Activity dependent mechanisms of olfactory system development

嗅觉系统发育的活动依赖机制

• 批准号: 8368090
• 负责人: Arie Sitthichai Mobley
• 金额: $ 16.6万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8368090
• 关键词: Affect; Afferent Neurons; Animals; Area; Axon; Behavior; Binding; Biological Assay; Brain; Cardiac; Cyclic AMP; Cyclic Nucleotides; Cytoskeleton; Development; Electroporation; Embryo; Epilepsy; Event; Filopodia; Future; GTP-Binding Proteins; Goals; Growth; Growth Cones; Image; Imagery; In Vitro; Investigation; Ion Channel; Kinetics; Location; Measures; Mediating; Membrane Potentials; Mesenchyme; Mus; Nasal cavity; Neuraxis; Neurites; Odorant Receptors; Odors; Olfactory Epithelium; Peripheral; Phase; Plasmids; Process; Regulation; Reporting; Research; Role; Signal Transduction; Sinus Bradycardia; Sorting - Cell Movement; Stimulus; Syndrome; System; Systems Development; Techniques; Testing; Up-Regulation; age related; axon growth; channel blockers; critical period; cyclic-nucleotide gated ion channels; design; immunocytochemistry; in utero; in vivo; interest; neuronal cell body; novel strategies; olfactory bulb; painful neuropathy; postnatal; research study; small hairpin RNA

DESCRIPTION (provided by applicant): During development, the olfactory sensory neuron (OSN) axons navigate through the mesenchyme, coalesce with other axons expressing the same odorant receptor, and converge into a glomerulus in a topographically defined region of the olfactory bulb (OB). Although considerable effort has been made, the mechanisms regulating these events are not well understood. My long- term goal is to understand the mechanisms mediating this progression. Currently I am interested in the role that functional activity has in these events. This application focuses on the contribution of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel activity to olfactory development. The HCN channel current Ih was first characterized in cardiac pacemaking activity; the channels are found extensively in the central nervous system. HCN channels are gated by cAMP, which can affect channel kinetics depending on which of the four HCN subunits (HCN1-4) are present. The cyclic nucleotide binding domain of HCN channels make them good candidates for affecting the cAMP activity recently implicated in olfactory system development (Imai et al., 2006, Zou et al., 2007, Chesler et al., 2007). The following aims are designed to determine the role of HCN channels in OSN axon outgrowth, growth cone activity, coalescence and glomerular formation. We previously showed an upregulation of the HCN2 subunit during development (Mobley et al., 2010). Specific Aim 1 examines the mechanisms underlying the role of HCN2, which is strongly modulated by cAMP, in development. I will employ in utero electroporation of the olfactory epithelium to introduce a plasmid expressing GFP and HCN2 shRNA targeting OSNs. This original technique adapted to the nasal cavity will enable visualization of OSN axon behavior in vivo and may have a lasting influence on future studies of OSN dynamics. Specific Aim 2 examines the role of HCN channels and membrane potential in growth cone lamellipodia formation. Guidance signal-induced cAMP signaling is important for OSN axon growth cone targeting and coalescence. Elsewhere in brain cyclic nucleotide signaling is implicated in growth cone dynamics, though the role of HCN channels has not been explored (Ming et al., 1997; Maritan et al., 2009; Murray et al., 2009). Thus, our analyses of HCN mediated growth cone dynamics in OSNs would not only impact olfactory system development but on developmental mechanisms throughout the brain. Specific Aim 2 will challenge OSNs in vitro with membrane potential changes and HCN channel blockers to measure changes in growth cone lamellipodia and filopodia. Demonstrating a role for HCN channels in growth cone dynamics would impact a much larger field than olfactory research and provide new avenues for investigating activity dependent mechanisms of olfactory system development. The results of these studies may provide mechanisms for OSN axon targeting during development such as age-dependent subunit regulation and membrane potential-induced growth cone dynamics. The experiments proposed here may have broad implications for Ih channelopathies including sinus bradycardia, epilepsy, and peripheral neuropathic pain. PUBLIC HEALTH RELEVANCE: This proposal seeks to determine how hyperpolarization-activated cyclic nucleotide-gated (HCN) channels influence development of the olfactory system. This study will expand our understanding of how activity- dependent mechanisms influence axon growth cone dynamics, coalescence and glomerular formation during development, and may apply to the study of olfactory deficiencies such as Kallman syndrome. In turn, these results may apply broadly to cardiac pacemaking, sinus bradycardia, some forms of epilepsy and neuropathic pain, in which HCN channels are implicated.

