Neuronal Nicotinic Receptors- Expression and Regulation

描述（由申请人提供）：尼古丁信号在神经系统中广泛存在，影响许多行为和神经病理。该信号依赖于烟碱受体，烟碱受体通常促进钙内流并调节钙依赖事件。因此，信号传导的结果主要取决于受体的位置和相关细胞内成分的身份。最近有研究表明，PSD-95/SAP90家族成员在神经元上形成与尼古丁受体相关的突触后pdz支架。支架不仅帮助介导下游信号传导，而且在支持突触输入神经元方面起着重要但未知的作用。初步结果表明突触后神经素、EphB2受体和快速活动驱动的snare依赖受体运输也聚集在突触后神经元中调节尼古丁信号。研究人员提出了四个具体目标：(1)确定突触后pdz支架如何影响突触输入，并确定可能发挥作用的中间成分。(2)验证突触后神经素独立于pdz支架支持尼古丁输入的假设，并确定其如何与尼古丁受体相互作用。(3)研究促进尼古丁受体在神经元上快速转运的机制，并评估其在突触信号传导中的生理意义。(4)验证EphB2受体增强烟碱效应的假设，并研究控制烟碱信号通路在神经元中的相互依赖性。荧光成像将用于可视化突触成分；转染将用于操纵相互作用的伙伴；电生理分析将用于研究功能后果；生物化学和分子生物学技术将用于探测相互作用。实验将主要集中在鸡纤毛神经节神经元上，但也将使用转染细胞系来定义分子相互作用，并使用大鼠海马神经元来评估研究结果的普遍性。这些结果将为形成尼古丁信号的调节机制提供新的见解，这一点很重要，因为信号是普遍存在的，并具有深远的生物医学后果。

DESCRIPTION (provided by applicant): Nicotinic signaling is widespread in the nervous system and influences numerous behaviors and neuropathologies. The signaling depends on nicotinic receptors which often promote calcium influx and regulate calcium-dependent events. The consequence of signaling, therefore, can depend critically both on receptor location and on the identity of associated intracellular components. Recently it has been shown that members of the PSD-95/SAP90 family form a postsynaptic PDZ-scaffold associated with nicotinic receptors on neurons. The scaffold not only helps mediates downstream signaling but also plays an important but unknown role in supporting synaptic input to the neuron. Preliminary results suggest that postsynaptic neuroligin, EphB2 receptors, and rapid activity-driven SNARE-dependent receptor trafficking also converge in the postsynaptic neuron to regulate nicotinic signaling. Four specific aims are proposed to pursue these findings: (1) Determine how postsynaptic PDZ-scaffolds influence synaptic input and identify possible intermediate components exerting the effect. (2) Test the hypothesis: that postsynaptic neuroligin supports nicotinic input independently from PDZ-scaffolds and determine how it interacts with nicotinic receptors. (3) Examine the mechanisms promoting rapid trafficking of nicotinic receptors on neurons and assess their physiological significance for synaptic signaling. (4) Test the hypothesis that EphB2 receptors enhance nicotinic effects and examine the interdependence of convergent pathways controlling nicotinic signaling in neurons. Fluorescence imaging will be used to visualize synaptic components; transfections will be used to manipulate interacting partners; electrophysiological analysis will be used to study functional consequences; and biochemical and molecular biological techniques will be used to probe interactions. Experiments will focus mainly on chick ciliary ganglion neurons but will also employ transfected cell lines to define molecular interactions, and rat hippocampal neurons to assess the generality of the findings. These results will provide new insight into regulatory mechanisms that shape nicotinic signaling, important because the signaling is pervasive and has profound biomedical consequences.