Investigations into the mouse olivocochlear system

描述(申请人提供)：最近发现促肾上腺皮质激素释放激素(CRH)受体和促肾上腺皮质激素释放激素(CRH)家族的成员urocortin分别在外毛细胞和橄榄耳蜗末梢表达。Urocorn是目前已知的唯一在内耳表达的能够激活CRH受体的配体，虽然已经在urocortin缺陷小鼠身上分析了它所起的作用，但对内耳CRH受体的发育表达模式或作用尚不清楚。CRH受体是G蛋白偶联受体，刺激cAMP第二信使-信号级联反应。我们假设CRH受体的激活可能代表了一种潜在的保护噪声引起的听力损失的现象。其作用机制可能包括SK2钙激活的阿帕明敏感钾通道的磷酸化和失活。为了进一步分析耳蜗CRH系统的形态特征、在听力中的功能作用以及对噪声性听力损失的保护作用，最后，为了评估与CRH受体激活相关的细胞作用机制，提出了三个特定的目标。首先，我们将建立CRH受体在内耳的发育和成体表达模式。这一特定目的的实验的成功完成将建立内耳内表达CRH受体的细胞的准确身份，并在超微结构水平上识别Urocortin免疫阳性纤维的突触后成分。其次，我们将确定CRH在听力中的功能作用，以及它们是否参与保护内耳免受噪声性听力损失的影响。这一目标将通过缺乏1型或2型CRH受体基因或两者都缺乏的小鼠来实现。最后，我们将利用这些小鼠来确定在CRH受体基因去除后，cAMP诱导的外毛细胞靶标的磷酸化是否有变化。这一目标的成功将使我们能够识别由于CRH受体激活而被磷酸化的单个蛋白质，以及它们在调节正常的橄榄耳蜗突触活动中的作用。这将在功能上将urocortin激素/CRH受体和经典的ACh神经递质系统联系在一起，在一个统一的模型中解释基于内耳的保护，防止噪音导致的听力损失。

DESCRIPTION (provided by applicant): Corticotropin releasing hormone (CRH) receptors, and urocortin, a member of the CRH family of peptides, has recently been discovered expressed in outer hair cells and olivocochlear terminals, respectively. While analysis of the role played by urocorUn, the only known ligand expressed in the inner ear capable of activating the CRH receptors, has been analyzed using urocortin deficient mice, nothing is known of the developmental expression pattern or role of the CRH receptors in the inner ear. The CRH receptors are G protein coupled receptors, and stimulate the cAMP second messenger-signaling cascade. We hypothesize that activation of the CRH receptors may represent one phenomenon underlying protection from noise induced hearing loss. The mechanisms of action may include phosphorylation and inactivation of the sK2 calcium-activated apamin-sensitive potassium channel. In order to further analyze the morphological aspects of the CRH system in the cochlea, its functional role in hearing and protection from noise induced hearing loss, and finally, to assess the cellular mechanisms of action associated with activation of the CRH receptors, three specific aims are proposed. First, we will establish the developmental and adult expression pattern of the CRH receptors in the inner ear. Successful completion of the experiments of this specific aim will establish the precise identity of the cells within the inner ear that express the CRH receptors, and identify the postsynaptic elements of the urocortin immunopositive fibers at the ultra structural level. Second, we will establish the functional roles CRH plays in hearing, and whether they participate in protection of the inner ear from noise induced hearing loss. This aim will be accomplished using mice that lack the gene for either the type 1 or the type 2 CRH receptor, or that lack both. Finally, we will use these mice to establish whether there are alterations in cAMP induced phosphorylation of targets in the outer hair cells following CRH receptors gene ablation. Success in this aim will allow us to identify individual proteins phosphorylated due to activation of the CRH receptors, as well as their role in modulating normal olivocochlear synaptic activity. This will functionally link the urocortin hormone/CRH receptor and classical ACh neurotransmitter systems together in a unified model explaining inner ear based protection from noise induced hearing loss.