Doctor en Farmacología / Autorizada por el autor, pero con restricción para ser publicada a texto completo en el Portal de tesis, hasta enero de 2017 / La Serotonina (5-HT) es un neurotransmisor detectado tempranamente durante el desarrollo del sistema nervioso central y recientemente se ha descrito su papel sobre los procesos de migración y morfología neuronal. Mediante la inhibición farmacológica de la síntesis de 5-HT durante el desarrollo embrionario, se ha determinado que la reducción en este neurotransmisor produce efectos a largo plazo reflejado en una disminución del peso del cerebro, alteración en la maduración neuronal y una disminución de la complejidad dendrítica. La 5-HT ejerce sus acciones a través de sus receptores ionotrópicos (5HT3) y metabotrópicos (5HT1, 5HT2, 5HT4-5HT7). Algunos de ellos se expresan tempranamente durante el desarrollo embrionario y más aún puede variar su distribución y/o niveles en forma dinámica durante el desarrollo pre y posnatal. Para efectos de esta Tesis se destacan los receptores 5HT1A y 5HT7 los cuales están presentes en estructuras cerebrales como el hipocampo. Interesantemente, el 5HT1A-R se expresa tempranamente durante el desarrollo embrionario; por lo cual se postula que la 5-HT tendría un efecto modulador sobre la migración, diferenciación y viabilidad celular. En cambio, solo se ha descrito una expresión dinámica del 5HT7-R durante el desarrollo posnatal; lo que sugiere una acción de este receptor en estadíos bien definidos. La sobre-expresión de los receptores serotoninérgicos 5HT1A y 5HT7 en líneas celulares, ha permitido estudiar su acoplamiento a distintas cascadas transduccionales. Se ha descrito el acoplamiento a diversas proteínas G que permiten la activación o inhibición de proteínas efectoras como AC, fosfolipasas, regulación de canales iónicos y activación de las vías MAPK y PI3K-Akt, entre otras. Estas vías participan promoviendo la formación de neuritas y dendritas y han sido involucradas con la regulación de la plasticidad sináptica. En contraste, los efectores de estos receptores descritos en líneas celulares no han sido bien definidos en neuronas. Si bien existen algunos antecedentes que apoyan la idea de que la 5-HT regula la morfología neuronal, no se ha establecido la participación de un subtipo de receptor en particular. La activación del 5HT1A-R induce un aumento en el número y largo de neuritas en neuroblastoma de ratón. En relación a estos cambios morfológicos, se ha descrito que la vía de las Rho GTPasas regula el citoesqueleto a través de la modulación de la cofilina, importante regulador del citoesqueleto de actina. Se ha demostrado un acoplamiento entre el 5HT7-R y Gα12 y el aumento del crecimiento de neuritas en neuronas hipocampales, aunque no se ha dilucidado cuáles son sus efectores río abajo. De acuerdo a estos antecedentes se postuló la siguiente hipótesis: “Agonistas de los receptores 5HT1A y 5HT7 activan vías transduccionales que modulan el citoesqueleto, efectos que se asocian a cambios morfológicos de neuronas hipocampales en cultivos”. Los objetivos fueron: 1) Determinar en neuronas hipocampales con distinta madurez, el efecto de agonistas de los receptores serotoninérgicos 5HT1A y 5HT7 sobre proteínas que participan en vías transduccionales, que tienen como blanco el citoesqueleto de microtúbulos y de actina, como Akt-GSK-3β, ERK y cofilina, respectivamente. 2) Determinar el efecto de agonistas de los receptores serotoninérgicos 5HT1A y 5HT7 sobre la morfología de neuronas hipocampales con distinta madurez. Estos objetivos se desarrollaron utilizando antagonistas específicos para los receptores 5HT1A (WAY-100635) y 5HT7 (SB269970). En esta tesis se demostró que la distribución del receptor 5HT1A experimenta una polarización a mayor madurez del cultivo, encontrándose tempranamente en el soma y más tardíamente con una localización somato-dendrítica, sin variar el nivel del receptor. En contraste, la distribución somato-dendrítica del 5HT7-R fue independiente de la madurez del cultivo. A su vez, se detectaron las isoformas 5HT7a-R y 5HT7b-R y cuya relación no varió durante los días del cultivo in vitro (DIV). No obstante, ambas isoformas aumentan significativamente entre los 2 y 14 DIV. El 8OH-DPAT, agonista de los receptores 5HT1A (Ki 10 nM) y 5HT7 (Ki 52 nM), promueve la activación de