描述（由申请人提供）：在发育过程中，嗅觉感觉神经元（OSN）轴突穿过间充质，与表达相同气味受体的其他轴突结合，并汇聚成嗅球（OB）地形定义区域中的肾小球。尽管已经付出了相当大的努力，但调节这些事件的机制尚不清楚。我的长期目标是了解介导这一进展的机制。目前我对功能活动在这些事件中的作用感兴趣。该应用重点关注超极化激活的环核苷酸门控 (HCN) 通道活性对嗅觉发育的贡献。 HCN通道电流Ih首先在心脏起搏活动中被表征；这些通道广泛存在于中枢神经系统中。 HCN 通道由 cAMP 门控，cAMP 会影响通道动力学，具体取决于存在四个 HCN 亚基 (HCN1-4) 中的哪一个。 HCN 通道的环核苷酸结合域使其成为影响最近与嗅觉系统发育有关的 cAMP 活性的良好候选者（Imai 等人，2006；Zou 等人，2007；Chesler 等人，2007）。 以下目标旨在确定 HCN 通道在 OSN 轴突生长、生长锥活性、聚结和肾小球形成中的作用。我们之前表明 HCN2 亚基在发育过程中上调（Mobley 等人，2010）。具体目标 1 检查 HCN2 在发育过程中受 cAMP 强烈调节的作用机制。我将在子宫内对嗅上皮进行电穿孔，以引入表达靶向 OSN 的 GFP 和 HCN2 shRNA 的质粒。这种适用于鼻腔的原创技术将使 OSN 轴突在体内的行为可视化，并可能对 OSN 动力学的未来研究产生持久的影响。具体目标 2 检查 HCN 通道和膜电位在生长锥片状伪足形成中的作用。引导信号诱导的 cAMP 信号对于 OSN 轴突生长锥靶向和聚结非常重要。在大脑的其他地方，环核苷酸信号传导与生长锥动力学有关，尽管 HCN 通道的作用尚未被探索（Ming 等人，1997 年；Maritan 等人，2009 年；Murray 等人，2009 年）。因此，我们对 OSN 中 HCN 介导的生长锥动力学的分析不仅会影响嗅觉系统的发育，还会影响整个大脑的发育机制。具体目标 2 将通过膜电位变化和 HCN 通道阻滞剂在体外挑战 OSN，以测量生长锥片状伪足和丝状伪足的变化。证明 HCN 通道在生长锥动力学中的作用将影响比嗅觉研究更大的领域，并为研究嗅觉系统发育的活动依赖机制提供新途径。 这些研究的结果可能提供发育过程中 OSN 轴突靶向的机制，例如年龄依赖性亚基调节和膜电位诱导的生长锥动力学。这里提出的实验可能对 Ih 通道病（包括窦性心动过缓、癫痫和周围神经性疼痛）具有广泛的影响。 公共健康相关性：该提案旨在确定超极化激活的环核苷酸门控 (HCN) 通道如何影响嗅觉系统的发育。这项研究将扩大我们对活动依赖性机制如何影响发育过程中轴突生长锥动力学、合并和肾小球形成的理解，并可能应用于嗅觉缺陷（例如卡尔曼综合征）的研究。反过来，这些结果可能广泛适用于与 HCN 通道有关的心脏起搏、窦性心动过缓、某些形式的癫痫和神经性疼痛。