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Striatal plasticity upon learning of a lateralized motor task
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RESUMO: Os circuitos dos núcleos da base estão crucialmente envolvidos na aquisição, aprendizagem e
consolidação de habilidades motoras. O corpo estriado é a principal região de input dos núcleos
da base e é composto principalmente por neurónios médios do estriado (medium spiny neurons,
MSNs). As espículas dendríticas dos MSNs representam um importante local de plasticidade
sináptica nos núcleos da base. Embora tenha sido demonstrado que a plasticidade sináptica
funcional do MSN ocorre num contexto de aprendizagem motora, não se sabe se esta é
acompanhada por plasticidade estrutural, ou seja, por mudanças no número, tamanho e/ou
padrão espacial das espículas dendríticas.
Neste estudo, o nosso objetivo consistiu em caracterizar o desempenho motor e a atividade do
corpo estriado na aprendizagem de habilidades motoras, com o objetivo final de estudar a
plasticidade estrutural na aprendizagem motora. Treinamos ratinhos numa tarefa operante
individualizada, em que uma recompensa é obtida após pressionar uma alavanca quatro vezes. À
medida que o treino avança, a posição e retração da alavanca impõem lateralidade, permitindo o
estudo das sequências de movimento realizadas por um único membro anterior. Durante o treino,
os ratinhos melhoraram o seu desempenho e aprenderam a realizar a tarefa com apenas uma das
patas dianteiras, aumentando o número total de movimentos de pressão sobre a alavanca por
minuto e organizando o comportamento em sequências de movimentos. Através da deteção da
trajetória da pata baseada em vídeo, examinamos ainda mais a cinemática do movimento durante
a aprendizagem da tarefa e descobrimos que a variabilidade da trajetória da pata durante a
pressão sobre a alavanca não diminuiu significativamente durante o treino.
Para identificar as células que estiveram ativas na última sessão da tarefa motora e, portanto, com
maior probabilidade de terem sofrido plasticidade sináptica, realizamos uma imunohistoquímica
contra c-Fos, um gene de expressão rápida frequentemente usado como marcador de plasticidade
neuronal. Usando um pipeline de deteção de células no cérebro inteiro, conseguimos detetar
células que expressam c-Fos e mapeá-las no atlas de uma maneira imparcial, usando fatias de
cérebro fixadas de ratinhos sacrificados 1h após a última sessão de treino. Os nossos resultados
preliminares sugerem que animais treinados na tarefa apresentam mudanças ligeiras na
expressão de c-Fos no hemisfério contralateral à pata treinada. Estas mudanças parecem ser
consistentes em regiões envolvidas na aprendizagem e desempenho motor nos animais
treinados, e incluem o corpo estriado, a camada 5 do córtex motor primário e a região do membro
superior do córtex somatossensorial primário.
Finalmente, procuramos estabelecer as ferramentas e técnicas necessárias para estudar a
plasticidade estrutural em fatias fixadas do cérebro. Otimizamos uma nova abordagem viral para
obter uma marcação pouco densa de MSNs, permitindo a visualização e reconstrução de toda a
árvore dendrítica de MSNs individuais, incluindo espículas dendríticas. Usando microscopia
confocal de alta resolução, em conjunto com softwares de deconvolução e análise de espículas
dendríticas, adquirirmos imagens e reconstruimos as espículas dendríticas do MSN e
caracterizamos a morfologia das mesmas. Descobrimos que os D1-MSNs têm uma
preponderância de espículas do tipo mushroom, seguidas por espículas do tipo thin e stubby, com
os filopodia constituindo apenas uma pequena fração do número total das protuberâncias
dendríticas.
O nosso trabalho estabeleceu as técnicas e metodologias básicas que permitirão estudos futuros
sobre a densidade, volume e distribuição das espículas dendríticas em neurónios que
recentemente sofreram plasticidade após aprendizagem motora. Essas experiências, por sua vez,
irão avançar a nossa compreensão de como a computação de inputs pelos MSNs muda durante a
aprendizagem motora, com implicações importantes para o estudo e tratamento de doenças
motoras.