la vía Akt-GSK-3β a los 2 DIV, efecto que involucraría la participación del 5HT7-R y, probablemente al 5HT1A-R. Adicionalmente, el 8OH-DPAT en neuronas más maduras (7 DIV), aumentó la activación de la Akt, efecto independiente de los receptores 5HT1A y 5HT7. Estos resultados sugieren que este agonista puede activar a otros receptores, lo que plantea una reevaluación de su acción como agonista selectivo del 5HT1A. Por otra parte, a pesar de la activación de la Akt, no se observaron cambios en la fosforilación de la GSK-3β, sugiriendo una disociación de ambas quinasas a este estadío. Adicionalmente, el 8OH-DPAT ó la 5-HT no modificaron significativamente el estado de fosforilación de las ERK1/2. La estimulación durante 24 horas con 8OH-DPAT en neuronas con 2 DIV aumentó significativamente el número de las neuritas primarias, acompañado de un aumento en el largo neurítico total; efectos no asociados a los receptores 5HT1A y 5HT7. Estos resultados sugieren que el 8OH-DPAT activa un receptor que incrementa el número de neuritas sin afectar su largo. Interesantemente, se observó que la estimulación con 8OH-DPAT disminuyó significativamente la fosforilación inactivante de la cofilina en Ser3; efecto insensible a la acción de los antagonistas de los receptores 5HT1A y 5HT7. Similarmente, a los 7 DIV se observó una disminución significativa de los niveles fosforilados de cofilina luego de la estimulación con 5-HT, efecto no asociado a los receptores 5HT1A y 5HT7, sugiriendo la participación de un receptor serotoninérgico desconocido sobre la dinámica de actina. Adicionalmente, la activación del 5HT7-R con 8OH-DPAT y 5HT a los 2 DIV durante 24 h disminuye el número de las dendritas primarias pero promueve el crecimiento. A este mismo estadío neuronal, la 5-HT a través de 5HT1A-R y 5HT7-R aumentó el crecimiento de dendritas secundarias. Por otro lado, se demostró a los 8 DIV que la 5-HT aumenta el número de dendritas primarias más cortas a través de un receptor serotoninérgico desconocido. Adicionalmente, los cambios inducidos por la 5-HT a los 8 DIV, indican que el 5HT1A-R promueve una reducción en el largo de las dendritas primarias, fenómeno observado por la reducción en el número de las dendritas primarias más largas (> 40 μm); efecto también observado en las secundarias más largas (> 20 μm). En apoyo, el análisis de Sholl muestra una disminución de la complejidad neuronal asociada a este receptor. Es decir, el 5HT1A-R inhibe el crecimiento y la complejidad dendrítica, probablemente al frenar el crecimiento o elongación de éstas, quedando por establecer los mecanismos moleculares asociados. En apoyo a este hallazgo, se ha descrito recientemente en hipocampo de ratón adulto que el 5HT1A-R produce una disminución de las dendritas terciarias. En contraste, a los 8 DIV el 5HT7-R promueve un aumento del crecimiento de dendritas primarias y secundarias, siendo este último efecto también observado por la estimulación del 5HT1A-R. En resumen, el 5HT1A-R promueve una disminución de la complejidad dendrítica, en contraste al efecto positivo de la activación del 5HT7-R observado a nivel de las neuritas y dendritas; sugiriendo que estos receptores pueden modular la morfología neuronal de forma diferencial dependiendo de la madurez del cultivo. Además, por primera vez se involucra la participación de los receptores 5HT1A y 5HT7 sobre la morfología neuronal en cultivo primario de hipocampo de rata. Además, este estudio muestra los efectos de agonistas serotoninérgicos sobre vías transduccionales relacionadas a la modulación del citoesqueleto y cambios en la morfología que depende de la madurez de neuronas hipocampales. / Serotonin (5-HT) is a neurotransmitter found early during development of the central nervous system and recently it has been involved in cell migration and neuronal morphology. The pharmacological inhibition of 5-HT synthesis during embryonic development suggests that its reduction produces long-term effects reflected as decreased brain weight, altered neuronal maturation and reduction of dendritic complexity. The 5-HT exerts its actions through ionotropic (5HT3) and metabotropic (5HT1, 5HT2, 5HT4-5HT7) receptors. Some of these receptors are expressed early during embryonic development and their distribution or levels can change dynamically during pre-and postnatal development. This