Molecular mechanisms of activity-dependent changes in neuronal connectivity

描述(申请人提供)：在发育期间和整个成年期，神经系统将环境中的感觉体验转化为神经元活动的变化，进而导致突触连接和树突树枝的长期变化。这一过程的中断可能导致长期的不良神经后果。例如，突触数量和功能可塑性反应的改变是包括抑郁症和精神分裂症在内的许多精神健康障碍的特征[1-3]。尽管活动依赖的过程在塑造神经元结构方面很重要，但人们对神经元兴奋性的变化转化为连接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，我们发现GTPase Rem2是兴奋性突触发育的新调节因子[4，5]。我们还证明了Rem2是一个新的即刻早期基因，它的转录通过神经元去极化反应钙内流而迅速上调[6]。因此，Rem2可能是一个关键分子，外部刺激通过该分子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说，我提议敲除啮齿动物视皮层锥体神经元中的Rem2，并在体内对这些神经元的突触接触和整体电路可塑性进行双光子成像 神经元同时调节视觉体验。这种依赖经验的体内方法来确定Rem2在活动依赖的神经回路发育中的作用，为我们提供了一个极好的机会来增加我们对遗传编码的活动依赖的神经回路的理解。通过拥有体内电路分析、突触发育和基因调控分子生物学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方面的专家，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控基因的功能。

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.