Publicação
In vivo and in real-time mechanisms of synaptic bouton formation
| Resumo: | A functional nervous system relies on the communication between neurons via highly specialized structures, called synapses. During development, neuronal morphology and wiring establishment are genetically determined but neuronal structural and function can be altered by changing levels of activity, a process known as synaptic plasticity. Adult neuronal circuits remain plastic and this feature is what allows us to store information and adapt to the environment. Defects in synaptic connectivity and activity-dependent plasticity are characteristic of neurodevelopmental and neurodegenerative disorders. Notoriously, recent studies highlight a link between functional defects in the presynaptic elements of neurons, called synaptic boutons, and the origin of several of these diseases. Presynaptic boutons, round varicosities conserved from invertebrates to man, are highly dynamic structures where synapses are located and where neurotransmission occurs. Despite being one of the main synaptic compartments, very little is known about the mechanism and dynamics of their genesis. Several studies contributed to the understanding of this question, but detailed mechanistic information regarding bouton outgrowth is still lacking. The purpose of this work is to acquire fundamental knowledge regarding how presynaptic boutons are formed and integrated into wired neurons. The powerful model synapse of Drosophila neuromuscular junction (NMJ) was adopted to dissect the details of bouton outgrowth in vivo and in real-time since it exhibits robust structural plasticity and the synapses formed on particular muscles have characteristic shapes and discernible boutons. The analysis was performed on the 3rd instar larvae because developmental bouton addition is nearly complete, and at this stage acute structural plasticity can be induced by patterned stimulation of motor neurons (MNs) using a variety of stimuli (high K+ solution, electrical activity and optogenetics). Well-established protocols using spaced high K+ paradigms were adopted to induce the rapid addition of synaptic boutons at this synapse. Neuronal migration and growth are critical events for the correct development and wiring of the nervous system. To date, the mechanisms described to give rise to presynaptic boutons involve the formation of filopodia or lamellipodia structures. However, performing high temporal resolution time-lapse imaging of unanesthetized Drosophila larval NMJs revealed a new, unreported, mechanism of presynaptic bouton addition into mature neurons. It was found that addition of synaptic boutons in response to acute activity does not occur like in the embryonic stage, where a growth cone differentiates into round boutons. Instead, new boutons rapidly emerge in a manner strongly resembling a mechanism known-to-be used by some cells in migration and tissue invasion. Considering that the NMJ is deeply inserted into the muscle, for the MN to include new boutons it must further invade the muscle, which mechanistically is not very different from migration across other tissues. It is suggested that MNs have possibly adopted a strategy that combines this form of migration with activity-dependent signaling pathways to modulate the formation of synaptic boutons during intense muscular activity. Additionally, manipulating the pathways that regulate this mechanism exposed an intricate interplay between MNs and the muscle in the regulation of the number of activity-dependent boutons. Interestingly, the movies also showed a strong correlation between muscle contraction and bouton formation. This finding implied that the muscle mechanics probably has an active role on the MN, that can be either by setting up de formation of new boutons in primed sites or by increasing the dynamics of this process in situations of increased stress. It is proposed that a balance of mechanical forces and biochemical signaling are probably coordinated during structural plasticity upon intense muscle activity. This research further expands our understanding of the mechanisms that control presynaptic growth and assembly in mature neurons. Further dissection of this phenomenon will contribute to uncover general principles that link normal development and function to dysfunction and disease, providing new insights into neuronal disease etiology and opening new avenues for the development of strategies to promote neuronal complexity and possibly delay symptoms associated with neuronal diseases. |
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| Autores principais: | Fernandes, Andreia Rubina Rodrigues |
| Assunto: | Botão pré-sináptico Atividade Plasticidade estrutural Drosophila Junção neuromuscular Teses de mestrado - 2017 |
| Ano: | 2017 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | A functional nervous system relies on the communication between neurons via highly specialized structures, called synapses. During development, neuronal morphology and wiring establishment are genetically determined but neuronal structural and function can be altered by changing levels of activity, a process known as synaptic plasticity. Adult neuronal circuits remain plastic and this feature is what allows us to store information and adapt to the environment. Defects in synaptic connectivity and activity-dependent plasticity are characteristic of neurodevelopmental and neurodegenerative disorders. Notoriously, recent studies highlight a link between functional defects in the presynaptic elements of neurons, called synaptic boutons, and the origin of several of these diseases. Presynaptic boutons, round varicosities conserved from invertebrates to man, are highly dynamic structures where synapses are located and where neurotransmission occurs. Despite being one of the main synaptic compartments, very little is known about the mechanism and dynamics of their genesis. Several studies contributed to the understanding of this question, but detailed mechanistic information regarding bouton outgrowth is still lacking. The purpose of this work is to acquire fundamental knowledge regarding how presynaptic boutons are formed and integrated into wired neurons. The powerful model synapse of Drosophila neuromuscular junction (NMJ) was adopted to dissect the details of bouton outgrowth in vivo and in real-time since it exhibits robust structural plasticity and the synapses formed on particular muscles have characteristic shapes and discernible boutons. The analysis was performed on the 3rd instar larvae because developmental bouton addition is nearly complete, and at this stage acute structural plasticity can be induced by patterned stimulation of motor neurons (MNs) using a variety of stimuli (high K+ solution, electrical activity and optogenetics). Well-established protocols using spaced high K+ paradigms were adopted to induce the rapid addition of synaptic boutons at this synapse. Neuronal migration and growth are critical events for the correct development and wiring of the nervous system. To date, the mechanisms described to give rise to presynaptic boutons involve the formation of filopodia or lamellipodia structures. However, performing high temporal resolution time-lapse imaging of unanesthetized Drosophila larval NMJs revealed a new, unreported, mechanism of presynaptic bouton addition into mature neurons. It was found that addition of synaptic boutons in response to acute activity does not occur like in the embryonic stage, where a growth cone differentiates into round boutons. Instead, new boutons rapidly emerge in a manner strongly resembling a mechanism known-to-be used by some cells in migration and tissue invasion. Considering that the NMJ is deeply inserted into the muscle, for the MN to include new boutons it must further invade the muscle, which mechanistically is not very different from migration across other tissues. It is suggested that MNs have possibly adopted a strategy that combines this form of migration with activity-dependent signaling pathways to modulate the formation of synaptic boutons during intense muscular activity. Additionally, manipulating the pathways that regulate this mechanism exposed an intricate interplay between MNs and the muscle in the regulation of the number of activity-dependent boutons. Interestingly, the movies also showed a strong correlation between muscle contraction and bouton formation. This finding implied that the muscle mechanics probably has an active role on the MN, that can be either by setting up de formation of new boutons in primed sites or by increasing the dynamics of this process in situations of increased stress. It is proposed that a balance of mechanical forces and biochemical signaling are probably coordinated during structural plasticity upon intense muscle activity. This research further expands our understanding of the mechanisms that control presynaptic growth and assembly in mature neurons. Further dissection of this phenomenon will contribute to uncover general principles that link normal development and function to dysfunction and disease, providing new insights into neuronal disease etiology and opening new avenues for the development of strategies to promote neuronal complexity and possibly delay symptoms associated with neuronal diseases. |
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