ABSTRACT The basal ganglia circuits are critically involved in the acquisition, learning and consolidation of motor skills. The striatum is the major input region of the basal ganglia and is mainly composed of medium spiny neurons (MSNs). MSN dendritic spines represent a major site of synaptic plasticity in the basal ganglia. While MSN functional synaptic plasticity has been shown to occur in the context of motor learning, it remains unknown whether it is accompanied by structural plasticity, i.e., by changes in the number, size and/or spatial pattern of dendritic spines. In this study, we aimed to characterize motor performance and striatal activity upon motor skill learning, with the ultimate goal of studying structural plasticity upon motor learning. We trained mice in a self-paced operant task where a reward is obtained after pressing a lever four times. As training progresses, the position and retractability of the lever impose laterality, allowing the study of movement sequences performed by a single forelimb. During training, mice improved their performance and learned to perform the task with only one forepaw, increasing the total number of lever presses per minute and organizing their behavior in sequences of lever presses. Using video-based paw trajectory detection, we further dissected movement kinematics during task learning, and found that paw trajectory variability during lever press did not significantly decrease throughout training. To identify cells that were active in the last session of the motor task, and therefore more likely to have undergone synaptic plasticity, we performed immunostaining against c-Fos, an immediate early gene commonly used as a neuronal plasticity marker. Using a whole-brain cell detection pipeline, we were able to achieve unbiased cell detection and atlas mapping of c-Fos expressing cells, using fixed brain slices of mice sacrificed 1h after the last training session. Our preliminary results suggest that task-trained animals have subtle changes in c-Fos expression in the contralateral hemisphere to the trained paw. These changes appeared to be consistent across regions involved in motor learning and performance in task-trained animals that include the striatum, primary motor cortex layer 5, and upper limb region of the primary somatosensory cortex. Finally, we sought to establish the tools and techniques needed to study structural plasticity in fixed brain slices. We optimized a novel viral approach to achieve sparse labelling of MSNs, allowing the visualization and reconstruction of the whole dendritic arbor of single MSNs, including dendritic spines. Using high-resolution confocal microscopy, together with deconvolution and spine analysis software, we were able to image and reconstruct MSN dendritic spines and characterize spine morphology. We found that D1-MSNs have a preponderance of mushroom spines, followed by thin and stubby spines, with filopodia making up just a small fraction of overall dendritic protrusions. Our work has established the basic techniques and methodologies that will allow future studies on dendritic spine density, volume and distribution in neurons that recently underwent plasticity upon motor learning. Those experiments will, in turn, advance our understanding of how MSN input computation changes during motor learning, with important implications for the study and treatment of movement disorders.
ABSTRACT The basal ganglia circuits are critically involved in the acquisition, learning and consolidation of motor skills. The striatum is the major input region of the basal ganglia and is mainly composed of medium spiny neurons (MSNs). MSN dendritic spines represent a major site of synaptic plasticity in the basal ganglia. While MSN functional synaptic plasticity has been shown to occur in the context of motor learning, it remains unknown whether it is accompanied by structural plasticity, i.e., by changes in the number, size and/or spatial pattern of dendritic spines. In this study, we aimed to characterize motor performance and striatal activity upon motor skill learning, with the ultimate goal of studying structural plasticity upon motor learning. We trained mice in a self-paced operant task where a reward is obtained after pressing a lever four times. As training progresses, the position and retractability of the lever impose laterality, allowing the study of movement sequences performed by a single forelimb. During training, mice improved their performance and learned to perform the task with only one forepaw, increasing the total number of lever presses per minute and organizing their behavior in sequences of lever presses. Using video-based paw trajectory detection, we further dissected movement kinematics during task learning, and found that paw trajectory variability during lever press did not significantly decrease throughout training. To identify cells that were active in the last session of the motor task, and therefore more likely to have undergone synaptic plasticity, we performed immunostaining against c-Fos, an immediate early gene commonly used as a neuronal plasticity marker. Using a whole-brain cell detection pipeline, we were able to achieve unbiased cell detection and atlas mapping of c-Fos expressing cells, using fixed brain slices of mice sacrificed 1h after the last training session. Our preliminary results suggest that task-trained animals have subtle changes in c-Fos expression in the contralateral hemisphere to the trained paw. These changes appeared to be consistent across regions involved in motor learning and performance in task-trained animals that include the striatum, primary motor cortex layer 5, and upper limb region of the primary somatosensory cortex. Finally, we sought to establish the tools and techniques needed to study structural plasticity in fixed brain slices. We optimized a novel viral approach to achieve sparse labelling of MSNs, allowing the visualization and reconstruction of the whole dendritic arbor of single MSNs, including dendritic spines. Using high-resolution confocal microscopy, together with deconvolution and spine analysis software, we were able to image and reconstruct MSN dendritic spines and characterize spine morphology. We found that D1-MSNs have a preponderance of mushroom spines, followed by thin and stubby spines, with filopodia making up just a small fraction of overall dendritic protrusions. Our work has established the basic techniques and methodologies that will allow future studies on dendritic spine density, volume and distribution in neurons that recently underwent plasticity upon motor learning. Those experiments will, in turn, advance our understanding of how MSN input computation changes during motor learning, with important implications for the study and treatment of movement disorders.
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Palavras-chave
Striatal Plasticity Motor Skills Dendritic Spine Neurons Movement Disorders Treatment