comportamentos defensivos relacionados ao transtorno de ansiedade generalizada, mas não ao transtorno do pânico. / Studies using intracerebral microinfusion of drugs have been performed to unveil the role of the hippocampus serotonergic system in the modulation of anxiety-related defensive behaviors. For instance, it has been shown that activation of 5-HT1A receptors in the dorsal hippocampus (DH) facilitated inhibitory avoidance acquisition, suggesting an anxiogenic effect, without altering escape expression in rats tested in the elevated T-maze (ETM). This selective effect on inhibitory avoidance response is consistent with the notion that the hippocampus is critically involved in the pathophysiology of generalized anxiety disorder. Besides 5-HT1A receptors, expressive levels of 5-HT2C receptors are also reported in hippocampus. Compelling evidence from animal studies shows that facilitation of 5-HT2C receptor-mediated neurotransmission increases anxiety. In this study we evaluated the involvement the 5-HT2C receptors of the DH in the regulation of defensive behaviors that have been associated with generalized anxiety and panic. Male Wistar rats were tested in the ETM after intra-DH injection of the 5-HT2C receptor agonists MK-212, RO-600175, or the preferential 5-HT2A receptor agonist DOI, or the 5-HT2C receptor antagonist SB242084. For comparative reason, the effect of MK-212, RO-600175 and SB-242084 was also evaluated on another generalized anxiety-associated model, the Vogel conflict test. Our results showed that while intra-DH microinjection of both MK-212 and RO-600175 facilitated inhibitory avoidance acquisition, suggesting an anxiolytic effect, SB-242084 had the opposite effect. Injections of DOI did not affected performance in ETM. None of these drugs affected escape performance in the ETM. The anxiolytic-like effects of the 5-HT2C receptor agonists and anxiogenic-like effect of the SB-242084 were also observed in the Vogel conflict test. Our findings indicate that 5-HT2C receptors in DH are selectively involved in the regulation of defensive behaviors associated with generalized anxiety, but not panic.

Estudos com microinjeções de drogas vêm sendo realizados na tentativa de se compreender a participação da neurotransmissão serotonérgica do hipocampo na modulação de comportamentos defensivos relacionados à ansiedade. Nesse sentido, observou-se que a ativação dos receptores do tipo 5-HT1A do hipocampo dorsal (HD) promoveu efeito do tipo ansiogênico sobre a resposta de esquiva inibitória, sem alterar a resposta de fuga, em ratos submetidos ao labirinto em T elevado (LTE). Essa alteração seletiva na resposta de esquiva inibitória sustenta a hipótese da participação do hipocampo na fisiopatologia do transtorno de ansiedade generalizada, uma vez que, as respostas defensivas de esquiva inibitória e fuga, expressas no LTE, têm sido relacionadas respectivamente ao transtorno de ansiedade generalizada e ao transtorno do pânico. Além de receptores do tipo 5- HT1A, destaca-se no HD a presença de receptores do tipo 5-HT2C. Tem sido observado que a estimulação desses últimos em áreas límbicas associadas à ansiedade, como a amígdala, promove efeito do tipo ansiogênico em diferentes modelos animais de ansiedade. Porém, ainda é desconhecida a participação desses receptores presentes no HD sobre as resposta de esquiva inibitória e fuga, geradas no LTE. Assim, o presente estudo procurou avaliar a participação dos receptores serotonérgicos do tipo 5-HT2C na modulação de respostas defensivas relacionadas à ansiedade generalizada e ao transtorno do pânico. Os resultados mostram que injeções bilaterais intra-HD dos agonistas de receptores 5-HT2C MK-212 ou RO600175 prejudicaram a aquisição da resposta de esquiva inibitória, em ratos testados no LTE, indicando um efeito do tipo ansiolítico. Por outro lado, a administração do antagonista de receptores 5-HT2C SB-242084 promoveu efeito oposto sobre essa mesma resposta. Adicionalmente administração do agonista preferencial de receptores 5-HT2A DOI não foi capaz de promover efeito em nenhuma das doses utilizadas. Nenhum dos tratamentos empregados alterou a resposta de fuga no LTE. O efeito ansiolítico da ativação dos receptores 5-HT2C, bem como o efeito ansiogênico resultante do seu bloqueio, foram confirmados no teste do beber punido de Vogel. Em suma, nossos resultados sugerem que os receptores do tipo 5-HT2C do hipocampo dorsal estão envolvidos na modulação de comportamentos defensivos relacionados ao transtorno de ansiedade generalizada, mas não ao transtorno do pânico. / Studies using intracerebral microinfusion of drugs have been performed to unveil the role of the hippocampus serotonergic system in the modulation of anxiety-related defensive behaviors. For instance, it has been shown that activation of 5-HT1A receptors in the dorsal hippocampus (DH) facilitated inhibitory avoidance acquisition, suggesting an anxiogenic effect, without altering escape expression in rats tested in the elevated T-maze (ETM). This selective effect on inhibitory avoidance response is consistent with the notion that the hippocampus is critically involved in the pathophysiology of generalized anxiety disorder. Besides 5-HT1A receptors, expressive levels of 5-HT2C receptors are also reported in hippocampus. Compelling evidence from animal studies shows that facilitation of 5-HT2C receptor-mediated neurotransmission increases anxiety. In this study we evaluated the involvement the 5-HT2C receptors of the DH in the regulation of defensive behaviors that have been associated with generalized anxiety and panic. Male Wistar rats were tested in the ETM after intra-DH injection of the 5-HT2C receptor agonists MK-212, RO-600175, or the preferential 5-HT2A receptor agonist DOI, or the 5-HT2C receptor antagonist SB242084. For comparative reason, the effect of MK-212, RO-600175 and SB-242084 was also evaluated on another generalized anxiety-associated model, the Vogel conflict test. Our results showed that while intra-DH microinjection of both MK-212 and RO-600175 facilitated inhibitory avoidance acquisition, suggesting an anxiolytic effect, SB-242084 had the opposite effect. Injections of DOI did not affected performance in ETM. None of these drugs affected escape performance in the ETM. The anxiolytic-like effects of the 5-HT2C receptor agonists and anxiogenic-like effect of the SB-242084 were also observed in the Vogel conflict test. Our findings indicate that 5-HT2C receptors in DH are selectively involved in the regulation of defensive behaviors associated with generalized anxiety, but not panic.