Molecular mechanisms of activity-dependent changes in neuronal connectivity

描述（由申请人提供）：在发育期间和整个成年期，神经系统将来自环境的感觉体验转化为神经元活动的变化，这反过来又导致突触连接和树突分支的长期改变。这一过程的中断可能导致长期不良的神经后果。例如，改变的突触数量和功能可塑性反应是许多精神健康障碍的标志，包括抑郁症和精神分裂症[1-3]。尽管活动依赖性过程在塑造神经元结构中的重要性，但对神经元兴奋性变化转化为连接性改变的分子机制知之甚少。在培养的神经元中使用基于RNAi的方法，我们鉴定了GTdR Rem 2作为兴奋性突触发育的新型调节剂[4，5]。我们还证明了Rem 2是一种新的立即早期基因，其转录响应于通过神经元去极化的钙内流而快速上调[6]。因此，Rem 2可能代表了一个关键分子，外部刺激通过它介导对神经元连接的直接影响。为了在脊椎动物模型生物体的完整神经系统中验证这一假设，我建议敲除啮齿动物视皮层锥体神经元中的Rem 2，并对这些神经元的突触接触和整体回路可塑性进行体内双光子成像。 神经元，同时调节视觉体验。这种鉴定Rem 2在活动依赖性神经回路发育中的作用的经验依赖性体内方法提供了极好的机会来增加我们对遗传编码的活动依赖性神经回路的理解。通过在体内电路分析，突触发育和基因调控的分子生物学方面具有专业知识的导师之间的独特合作，我将成为一系列令人印象深刻的实验技术的专家，这些技术将使我能够在未来探索完整神经系统中其他活动调节基因的功能。

DESCRIPTION (provided by applicant): During development and throughout adulthood, the nervous system transforms sensory experience from the environment into changes in neuronal activity which, in turn, cause long-lasting alterations in synaptic connections and dendritic arborization. Disruption to this process can result in long-term undesirable neurological consequences. For example, altered synapse number and functional plasticity responses are hallmarks of a number of mental health disorders including depression and schizophrenia [1-3]. Despite the importance of activity-dependent processes in shaping neuronal architecture, little is known about the molecular mechanisms by which changes in neuronal excitability are translated into altered connectivity. Using an RNAi-based approach in cultured neurons, we identified the GTPase Rem2 as a novel regulator of excitatory synapse development [4, 5]. We have also demonstrated that Rem2 is a novel immediate early gene whose transcription is rapidly up-regulated in response to calcium influx via neuronal depolarization [6]. Thus, Rem2 may represent a key molecule through which external stimuli mediate direct effects on neuronal connectivity. To test this hypothesis in the intact nervous system of a vertebrate model organism, I propose to knockdown Rem2 in pyramidal neurons in rodent visual cortex and perform in vivo two-photon imaging of the synaptic contacts and overall circuit plasticity of these neurons while modulating visual experience. This experience-dependent in vivo approach to identify the role of Rem2 in activity-dependent neural circuit development provides an excellent opportunity to increase our understanding of genetically encoded, activity- dependent neural circuitry. Through a unique collaboration between mentors with expertise in in vivo circuit analysis, synapse development, and the molecular biology of gene regulation, I will become an expert in an impressive array of experimental techniques which will allow me to probe the function of other activity- regulated genes in the intact nervous system in the future.