thesis focuses on 5HT1A and 5HT7 receptors, which are present in brain structures such as the hippocampus. Interestingly, the 5HT1A-R is expressed early during embryonic development, suggesting that 5-HT could have a modulating effect on cell migration, differentiation and viability. In contrast, a dynamics expression of 5HT7-R has been described during postnatal development, suggesting a role in well-defined neuronal stages. Overexpression of 5HT1A and 5HT7 serotonin receptors in cell lines has allowed studying their coupling to several transduction pathways. These receptors are coupled to several G protein allowing the activation or inhibition of effector proteins such as AC, phospholipases, regulation of ion channels and activation of MAPK and PI3K-Akt, among others. These pathways promote neurites and dendrites formation, and have been implicated in the regulation of synaptic plasticity. In contrast, the effectors of these receptors described in cell lines have not been well defined in neurons. Although some evidences support the idea that 5-HT regulates neuronal morphology, it has not been established which serotonergic receptors are involved. The 5HT1A-R activation induces an increase in both the number and length of neurites in mouse neuroblastoma. In relation to these morphological changes, it has been described that Rho GTPases pathway regulates the cytoskeleton through the modulation of cofilin, an important regulator of actin dynamics. Additionally, a link between 5HT7-R, activation of Gα12 and the increase of neurite outgrowth in hippocampal neurons has been described, but it has not been elucidated the transduction pathway involved. According to these evidences, the following hypothesis was proposed: “Agonists of 5HT1A and 5HT7 receptors activate pathways that modulate the cytoskeleton, effects that are associated with morphological changes in cultured hippocampal neurons." The aims were to: 1) Determine, the effect of 5HT1A-R and 5HT7-R agonist on proteins involved in transduction pathways, targeting microtubule and actin cytoskeleton like Akt-GSK- 3β, ERK and cofilin, respectively, on hippocampal neurons with different maturity. 2) Determine the effect of 5HT1A-R and 5HT7-R agonist on morphology of hippocampal neurons with different maturity. These objectives were developed using specific antagonists of 5HT1A (WAY-100635) and 5HT7 (SB269970) receptors. In this thesis it was shown that the distribution of 5HT1A receptor undergoes a polarization during the maturity of neurons, being early found in the soma and late with a somato-dendritic distribution but without changes on receptor levels. In contrast, the somato-dendritic distribution of 5HT7-R was independent of neuronal maturity. Besides, the isoforms “a” and “b” of 5HT7-R were detected without changes in their ratio during days in vitro (DIV). Nonetheless, both isoforms increased significantly from 2 to 14 DIV. The 8OH-DPAT, a 5HT1A (Ki 10 nM) and 5HT7 (Ki 52 nM) receptor agonist, promotes the activation of the Akt-GSK-3β pathway at 2 DIV, effect which involves the participation of 5HT7-R and probably the 5HT1A-R. Additionally, in more mature neurons (7 DIV) stimulation with 8OH-DPAT increased activation of Akt, an effect independent of 5HT1A and 5HT7 receptors. These results suggest that this agonist may activate other receptors, and hence demands a reconsideration of its action as selective 5HT1A agonist. Moreover, in spite of Akt activation, no changes were observed in the phosphorylation levels of GSK-3β, suggesting dissociation of both kinases at this stage. Additionally, neither 8OH-DPAT nor 5-HT altered the phosphorylation levels of ERK1/2. Stimulation for 24 hours with 8OH-DPAT at 2 DIV significantly increased the number of primary neurites, accompanied by an increase in the overall length of neurites; effects which are not related to 5HT1A and 5HT7 receptors. These evidences suggest that 8OH-DPAT targets an unknown receptor whose activation increases the number of neurites without affecting their length. Interestingly, it was observed that stimulation with 8OH-DPAT significantly decreased the inactivating phosphorylation of cofilin in Ser3; an effect insensitive to the antagonists of the 5HT1A and 5HT7 receptors. Similarly, a significant decrease of phosphorylated cofilin levels after