Noise Trauma-Induced Cortical PV Neuron Dysfunction

项目总结 噪声损伤可导致听皮层小白蛋白阳性抑制性中间神经元的丢失，这是 在啮齿动物模型中与听觉处理缺陷和耳鸣有关。噪音背后的机制- 诱导的PV神经元丢失尚不清楚。我们建议对噪声诱导的PV神经元进行系统的表征 缺失并检验皮层PV神经元中TNFR1和TNFR2的不同激活 决定噪声损伤后PV神经元的命运，TNFR1偏向和TNFR2偏向 反对，神经元丢失和功能障碍。 具体目的1.研究细胞死亡作为噪声诱导的PV神经元丢失的一个机制。PV-Cre-td番茄 小鼠将经历一个诱发噪音创伤的程序。将分四次收集听觉皮质组织 点(1、3、5和10d后)覆盖整个细胞死亡过程。皮质组织切片将增加一倍 PV和细胞死亡标记的染色(TUNEL染色用于细胞凋亡，p-RIP3的抗体染色用于 坏死性下垂)。Td番茄的共定位(由结构性活性启动子驱动以标记所有PV神经元 即使停止表达PV)，PV和细胞死亡标记物也将被分析以量化PV神经元死亡 与缺少光伏。 特定目标2.确定存活的皮质PV神经元的输入和输出突触的特征 噪音创伤。PV-Cre-td番茄和PV-Cre-ChR2-td番茄小鼠将接受噪声创伤程序。 10天后，将准备好急性听觉皮质切片。膜片钳记录PV-CRE中的PV神经元 本课程将制作番茄切片，并研究兴奋性和抑制性突触在PV神经元上的输入。在……里面 此外，还将研究光基因激活的PV神经元在层2/3锥体神经元上的输出。这个 将描述输入-输出功能、短期可塑性和突触耗竭/恢复动力学。全 将噪声创伤组和假创伤组的结果进行比较。 具体目的3.研究TNFR1和TNFR2在噪声诱导的PV神经元损伤中的作用 突触功能障碍。我们将敲除PV-Cre-td番茄皮层PV神经元中的TNFR1或TNFR2，并 使用我们的定制病毒载体表达依赖于Cre的TNFR1或 TNFR2 shRNA。病毒载体表达绿色荧光蛋白，以便于比较转基因和未转基因 PV神经元。我们将研究TNFR1或TNFR2基因敲除对噪声诱导的PV神经元丢失的影响(如 目标1)、突触功能障碍(如目标2)和行为缺陷(如Masri等人，20218)。

PROJECT SUMMARY Noise trauma can lead to loss of parvalbumin-positive inhibitory interneurons in the auditory cortex, which is associated with audiotory processing deficit and tinnitus in rodent models. The mechanisms underlying noise- induced PV neuron loss are unknown. We propose to systematically characterize noise-induced PV neuron deficits and examine the hypothesis that differential activation of TNFR1 and TNFR2 in cortical PV neurons determines the fate of the PV neurons following noise trauma, with TNFR1 biasing for, and TNFR2 biasing against, neuronal loss and dysfunction. Specific Aim 1. Examine cell death as a mechanism of noise-induced PV neuron loss. PV-Cre-tdTomato mice will undergo a procedure to induce noise trauma. Auditory cortical tissue will be collected at four time points (1, 3, 5 and 10 d later) to cover the entire cell death process. Sections of cortical tissue will be double stained for PV and cell death markers (TUNEL staining for apoptosis, and antibody staining of p-RIP3 for necroptosis). Colocalization of tdTomato (driven by a constitutively active promoter to mark all PV neurons even if they stop expressing PV), PV and the cell death markers will be analyzed to quantify PV neuron death vs. lack of PV. Specific Aim 2. Characterize input and output synapses of the surviving cortical PV neurons following noise trauma. PV-Cre-tdTomato and PV-Cre-ChR2-tdTomato mice will undergo the noise trauma procedure. Ten days later, acute auditory cortical slices will be prepared. Patch clamp recording of PV neurons in PV-Cre- tdTomato slices will be made, and excitatory and inhibitory synaptic inputs on PV neurons will be examined. In addition, optogenetically activated PV neuron output onto Layer2/3 pyramidal neurons will be examined. The input-output function, short-term plasticity, and synaptic depletion/recovery dynamics will be characterized. All results will be compared between noise trauma and sham trauma groups. Specific Aim 3. investigate the roles of TNFR1 and TNFR2 in noise-induced PV neuron loss and synaptic dysfunction. We will knock down TNFR1 or TNFR2 in cortical PV neurons of PV-Cre-tdTomato and PV-Cre-ChR2-tdTomato mice using our custom-made viral vectors for Cre-dependent expression of TNFR1 or TNFR2 shRNA. The viral vector expresses GFP to allow comparison between transfected and untransfected PV neurons. We will examine effects of TNFR1 or TNFR2 knockdown on noise-induced PV neuron loss (as in Aim 1), synaptic dysfunction (as in Aim 2) and behavioral deficits (as in Masri et al., 20218).