Type II Afferents and Cochlear Damage

II 型传入神经和耳蜗损伤

• 批准号: 9920127
• 负责人: Paul A Fuchs
• 金额: $ 39.25万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-16 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9920127
• 关键词: Acoustic Trauma; Action Potentials; Acute; Age; Anatomy; Antibiotics; Antibodies; Biological Markers; C Fiber; Calcium; Cochlea; Cre driver; Cues; DRD2 gene; Dose; Drug Delivery Systems; Electron Microscopy; Electrophysiology (science); Enterobacteria phage P1 Cre recombinase; Fiber; Future; G-Protein-Coupled Receptors; Genetic; Genetic Models; Glutamates; Goals; Hearing; Human; Hyperactive behavior; Hyperacusis; Hyperalgesia; Hypersensitivity; Injections; Label; Labyrinth; Ligands; Membrane; Methods; Microtomy; Modeling; Molecular; Mus; Neurobiology; Nociception; Nociceptors; Outer Hair Cells; Pattern; Perception; Pharmacology; Physiological; Property; Proteins; Protocols documentation; Rattus; Reporter; Role; Site; Specificity; Structure; Synapses; Testing; Tissues; Toxin; Transgenic Model; Trauma; Tyrosine 3-Monooxygenase; Validation; Work; animal tissue; base; cell injury; designer receptors exclusively activated by designer drugs; experimental study; extracellular; hearing impairment; light microscopy; mouse model; nerve supply; novel; noxacusis; optogenetics; ototoxicity; post-trauma; postsynaptic; prevent; programs; promoter; response; round window; sound; therapeutic target; tool; voltage

This proposal aims to test the hypothesis that type II afferents serve as cochlear nociceptors. Taking cues from the human complaint of hyperacusis after hearing loss, we will examine the structure and function of type II afferents in normal and post‐trauma cochleas. The working hypothesis is that painful hyperacusis, noxacusis, includes hyperactivity of type II afferents, by analogy to hyperalgesia of somatic nociceptive C‐fibers. Thus we will examine type II structure and function in normal and post‐trauma cochleas of rats and mice. In parallel we will investigate the properties of surviving outer hair cells in post‐ trauma cochleas. Our methods include: ex vivo electrophysiology, light and electron microscopy, utilization of optogenetic and chemogenetic tools,and validation and quantification of mouse models in which type II specific bio‐markers are expressed. A necessary first step is to extend our ex vivo experimental approach to older cochleas so that changes wrought by acoustic trauma can be compared to the normal condition. We will compare damaging sound, ototoxic antibiotics and genetically encoded biotoxins to produce experimentally tractable effects on tissue for ex vivo experiments. The properties and synaptic connections of type II afferents and outer hair cells will be examined in the excised cochlear tissue of these animals. We will continue to explore type II specific genetic mouse models. Genetically‐encoded reporter proteins, voltage‐ and calcium‐sensitive indicators, biotoxins, and opto‐ and chemo‐genetic modulators have become highly informative tools in neurobiology generally and for the inner ear specifically. Our ongoing work has characterized one mouse line, tyrosine hydroxylase promoter driven Cre‐recombinase expression. Three other candidate type II specific Cre lines will be validated and quantified. With such transgenic models it becomes possible to study innervation patterns by expression of fluorescent reporter proteins, and to activate, eliminate, or modulate type II activity for anatomical and physiological studies. Cre‐dependent expression of genetically‐modified G‐protein‐coupled receptors (DREADDS) will provide mice in which type II activity can be increased or decreased by injection of a novel synthetic ligand, depending on the specific construct. Varying combinations of systemic and round window drug delivery will be employed to increase the specificity of experimental manipulations. The over‐arching goal of this program of experiments is to complete the description of type II afferents, a still‐unresolved component of cochlear innervation. The working hypothesis is that these serve as cochlear nociceptors. If correct these are a likely neurobiological substrate for noxacusis (painful hyperacusis). By defining the basic cellular and molecular mechanisms of type II function and plasticity, future therapeutic targets can be identified to ameliorate or prevent noxacusis.

该提案旨在检验 II 型传入神经作为耳蜗伤害感受器的假设。 从人类听力损失后对听觉过敏的抱怨中汲取线索，我们将检查其结构 正常和创伤后耳蜗中 II 型传入神经的功能。工作假设是 疼痛性听觉过敏，noxacusis，包括 II 型传入神经的过度活跃，类似于 躯体伤害性 C 纤维。因此，我们将检查正常和创伤后的 II 型结构和功能 大鼠和小鼠的耳蜗。 同时，我们将研究存活后的外毛细胞的特性。 耳蜗外伤。我们的方法包括：离体电生理学、光学和电子显微镜、利用 光遗传学和化学遗传学工具的开发，以及 II 型小鼠模型的验证和定量 表达特定的生物标志物。 必要的第一步是将我们的离体实验方法扩展到老年耳蜗，以便 声损伤造成的变化可以与正常情况进行比较。 我们会比较 实验性生产破坏性声音、耳毒性抗生素和基因编码生物毒素 对离体实验的组织产生易于处理的影响。 II型的特性和突触连接 将在这些动物的切除耳蜗组织中检查传入细胞和外毛细胞。 我们将 继续探索II型特异性基因小鼠模型。 基因编码的报告蛋白， 电压和钙敏感指标、生物毒素以及光和化学遗传调节剂 成为神经生物学领域信息丰富的工具，特别是内耳领域的信息丰富的工具。 我们的 正在进行的工作已经确定了一种小鼠品系，即酪氨酸羟化酶启动子驱动的 Cre 重组酶 表达。 其他三个候选 II 型特异性 Cre 品系将得到验证和量化。 有了这样的 转基因模型可以通过荧光表达来研究神经支配模式 报告蛋白，并激活、消除或调节解剖学和生理学的 II 型活性 研究。转基因 G 蛋白偶联受体 (DREADDS) 的 Cre 依赖性表达将 提供可以通过注射新型合成物来增加或减少 II 型活性的小鼠 配体，取决于具体的构建体。 全身药物和圆窗药物的不同组合 将采用递送来增加实验操作的特异性。 该实验计划的首要目标是完成 II 型的描述 传入神经，耳蜗神经支配的一个尚未解决的组成部分。工作假设是这些 作为耳蜗伤害感受器。如果正确的话，这些可能是诺沙库斯病的神经生物学基础 （痛苦的听觉过敏）。通过定义 II 型功能的基本细胞和分子机制 通过可塑性，可以确定未来的治疗目标以改善或预防吸毒症。