Noise Trauma-Induced Cortical PV Neuron Dysfunction

项目摘要 噪声创伤可导致听觉皮层中小白蛋白阳性抑制性中间神经元的丢失， 与啮齿动物模型中的听觉处理缺陷和耳鸣相关。噪音背后的机制- 引起的PV神经元损失是未知的。我们建议系统地描述噪声诱导的PV神经元 缺陷和检查的假设，差异激活TNFR 1和TNFR 2在皮层PV神经元 决定了噪声损伤后PV神经元的命运，TNFR 1偏向于，TNFR 2偏向于 防止神经元丢失和功能障碍。 具体目标1。检查细胞死亡作为噪声诱导PV神经元损失的机制。PV-Cre-td番茄 小鼠将经历诱导噪声创伤的程序。将在四个时间点收集听觉皮层组织 点（1，3，5和10天后），以涵盖整个细胞死亡过程。皮质组织的切片将是双层的 针对PV和细胞死亡标记物染色（针对凋亡的TUNEL染色，以及针对凋亡的p-RIP 3的抗体染色）。 坏死性凋亡）。tdTomato的共定位（由组成型活性启动子驱动以标记所有PV神经元 即使它们停止表达PV），将分析PV和细胞死亡标志物以定量PV神经元死亡 vs.没有PV。 具体目标2。表征以下存活的皮质PV神经元的输入和输出突触 噪音创伤PV-Cre-tdTomato和PV-Cre-ChR 2-tdTomato小鼠将经历噪声创伤程序。 10天后，制备急性听觉皮层切片。PV-Cre-PV神经元的膜片钳记录 制作tdTomato切片，并检查PV神经元上的兴奋性和抑制性突触输入。在 此外，将检查到第2/3层锥体神经元上的光遗传学激活的PV神经元输出。的 输入-输出功能、短期可塑性和突触耗尽/恢复动力学将被表征。所有 结果将在噪声创伤和假创伤组之间进行比较。 具体目标3。研究TNFR 1和TNFR 2在噪声诱导的PV神经元损失中的作用， 突触功能障碍我们将敲低PV-Cre-td Tomato的皮质PV神经元中的TNFR 1或TNFR 2， PV-Cre-ChR 2-tdTomato小鼠使用我们定制的病毒载体进行TNFR 1或TNFR 2的Cre依赖性表达。 TNFR2 shRNA。病毒载体表达GFP以允许转染和未转染之间的比较 PV神经元。我们将研究TNFR 1或TNFR 2敲低对噪声诱导的PV神经元损失的影响（如在 目的1）、突触功能障碍（如目的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).