SNARE蛋白对谷氨酸的调节及其在耳鸣大鼠听皮层中的作用

Sodium salicylate (aspirin) , as a common drug that is frequently used for long term treatment in a clinical setting, has the potential to cause reversible tinnitus. Salicylate-induced tinnitus in rats has served as a popular animal model for the study of tinnitus, however, the precise mechanism still remains unclear..Some studies suggest that tinnitus arises from an increase in excitatory neurotransmission, and is associated with N-methyl D-aspartate receptor (NMDAR) activity. Glutamate, as an important excitatory neurotransmitter,plays a central role in the central nervous system. Regulated membrane fusion of synaptic vesicles and subsequent glutamate release involve the assembly of ternary complex from soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Our recent studies showed the changes markedly, but reversibly, in the expression of the excitatory glutamate receptor subtype NR2B mRNA and protein levels, and also some changes in synaptic ultrastructure of neurons in auditory cortex. These data suggested that there are some plastic changes in auditory cortex of tinnitus animal. All of those provide evidence that the neural changes associated with tinnitus maybe become progressively “centralized” over time. However, whether the enhanced electrophysiological activity leads to enhancement of auditory central activity and / or induces to auditory central plasticity, still remain largely unclear. .In this project, we establish the animal model of tinnitus by long-term salicylate administration, moreover, using gap prepulse inhibition of acoustic startle (GPIAS) paradigm and electrophysiological test methods to verify the presence of tinnitus in the animal. To assess the auditory cortex activities and NMDA receptor activities of synapses by microPET (Positron emission tomography)molecular imaging technology, to find out the corresponding relationship between alterations of the auditory cortex activity and animal behavior( presence of tinnitus in the animal ) during the long-term salicylate administration process and after salicylate treatment ceased. To reveal the role of auditory center activity on mechanisms of tinnitus generation. Using transmission electron microscopy (TEM) and molecular biology technique to understand corresponding the ultrastructure of auditory cortex, and the expression of mRNA & protein of NMDA receptor, synaptobrevin，SNAP-25, syntaxin 1A and EAAC1et al. To elucidate mechanism of tinnitus on auditory central plasticity (Tinnitus centralization), which will provide theoretical basis for pathophysiological mechanisms and clinical interventions of tinnitus.

水杨酸盐致耳鸣确切机制尚不清楚，前期研究观察到听皮层兴奋性升高和突触超微结构发生改变。谷氨酸是中枢系统重要的兴奋性神经递质，而谷氨酸的释放与重摄取可能都与SNARE蛋白复合体相关。项目建立长期注射水杨酸盐诱导耳鸣动物模型，用听觉惊跳反射间隔前刺激抑制（GPIAS）和电生理学方法筛选耳鸣动物；采用microPET技术评估听皮层功能活动及其突触递质受体活性；通过免疫和透射电镜及分子生物学等方法，了解听皮层突触超微结构及检测SNARE蛋白复合体和谷氨酸受体mRNA及蛋白表达量。动态同步观察耳鸣建模过程中听皮层功能活动、谷氨酸转运功能和递质受体活性改变与动物行为学（出现耳鸣）在时间相位上的对应关系，通过生理、病理模型来验证SNARE蛋白是否调节谷氨酸水平从而参与了耳鸣，明确听皮层突触功能、结构和递质受体等改变的内在联系。本研究拟进一步完善耳鸣听皮层可塑性的生理机制。

耳鸣在临床上发病率高，治疗较为棘手，且其发生机制尚未明确。我们应用长期注射水杨酸盐的方法成功建立了耳鸣的动物模型，采用听觉惊跳反射间隔前刺激抑制实验（GPIAS）和动物行为学相结合的方法来筛选和验证动物耳鸣相关行为；利用microPET技术发现耳鸣大鼠听觉皮层功能活动升高，又在透射电镜下发现突触超微结构发生变化；应用高效液相色谱检测了多个时间点血清、脑脊液中水杨酸盐的浓度，以及听觉通路多个核团谷氨酸及γ氨基丁酸的水平；应用多种分子生物学方法检测到突触囊泡SNARE蛋白复合体（Syntaxin 1A、SNAP-25 和Synaptobrevin）在耳鸣大鼠不同时相的表达变化，以及谷氨酸受体及谷氨酸转运蛋白的表达。本项目明确了听皮层突触功能、结构和递质受体等改变的内在联系，验证了SNARE蛋白可以直接作用于谷氨酸转运蛋白（EAAC1），调节谷氨酸的释放和重摄取从而参与耳鸣，为耳鸣的预防及治疗开辟了新思路。