stimulation with 5-HT was observed at 7 DIV; effect not associated to 5HT1A and 5HT7 receptors, suggesting the participation of an unknown serotonin receptor modulating actin dynamics. Additionally, the 5HT7-R activation with 8OH-DPAT and 5-HT at 2 DIV during 24 h decreases the number of primary dendrites but promotes its growth. At this neuronal stage, 5-HT stimulates secondary dendrite growth through both 5HT1A-R and 5HT7-R. Furthermore, this thesis demonstrated at 8 DIV that 5-HT increases the number of short primary dendrites via an unknown serotonergic receptor. Additionally, the changes induced by 5-HT at 8 DIV, indicate that 5HT1A-R promotes a reduction in the length of the primary dendrites, a phenomenon observed as reduction in the number of longer primary dendrites (> 40 um); effect also observed in longer secondary dendrites (> 20 um). In accordance, Sholl analysis shows a decrease in neuronal complexity associated with this receptor. Thus, 5HT1A-R inhibits primary dendritic growth and complexity, probably by slowing the growth or elongation, and the molecular mechanisms associated remains to be explained. In support of this finding, it has been recently described in adult mouse hippocampus, that 5HT1A-R promotes a decrease of tertiary dendrites. In contrast, this Thesis described that 5HT7-R induces an increased growth of primary and secondary dendrites at 8 DIV; being the latter effect also observed by 5HT1A-R stimulation. To summarize, the 5HT1A promotes a reduction of dendritic complexity in contrast to the positive effect of 5HT7 receptors observed in both neurites and dendrites; suggesting that these receptors differentially modulate the neuronal morphology depending on neuron maturity. Furthermore, we described for first time the participation of 5HT1A and 5HT7 on neuronal morphology in primary culture of rat hippocampal neurons. Additionally this study shows the effects of serotonergic agonists on transduction pathways related to cytoskeleton modulation and morphological changes depending on maturity of hippocampal neurons. / Conicyt

A provocação social é um método utilizado em animais de laboratório para a indução de elevados níveis de agressividade, produzindo padrões comportamentais semelhantes ao de indivíduos violentos. Estudos prévios utilizando drogas que atuam seletivamente sobre os receptores 5-HT1A e 5-HT1B demonstraram uma redução pronunciada no comportamento agressivo. Um dos mais importantes sítios de ação para esses agentes serotonérgicos é o córtex pré-frontal, uma região cerebral particularmente relevante no controle inibitório da agressividade e da impulsividade. O objetivo do presente estudo foi verificar os possíveis efeitos anti-agressivos da microinjeção de 8-OH-DPAT e CP-93,129 (agonistas específicos dos receptores 5-HT1A e 5-HT1B, respectivamente) na região ventro orbital do córtex pré-frontal de camundongos machos provocados socialmente. Para confirmar a especificidade do receptor, antagonistas 5-HT1A e 5-HT1B (WAY-100,635 e SB-224,289, respectivamente) também foram microinjetados na mesma região cerebral. 8-OH-DPAT na dose de 1.0 µg reduziu significativamente a freqüência de mordidas. A menor dose de CP- 93,129 (0.1 µg) também diminuiu o número de mordidas e de ataques laterais. Tais efeitos anti-agressivos não foram acompanhados por alterações no restante do repertório comportamental. A participação específica desses receptores foi verificada pela reversão dos efeitos com a utilização de WAY-100,635 (10.0 µg) e SB-224,289 (1.0 µg). Nossos resultados confirmam o envolvimento da região VO CPF e dos receptores 5-HT1A e 5-HT1B na modulação de altos níveis de agressividade, sem conseqüentes alterações em outras rotinas motoras. / Social instigation is used in rodents to induce high levels of aggression, a pattern of behavior similar to that of violent individuals. This procedure consists of a brief exposure to a provocative stimulus male, before direct confrontation with an intruder. Studies using 5- HT1A and 5-HT1B agonist receptors show a reduction in aggressive behavior. An important site of action for these drugs is the ventral orbito frontal cortex (VO PFC), an area of the brain which is particularly relevant in the inhibitory control of aggressiveness and impulsiveness. The objective of the present study was to assess the anti-aggressive effects of 5-HT1A and 5- HT1B agonist receptors (8-OH-DPAT and CP-93,129) on the VO PFC of socially provoked male mice. To confirm the specificity of the receptor, 5-HT1A and 5-HT1B antagonist receptors (WAY-100,635 and SB-224,289) were microinjected into the same area, in order to reverse the agonist effects. 