The tachykinin NK-3 receptor is a G-protein coupled receptor activated by mammalian tachykinin neuropeptides, which can modulate dopaminergic neurotransmission, and alter dopamine-mediated behaviors. The NK-3 receptor is currently under investigation as a novel therapeutic target for cocaine addiction. Our studies, as outlined in this dissertation, sought to determine if NK-3 receptors have a functional role in the acute as well as long-term behavioral effects of cocaine. Administration of NK-3 receptor agonists or antagonists potentiates or attenuates dopamine-mediated behaviors, respectively. Based on these findings, we hypothesized that blockade of neurokinin-3 receptors would alter acute and long-term behavioral responses to cocaine. We investigated whether acute and repeated administration of the NK-3 receptor antagonist SB 222200 altered hyperactivity induced by cocaine, and determined a possible mechanism involving dopamine D1 receptors in the striatum. We also determined whether NK-3 receptor blockade altered the development and expression of behavioral sensitization after repeated cocaine administration. Lastly, we investigated whether modulation of behavioral effects of acute and repeated cocaine by NK-3 receptors involved GSK3 phosphorylation in the nucleus accumbens. As described in this dissertation, we show that acute administration of the NK-3 receptor antagonist SB 222200 before a cocaine injection attenuated stereotypic responses produced by cocaine. Repeated administration of SB 222200 enhanced stereotypic activity produced by either cocaine or a low dose of SKF 82958 (0.125 mg/kg, i.p.) when administered seven days later. Dopamine receptor binding studies were performed to determine the mechanism of enhanced stereotypic responses. Binding studies showed a 19.7% increase in dopamine D1 receptor density in the striatum seven days later after repeated SB 222200 administration. These findings demonstrate that acute blockade of NK-3 receptors attenuated cocaine-induced behaviors in agreement with previous studies. Furthermore, these studies also show novel effects of repeated blockade of NK-3 receptors, which causes subsequent enhancement of cocaine and dopamine D1 receptor-mediated behaviors, possibly resulting from dopamine D1 receptor up-regulation in the striatum. In order to determine a role of NK-3 receptors in the development of cocaine-induced behavioral sensitization, the NK-3 receptor antagonist SB 222200 (2.5 or 5 mg/kg, s.c.) was administered prior to daily cocaine injections for 5 days. After a 7-day drug-free period, behavioral responses to a cocaine challenge were measured. Repeated administration of cocaine for 5 days induced a sensitized response upon a cocaine challenge 7 days later. Administration of SB 222200 prior to daily cocaine attenuated the development of behavioral sensitization. Moreover, administration of SB 222200 prior to the cocaine challenge blocked the expression of behavioral sensitization. These findings demonstrate that NK-3 receptor activity is involved in the development and expression of behavioral sensitization to cocaine. Lastly, we examined GSK3 phosphorylation in the nucleus accumbens induced by acute and repeated cocaine administration and determined if phosphorylation was altered by NK-3 receptor blockade. Similar to the drug administration regimens used in the behavioral studies, the NK-3 receptor antagonist SB 222200 was administered 30 mins prior to an acute cocaine injection. The nucleus accumbens was examined for changes in GSK3 phosphorylation by Western blot analysis. Increases in phosphorylation of the isoforms, GSK3α and GSK3β in the nucleus accumbens were detected 20 mins after an acute injection of cocaine. NK-3 receptor blockade prior to cocaine administration did not alter the cocaine-induced increase in GSK3 phosphorylation. Similar to the behavioral sensitization studies, SB 222200 was administered prior to repeated cocaine for 5 days, and 7 days later GSK3 phosphorylation was measured after a subsequent cocaine challenge. In contrast to the increases in GSK3α and GSK3β in the nucleus accumbens after an acute cocaine injection, no regulation of GSK3 phosphorylation was found after prior repeated cocaine administration and cocaine challenge. Administration of SB 222200 prior to repeated cocaine produced an increase in GSK3α and GSK3β phosphorylation after a cocaine challenge. Collectively, these data point to involvement of NK-3 receptor activity in changes in the phosphorylation of GSK3 in the nucleus accumbens produced by cocaine. In summary, functional involvement of NK-3 receptors in acute and long-term behavioral effects of cocaine was investigated. In agreement with previous findings, studies in this dissertation demonstrate that