SSA: Presynaptic control of subunit composition of NMDA receptors mediating synaptic plasticity in the dorsal cochlear nucleus

突触前受体非常适合影响神经递质的释放和影响突触传递的效果。突触前受体影响神经递质释放的细胞机制在很大程度上是未知的。NMDAR是一种谷氨酸门控离子通道，具有独特的生物物理、药理和信号转导特性。该项目旨在研究突触前谷氨酸能NMDAR在调节耳蜗背核(DCN)突触前释放概率和短期可塑性方面的新功能。DCN是一种参与耳鸣发生的听觉脑干结构。这项研究将研究它们的解剖分布、亚单位组成、激活机制以及它们对小鼠DCN突触功能的下游影响。解剖学证据将来自免疫电子显微镜实验，这将允许在DCN的突触前元件中识别NMDAR。利用表达突触前荧光标记的转基因小鼠的钙成像和DCN主细胞的全细胞记录直接监测突触前功能，将证明功能性突触前NMDAR的存在。亚基组成的作用将使用药理学和遗传工具进行研究。特别是，单细胞逆转录聚合酶链式反应将被用来分析突触前细胞电生理特征之后NMDAR亚单位2A、2B和2C(NR2A-C)的表达模式。计算模型将决定DCN网络的生理行为。听觉脑干反应记录将允许评估由声过度暴露引起的听力阈值的变化。耳蜗内注射NMDAR拮抗剂将允许建立突触前NMDAR的生理意义。

Presynaptic receptors are ideally suited to affect neurotransmitter release and influence the efficacy of synaptic transmission. Cellular mechanisms by which presynaptic receptors can affect neurotransmitter release are largely unknown. NMDA receptors (NMDARs) are glutamate-gated ion channels with unique biophysical, pharmacological and signalling properties. The project aims at studying the novel function of presynaptic glutamatergic NMDARs at regulating presynaptic release probability and short-term plasticity in the dorsal cochlear nucleus (DCN), an auditory brainstem structure involved in the generation of tinnitus. The study will be investigating their anatomical distribution, subunit composition, activation mechanisms as well as their downstream effects on synapse function in the mouse DCN. Anatomical evidence will come from immuno-electron microscopy experiments that will allow identifying NMDARs in presynaptic elements in the DCN. Direct monitoring of presynaptic function using calcium imaging from transgenic mice expressing fluorescent presynaptic markers and whole cell recordings of DCN principal cells will demonstrate the existence of functional presynaptic NMDARs. The role of subunit composition will be investigated using pharmacological and genetic tools. In particular, single-cell reverse transcriptase-polymerase chain reaction will be used to analyse the expression pattern of NMDAR subunits 2A, 2B, and 2C (NR2A-C) subsequent to electrophysiological characterization in presynaptic cells. Computational modelling will determine the physiological behaviour of the DCN network. Auditory brainstem response recordings will allow assessing shifts of hearing thresholds induced by acoustic over-exposure. Cochlear injections of NMDAR antagonists will allow establishing the physiological significance of presynaptic NMDARs.