8-OH-DPAT at 1.0 µg dose reduced the frequency of attack bites. The lowest dose of CP-93,129 (0.1 µg) also decreased the number of attack bites and lateral threats. The anti-aggressive effects were not accompanied by impairment of non-aggressive activities. Specific participation of the 1A and 1B receptors was verified by reversal of antiaggressive effects using selective antagonists WAY-100,635 (10.0 µg) and SB-224,289 (1.0 µg). In conclusion, the decrease in aggressiveness observed with microinjections of 5-HT1A and 5-HT1B receptor agonists into the VO PFC of socially provoked mice, supports the hypothesis that activation of these receptors modulates high levels of aggression in a behaviorally specific manner.

A provocação social é um método utilizado em animais de laboratório para a indução de elevados níveis de agressividade, produzindo padrões comportamentais semelhantes ao de indivíduos violentos. Estudos prévios utilizando drogas que atuam seletivamente sobre os receptores 5-HT1A e 5-HT1B demonstraram uma redução pronunciada no comportamento agressivo. Um dos mais importantes sítios de ação para esses agentes serotonérgicos é o córtex pré-frontal, uma região cerebral particularmente relevante no controle inibitório da agressividade e da impulsividade. O objetivo do presente estudo foi verificar os possíveis efeitos anti-agressivos da microinjeção de 8-OH-DPAT e CP-93,129 (agonistas específicos dos receptores 5-HT1A e 5-HT1B, respectivamente) na região ventro orbital do córtex pré-frontal de camundongos machos provocados socialmente. Para confirmar a especificidade do receptor, antagonistas 5-HT1A e 5-HT1B (WAY-100,635 e SB-224,289, respectivamente) também foram microinjetados na mesma região cerebral. 8-OH-DPAT na dose de 1.0 µg reduziu significativamente a freqüência de mordidas. A menor dose de CP- 93,129 (0.1 µg) também diminuiu o número de mordidas e de ataques laterais. Tais efeitos anti-agressivos não foram acompanhados por alterações no restante do repertório comportamental. A participação específica desses receptores foi verificada pela reversão dos efeitos com a utilização de WAY-100,635 (10.0 µg) e SB-224,289 (1.0 µg). Nossos resultados confirmam o envolvimento da região VO CPF e dos receptores 5-HT1A e 5-HT1B na modulação de altos níveis de agressividade, sem conseqüentes alterações em outras rotinas motoras. / Social instigation is used in rodents to induce high levels of aggression, a pattern of behavior similar to that of violent individuals. This procedure consists of a brief exposure to a provocative stimulus male, before direct confrontation with an intruder. Studies using 5- HT1A and 5-HT1B agonist receptors show a reduction in aggressive behavior. An important site of action for these drugs is the ventral orbito frontal cortex (VO PFC), an area of the brain which is particularly relevant in the inhibitory control of aggressiveness and impulsiveness. The objective of the present study was to assess the anti-aggressive effects of 5-HT1A and 5- HT1B agonist receptors (8-OH-DPAT and CP-93,129) on the VO PFC of socially provoked male mice. To confirm the specificity of the receptor, 5-HT1A and 5-HT1B antagonist receptors (WAY-100,635 and SB-224,289) were microinjected into the same area, in order to reverse the agonist effects. 8-OH-DPAT at 1.0 µg dose reduced the frequency of attack bites. The lowest dose of CP-93,129 (0.1 µg) also decreased the number of attack bites and lateral threats. The anti-aggressive effects were not accompanied by impairment of non-aggressive activities. Specific participation of the 1A and 1B receptors was verified by reversal of antiaggressive effects using selective antagonists WAY-100,635 (10.0 µg) and SB-224,289 (1.0 µg). In conclusion, the decrease in aggressiveness observed with microinjections of 5-HT1A and 5-HT1B receptor agonists into the VO PFC of socially provoked mice, supports the hypothesis that activation of these receptors modulates high levels of aggression in a behaviorally specific manner.