acute blockade of NK-3 receptors attenuates cocaine-induced behaviors. In addition, we found novel effects of repeated blockade of NK-3 receptors on cocaine-induced hyperactivity. There is enhancement of subsequent cocaine and dopamine D1 receptor-mediated behaviors possibly due to dopamine D1 receptor up-regulation in the striatum. NK-3 receptor activity was shown to be involved in long-term behavioral effects of cocaine and molecular changes in GSK3 phosphorylation in the nucleus accumbens. Blockade of NK-3 receptors prevented the development and expression of behavioral sensitization to cocaine and also blocked the changes in the phosphorylation of GSK3 in the nucleus accumbens. This dissertation has demonstrated a role of NK-3 receptors in modulating acute as well long-term cocaine-induced behavioral hyperactivity. Therefore, there is potential clinical relevance of NK-3 receptors in cocaine abuse and dependence as a therapeutic target for treatment, which warrants further characterization in future preclinical and clinical investigations. / Pharmacology

A substância cinzenta periaquedutal dorsal (SCPd) é considerada uma das principais estruturas do teto mesencefálico envolvida no substrato neural da aversão a estímulos proximais. GABA e 5-HT são apontados como neurotransmissores envolvidos na modulação das respostas defensivas elaboradas na SCPd. Recentemente, mecanismos neurocininérgicos também têm sido propostos como mediadores das reações de defesa organizadas nessa estrutura. O objetivo do presente estudo foi avaliar o envolvimento dos receptores NK-1 e NK-3 da SCPd no comportamento defensivo induzido pela estimulação elétrica dessa região em ratos com e sem experiência prévia ao condicionamento contextual aversivo. Para isso, os limiares aversivos de congelamento e fuga foram medidos durante a estimulação elétrica da SCPd em ratos ingênuos e em animais submetidos previamente ao procedimento de condicionamento contextual aversivo. A mediação destas repostas defensivas pelos receptores NK-1 e NK-3 foi avaliada pela injeção local de spantide (100 pmol/0,2 L) e SB 222200 (50 e 100 pmol/0,2 L), antagonistas seletivos de receptores NK-1 e NK-3, respectivamente. Os resultados mostraram que a injeção intra-SCPd de spantide aumentou os limiares aversivos determinados pela estimulação elétrica da SCPd em animais ingênuos e com experiência aversiva prévia. Injeções similares de 100 pmol de SB 222200 na SCPd também causaram um aumento nos limiares de congelamento e fuga. Entretanto, esses efeitos devem ser atribuídos a um déficit motor causado por essa dose da droga, uma vez que SB 222200 na dose de 100 pmol, reduziu significativamente a atividade motora dos animais submetidos ao teste do campo aberto. Estes resultados sugerem que receptores NK-1, mas não NK-3, estão envolvidos na mediação do comportamento defensivo induzido pela estimulação elétrica da SCPd. / The dorsal periaqueductal gray (dPAG) constitutes the main structure of the neural substrate for the defensive response to proximal aversive stimuli. It is well established that GABA and 5-HT are important neuromediators of the defense reaction at the dPAG level but neurokinin-mediated mechanisms have also been recently implicated in these processes. The aim of the present investigation was to examine the involvement of NK-1 and NK-3 receptors of the dPAG in the unconditioned defensive behaviors induced by electrical stimulation of the dPAG of rats with and without previous experience with contextual fear conditioning. For this, aversive thresholds were measured as the lowest intensity of electric current to produce freezing and escape behaviors using a procedure of gradual increases of the electrical stimulation of the dPAG. These thresholds were also measured in rats previously submitted to a contextual fear conditioning procedure. The mediation of these defensive responses by NK-1 and NK-3 receptors of the dPAG was evaluated by local injections of spantide (100 pmol/0.2 L) and SB 222200 (50 and 100 pmol/0.2 L), selective antagonists of NK-1 and NK-3 receptors, respectively. The present results showed that spantide injections into the dPAG increased the aversive thresholds (anti-aversive effects) determined by stimulation of the dPAG in naive and in animals subjected previously to the contextual fear conditioning. Similar injections of SB 222200 100 pmol into the dPAG increased the freezing and escape thresholds. However, this effect can be attributable to a motor deficit since this dose of SB 222200 decreased the exploratory activity of the animals subjected to the open field test. These results suggest that NK-1 receptors, but not NK-3 receptors, are involved in the mediation of the defensive behaviors induced by electrical stimulation of the dPAG.

