Effects of nerve demyelination on auditory synaptic functions

描述（由申请人提供）：哺乳动物中枢神经系统中的髓鞘轴突具有独特的设计，可支持快速有效的跃式脉冲传播。由于基因突变或自身免疫性疾病导致的髓磷脂损失，如多发性硬化症（MS），可导致运动神经元疲劳增加和神经冲动传导失败。这种疾病最终可导致轴突变性和对运动和感觉功能的不可逆损害。有趣的是，一些多发性硬化症患者表现为耳蜗功能正常的听力敏锐度下降，称为听神经病变。大多数与脱髓鞘相关的研究都集中在PNS和脊髓轴突上，然而，关于髓鞘的丢失如何影响哺乳动物中枢神经系统（CNS）单突触水平的突触传递，我们知之甚少。本研究的目的是通过研究中枢神经系统听觉脑干单个突触的电生理特性，探讨脱髓鞘后单个听觉轴突和神经末梢的特性。突变大鼠（Long-Evans Shaker； LES）缺乏紧密的中枢神经系统髓鞘形成，模拟MS的一些标志性特征，并且它们的症状通常与MS患者相似。我的主要假设是，失去髓磷脂的轴突具有离子通道分布、动作电位时间和Ca2+和Na+离子内流处理的改变。由于轴突Ca2+和Na+离子浓度异常，与轴突相关的神经末梢具有异常的突触特性。为了验证这一假设，我将对听觉脑干的Held突触的花萼进行电生理记录和荧光染料成像研究。这个神经末梢与一个特殊的神经元回路有关，该回路为听觉系统计算声源的位置。因此，花萼突触为我们提供了一个更容易接近的模型来直接研究脱髓鞘对中枢神经系统神经末梢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影像学实验，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幼崽中获得的神经组织的轴突和突触特性。本研究将研究不同频率强直刺激下动作电位（AP）的时间和放电概率，以及正常和突变幼鼠的兴奋性突触后电流（EPSC）。并且，我将检查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Ca2+和Na+离子敏感荧光染料直接研究轴突和神经末梢Ca2+和Na+离子浓度的变化。

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.