Effects of nerve demyelination on auditory synaptic functions

描述(申请人提供)：哺乳动物中枢神经系统中的有髓轴突是独特的设计，以支持快速和有效的跳跃脉冲传播。由于基因突变或自身免疫性疾病造成的髓鞘丢失，如多发性硬化症(MS)，可能会导致运动神经元疲劳和神经冲动传导障碍的增加。这种疾病最终会导致轴突变性，并对运动和感觉功能造成不可逆转的损害。有趣的是，一些MS患者表现出听力损失，而耳蜗功能正常，称为听神经病。与脱髓鞘相关的研究大多集中在三叉神经节和脊髓轴突，而对于髓鞘缺失对哺乳动物中枢神经系统(CNS)单个突触水平的突触传递的影响却知之甚少。本研究的目的是通过研究中枢神经系统听性脑干中单个突触的电生理特性，来研究脱髓鞘后单个听神经轴突和神经末梢的特性。缺乏紧密中枢髓鞘形成的突变大鼠(Long-Evans Shaker；LES)模拟多发性硬化症的一些标志性特征，它们的症状通常与多发性硬化症患者的症状相似。我的主要假设是，失去髓鞘的轴突具有离子通道的混乱分布、动作电位的时序以及对钙离子和钠离子内流的处理方式的改变。由于轴突钙、钠离子浓度的异常，与轴突相关的神经末梢具有异常的突触性质。为了验证这一假设，我将对听觉脑干的突触进行电生理记录和荧光染料成像研究。这种神经末梢参与了一种特殊的神经元回路，该回路计算听觉系统的声源位置。因此，花萼突触为我们提供了一个更容易获得的模型来直接研究脱髓鞘对中枢神经系统神经末梢内稳态机制和突触传递的影响。具体目的：我计划首先进行电生理和成像实验，以研究LES大鼠的特性。我将比较从表现出严重行为表型(纯合子)或正常(杂合子)的LES幼崽获得的神经组织的轴突和突触属性。我将研究在几种不同频率的强直刺激下动作电位(AP)的时序和放电概率，以及正常和突变幼鼠的兴奋性突触后电流(EPSC)。此外，我还将研究脱髓鞘轴突和神经末梢的离子通道和转运体的重新分布。接下来，我将使用对钙离子和钠离子敏感的荧光染料直接研究轴突和神经末梢中钙离子和钠离子浓度的变化。 公共卫生相关性：拟议的研究将使我们深入了解听觉神经纤维脱髓鞘引发的基本生物学过程，以及这一提议对整个神经退行性疾病领域具有广泛的相关性。这项拟议的研究将为开发新的治疗策略奠定基础，以防止由于多发性硬化症或听神经病等脱髓鞘疾病的后果而导致的永久性儿童听力损失和成人听力障碍。

DESCRIPTION (provided by applicant): Myelinated axons in the mammalian CNS are uniquely designed to support rapid and efficient saltatory impulse propagation. Myelin loss due to genetic mutations or autoimmune disease, as in the case of multiple sclerosis (MS) can result in increased motor neurons fatigue and nerve impulse conduction failure. This disorder can eventually lead to axonal degeneration and irreversible damage to motor and sensory function. Interestingly, some of MS patients show hearing acuity loss with normal cochlear function, referred as auditory neuropathy. Most studies related to demyelination have focused on the PNS and spinal cord axons, however, little is known about how loss of myelin sheaths affects the synaptic transmission at the single synapse level in mammalian central nervous system (CNS). The object of this proposal is to investigate the properties of single auditory axons and the nerve terminals after demyelination by studying the electrophysiological properties of a single synapse in the auditory brainstem in the CNS. Mutant rats (the Long-Evans Shaker; LES) that lack compact CNS myelination, simulate some of the hallmark properties of MS and they have symptoms that often mimic those of MS patients. My main hypothesis is that axons that lose myelin have a disrupted distribution of ion channels, action potentials timing, and an altered handling of Ca2+ and Na+ ion influx. Because of abnormal axonal Ca2+ and Na+ ion concentrations, the nerve terminal associated with the axon has abnormal synaptic properties. To test this hypothesis I will perform electrophysiological recordings and fluorescent dye imaging studies in the calyx of Held synapse of the auditory brainstem. This nerve terminal is involved in a special neuronal circuit that computes the location of sound sources for the auditory system. The calyx synapse therefore provides us with a more accessible model to directly examine the effects of demyelination on homeostatic mechanisms and synaptic transmission in CNS nerve terminals. Specific Aims: I plan to first conduct electrophysiological and imaging experiments to study the properties of the LES rats. I will compare the axonal and synaptic properties of neural tissue obtained from the LES pups that show a severe behavior phenotype (homozygote) or are normal (heterozygote). I will study the timing and firing probability of the action potential (AP) during tetanic stimulation at several different frequencies, and the excitatory postsynaptic currents (EPSC) of the normal and mutant pups. And, I will examine the re-distribution of ion channels and transporters of demyelinated axons and nerve terminals. Next, I will directly study Ca2+ and Na+ ion concentration changes in the axon and nerve terminal using Ca2+ and Na+ ion sensitive fluorescent dyes. PUBLIC HEALTH RELEVANCE: The proposed research will give us insights into the basic biological processes triggered by demyelination in auditory nerve fibers, as well as this proposal has a broad relevance for the entire field of neurodegenerative diseases. The proposed study will lay the foundations for the development of novel therapeutic strategies for preventing a permanent childhood hearing loss and an adult hearing impairment due to the consequences of demyelinating diseases like MS or auditory neuropathy.