A substância cinzenta periaquedutal dorsal (SCPd) é considerada uma das principais estruturas do teto mesencefálico envolvida no substrato neural da aversão a estímulos proximais. GABA e 5-HT são apontados como neurotransmissores envolvidos na modulação das respostas defensivas elaboradas na SCPd. Recentemente, mecanismos neurocininérgicos também têm sido propostos como mediadores das reações de defesa organizadas nessa estrutura. O objetivo do presente estudo foi avaliar o envolvimento dos receptores NK-1 e NK-3 da SCPd no comportamento defensivo induzido pela estimulação elétrica dessa região em ratos com e sem experiência prévia ao condicionamento contextual aversivo. Para isso, os limiares aversivos de congelamento e fuga foram medidos durante a estimulação elétrica da SCPd em ratos ingênuos e em animais submetidos previamente ao procedimento de condicionamento contextual aversivo. A mediação destas repostas defensivas pelos receptores NK-1 e NK-3 foi avaliada pela injeção local de spantide (100 pmol/0,2 L) e SB 222200 (50 e 100 pmol/0,2 L), antagonistas seletivos de receptores NK-1 e NK-3, respectivamente. Os resultados mostraram que a injeção intra-SCPd de spantide aumentou os limiares aversivos determinados pela estimulação elétrica da SCPd em animais ingênuos e com experiência aversiva prévia. Injeções similares de 100 pmol de SB 222200 na SCPd também causaram um aumento nos limiares de congelamento e fuga. Entretanto, esses efeitos devem ser atribuídos a um déficit motor causado por essa dose da droga, uma vez que SB 222200 na dose de 100 pmol, reduziu significativamente a atividade motora dos animais submetidos ao teste do campo aberto. Estes resultados sugerem que receptores NK-1, mas não NK-3, estão envolvidos na mediação do comportamento defensivo induzido pela estimulação elétrica da SCPd. / The dorsal periaqueductal gray (dPAG) constitutes the main structure of the neural substrate for the defensive response to proximal aversive stimuli. It is well established that GABA and 5-HT are important neuromediators of the defense reaction at the dPAG level but neurokinin-mediated mechanisms have also been recently implicated in these processes. The aim of the present investigation was to examine the involvement of NK-1 and NK-3 receptors of the dPAG in the unconditioned defensive behaviors induced by electrical stimulation of the dPAG of rats with and without previous experience with contextual fear conditioning. For this, aversive thresholds were measured as the lowest intensity of electric current to produce freezing and escape behaviors using a procedure of gradual increases of the electrical stimulation of the dPAG. These thresholds were also measured in rats previously submitted to a contextual fear conditioning procedure. The mediation of these defensive responses by NK-1 and NK-3 receptors of the dPAG was evaluated by local injections of spantide (100 pmol/0.2 L) and SB 222200 (50 and 100 pmol/0.2 L), selective antagonists of NK-1 and NK-3 receptors, respectively. The present results showed that spantide injections into the dPAG increased the aversive thresholds (anti-aversive effects) determined by stimulation of the dPAG in naive and in animals subjected previously to the contextual fear conditioning. Similar injections of SB 222200 100 pmol into the dPAG increased the freezing and escape thresholds. However, this effect can be attributable to a motor deficit since this dose of SB 222200 decreased the exploratory activity of the animals subjected to the open field test. These results suggest that NK-1 receptors, but not NK-3 receptors, are involved in the mediation of the defensive behaviors induced by electrical stimulation of the dPAG.