Type II Afferents and Cochlear Damage

这一建议旨在检验类型II传入作为耳蜗痛感受器的假说。 从人类对听力损失后听力亢进的抱怨中得到线索，我们将检查这个结构 正常耳蜗区和创伤后耳蜗区II型传入神经的功能。工作假说是 痛性听觉过敏症包括II型传入神经活动亢进，类比为痛觉过敏。 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 大鼠和小鼠的耳蜗虫。同时，我们将研究后存活的外毛细胞的特性 外伤性耳蜗病。我们的方法包括：体外电生理学，光学和电子显微镜，利用 光遗传和化学遗传工具，以及对II型小鼠模型的验证和量化 表达了特定的生物标记。 必要的第一步是将我们的体外实验方法扩展到较老的耳蜗，以便 声音创伤所造成的变化可以与正常情况相比较。我们会比较一下 破坏声音、耳毒性抗生素和基因编码的生物毒素以实验生产 在体外实验中对组织的易于处理的影响。II型突触的性质和突触联系 将在这些动物的耳蜗组织中检查传入细胞和外毛细胞。我们会 继续探索II型特异性遗传小鼠模型。基因编码的报告蛋白， 电压和钙敏感指示剂、生物毒素以及光和化学遗传调节剂 成为神经生物学中信息量很大的工具，特别是内耳。我们的 正在进行的研究已经鉴定了一种小鼠系，酪氨酸羟化酶启动子驱动的Cre重组酶 表情。另外三个候选的II型特定CRE品系将被验证和量化。有了这样的 转基因模型通过表达荧光研究神经支配模式成为可能 报告蛋白，并激活、消除或调节II型的解剖和生理活性 学习。依赖Cre的转基因G蛋白偶联受体(DREADD)的表达将 提供可以通过注射一种新的合成物来增加或降低II型活动的小鼠 配基，取决于特定的结构。全身用药和圆窗用药的不同组合 传递将被用来增加实验操作的特异性。 这个实验计划的总体目标是完成对第二类的描述 传入，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 充当耳蜗性伤害性感受器。如果正确，这些可能是缺氧症的神经生物学底物。 (痛性听觉过敏症)。通过定义II型功能的基本细胞和分子机制和 可塑性，未来的治疗靶点可以被确定以改善或预防缺氧症。

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.