A provocação social é um método utilizado em animais de laboratório para a indução de elevados níveis de agressividade, produzindo padrões comportamentais semelhantes ao de indivíduos violentos. Estudos prévios utilizando drogas que atuam seletivamente sobre os receptores 5-HT1A e 5-HT1B demonstraram uma redução pronunciada no comportamento agressivo. Um dos mais importantes sítios de ação para esses agentes serotonérgicos é o córtex pré-frontal, uma região cerebral particularmente relevante no controle inibitório da agressividade e da impulsividade. O objetivo do presente estudo foi verificar os possíveis efeitos anti-agressivos da microinjeção de 8-OH-DPAT e CP-93,129 (agonistas específicos dos receptores 5-HT1A e 5-HT1B, respectivamente) na região ventro orbital do córtex pré-frontal de camundongos machos provocados socialmente. Para confirmar a especificidade do receptor, antagonistas 5-HT1A e 5-HT1B (WAY-100,635 e SB-224,289, respectivamente) também foram microinjetados na mesma região cerebral. 8-OH-DPAT na dose de 1.0 µg reduziu significativamente a freqüência de mordidas. A menor dose de CP- 93,129 (0.1 µg) também diminuiu o número de mordidas e de ataques laterais. Tais efeitos anti-agressivos não foram acompanhados por alterações no restante do repertório comportamental. A participação específica desses receptores foi verificada pela reversão dos efeitos com a utilização de WAY-100,635 (10.0 µg) e SB-224,289 (1.0 µg). Nossos resultados confirmam o envolvimento da região VO CPF e dos receptores 5-HT1A e 5-HT1B na modulação de altos níveis de agressividade, sem conseqüentes alterações em outras rotinas motoras. / Social instigation is used in rodents to induce high levels of aggression, a pattern of behavior similar to that of violent individuals. This procedure consists of a brief exposure to a provocative stimulus male, before direct confrontation with an intruder. Studies using 5- HT1A and 5-HT1B agonist receptors show a reduction in aggressive behavior. An important site of action for these drugs is the ventral orbito frontal cortex (VO PFC), an area of the brain which is particularly relevant in the inhibitory control of aggressiveness and impulsiveness. The objective of the present study was to assess the anti-aggressive effects of 5-HT1A and 5- HT1B agonist receptors (8-OH-DPAT and CP-93,129) on the VO PFC of socially provoked male mice. To confirm the specificity of the receptor, 5-HT1A and 5-HT1B antagonist receptors (WAY-100,635 and SB-224,289) were microinjected into the same area, in order to reverse the agonist effects. 8-OH-DPAT at 1.0 µg dose reduced the frequency of attack bites. The lowest dose of CP-93,129 (0.1 µg) also decreased the number of attack bites and lateral threats. The anti-aggressive effects were not accompanied by impairment of non-aggressive activities. Specific participation of the 1A and 1B receptors was verified by reversal of antiaggressive effects using selective antagonists WAY-100,635 (10.0 µg) and SB-224,289 (1.0 µg). In conclusion, the decrease in aggressiveness observed with microinjections of 5-HT1A and 5-HT1B receptor agonists into the VO PFC of socially provoked mice, supports the hypothesis that activation of these receptors modulates high levels of aggression in a behaviorally specific manner.