Synaptic physiology of unipolar brush cells in the dorsal cochlear nucleus

描述(申请人提供)：耳蜗背核(DCN)是哺乳动物脑干中处理声音、整合多感官和听神经输入的初始结构之一。听觉输入的多感觉调制是DCN的一个基本特征，可能是耳鸣的关键因素。耳鸣是一种常见的听力障碍疾病，其特征是在没有外部听觉刺激的情况下感知声音。虽然DCN中听觉输入的多感觉调制对DCN功能很重要，但DCN回路中多感觉纤维向其主要靶点的突触传递的突触和细胞机制尚不清楚。DCN的多感觉输入是由苔藓纤维(MF)传递的，其主要突触靶标是颗粒细胞(GRC)。然而，GRCs的一大子集通过称为单极刷状细胞(UBC)的兴奋性中间神经元接受输入。UBC有一个短的树突，以刷状结构终止，并与单个突触前终末交错，形成异常大的兴奋性突触接触。先前的一个模型表明，谷氨酸在不规则突触间隙中的长期捕获是UBC特有的慢衰突触后电流(EPSC)的基础。本研究的目的是研究UBC是否在时间上放大了从MF到GRC的输入以进一步整合听觉输入，如果是这样的话，是什么突触专门化促进了这种放大。我们认为，MF终末的多囊释放有助于延长裂隙中的谷氨酸，并允许UBC向GRCs提供信号的时间放大。目的1通过双光子显微镜和膜片钳技术对连接的UBC-GRC对的记录，首次采用成像和电生理相结合的方法，在GFP标记的UBC小鼠中，直接检测UBC对GRCs的生理突触后反应。目标2将确定MF-UBC突触的胞吐方式。初步数据显示，在持续突触抑制的情况下，在MF刺激下，UBC中诱发的EPSCs的幅度变化很小，因此表明释放MF-UBC突触的可能性很高。这些实验将通过膜片钳记录来研究这些突触的释放模式，并结合多概率波动分析来分析低亲和力竞争性拮抗剂对突触后受体的非平衡抑制。通过这些方法，可以计算出量子大小、独立释放点的数量和每个点的释放概率。通过研究通过向DCN传递多感觉输入的主要结构的突触传递，以及UBC对GRC活动的影响，本研究将为了解DCN对多感觉输入的早期处理如何影响主输出细胞与听觉输入的整合提供基础。这将表征第一个与DCN的体细胞耳鸣的调制和触发有关的细胞贡献者。

DESCRIPTION (provided by applicant): The dorsal cochlear nucleus (DCN) is one of the initial structures in the mammalian brainstem that processes sound, integrating multisensory and auditory nerve input. Multisensory modulation of auditory input is a basic feature of the DCN and may be a critical contributor to tinnitus, a common hearing impairment disorders characterized by the perception of sound without exterior auditory stimulus. Although multisensory modulation of auditory input in the DCN has been shown to be important for DCN function, the synaptic and cellular mechanisms underlying synaptic transmission from multisensory fibers to its primary targets within the DCN circuitry are not well known. Multisensory input to the DCN is relayed by mossy fibers (MF), whose primary synaptic targets are granule cells (GrCs). However, a large subset of GrCs receives input through an excitatory interneuron called unipolar brush cells (UBCs). UBCs have a single short dendrite that terminates in a brush-like structure and interdigitates with a single pre-synaptic terminal, formin an unusually large excitatory synaptic contact. A previous model suggests that prolonged entrapment of glutamate in the irregular synaptic cleft underlies the characteristic slow-decaying post-synaptic current (EPSC) of UBCs. The goal of this proposal is to investigate whether UBCs temporally amplify input from MF to GrCs for further integration with auditory input, and if so, what synaptic specializations promote such amplification. We propose that multivesicular release from MF terminals contributes to prolonged glutamate in the cleft and allows UBCs to provide temporal amplification of signals to GrCs. Aim 1 will determine the physiological postsynaptic response of GrCs to UBC mediated input These experiments will directly test for the first time the effect of UBCs input to GrCs using combined imaging and electrophysiological approaches in a GFP-tagged UBC mouse through 2- photon microscopy and patch-clamp recordings from connected UBC-GrC pairs. Aim 2 will determine the mode of exocytosis at the MF-UBC synapse. Preliminary data shows low variance in amplitude of evoked EPSCs in UBC upon MF stimulation with consistent synaptic depression, therefore suggesting a high probability of release the MF-UBC synapse. The proposed experiments will investigate the mode of release at these synapses with patch-clamp recordings and the analysis of the non-equilibrium inhibition of the post- synaptic receptors by low-affinity competitive antagonists coupled to multiple-probability fluctuation analysis. Through these methods the quantal size, the number of independent release sites and the probability of release at each site can be calculated. By investigating synaptic transmission through the main structure relaying multisensory input to the DCN, and the impact of UBCs to GrC activity, this proposal will provide the foundation for understanding how the DCN early processing of multisensory input affects integration with auditory input at the principal output cells. This will characterize the first cellular contributor pertaining to the modulation and triggers of somatic tinnitus at the DCN.