Novel Chamber for Axonal Glutamate Signal Transduction

描述（由申请人提供）：作为精细和高度极化的细胞，中枢神经系统的神经元经常将其轴突延伸到极端距离。在这种现象中固有的是，神经元过程遇到多种不同的细胞外微环境。β-淀粉样蛋白、谷氨酸和神经营养因子都是这些微环境暴露的例子，它们可能影响其过程遇到它们的神经元的功能甚至存活。研究这些微环境对细胞过程和信号级联的影响在很大程度上受到了在体外复制这种微环境暴露的困难的阻碍。我们的实验室最近开发了新的腔室，使用微加工和软光刻技术，可以可靠地和可重复地模拟CNS神经元遇到的微环境。进一步的证据表明，在体外培养9天后，分离的神经突仅为轴突。该建议旨在进一步发展这种新的多室模型，用于研究微环境对中枢神经元的影响。此外，该模型的效用将在一项研究中检查的信号级联介导的cAMP反应元件结合蛋白（CREB）的磷酸化响应轴突限制性谷氨酸暴露。这个建议将测试的假设，轴突谷氨酸暴露诱导CREB磷酸化通过特定的受体介导的启动细胞内信号转导级联。特别是，我们将集中在潜在的钙介导的激活Ras-ERK和CaM激酶途径。

DESCRIPTION (provided by applicant): As elaborate and highly polarized cells, neurons of the central nervous system frequently extend their axons over extreme distances. Inherent in this phenomenon, neuronal processes encounter multiple varying extracellular microenvironments. beta-amyloid, glutamate, and neurotrophins are all examples of such microenvironmental exposures that may influence the function or even survival of neurons whose processes encounter them. Studies that examine the impact of these microenvironments on cellular processes and signaling cascades have in large part been hindered by the difficulty in replicating in vitro such microenvironmental exposures. Our lab has recently developed novel chambers using microfabrication and soft lithography techniques that can reliably and reproducibly simulate microenviroments encountered by CNS neurons. Further evidence shows that the neurites isolated are exclusively axons after nine days in vitro. This proposal is designed to further develop this novel multicompartment model for the study of microenvironmental influences over central neurons. In addition, the utility of this model will be examined in a study of the signaling cascades that mediate the phosphorylation of cAMP-responsive element binding protein (CREB) in response to axonally-restricted glutamate exposure. This proposal will test the hypothesis that axonal glutamate exposure induces CREB-phosphorylation via specific receptor-mediated initiation of intracellular signal transduction cascades. In particular, we will focus on potential calcium-mediated activation of Ras-ERK and CaM kinase pathways.