Mechanisms and function of neurotransmitter corelease at an auditory synapse

虽然抑制性神经递质甘氨酸和GABA在许多听性脑干突触中共同释放，但这些递质到底是如何共同释放的，这种共同传递的生理功能仍然不清楚。在耳蜗背核(DCN)，甘氨酸/GABA共同释放的中间神经元(称为手轮细胞)的抑制严格控制靶神经元是否对多感觉输入做出反应。因此，缺乏对控制突触小泡中抑制性递质数量的细胞机制的了解严重限制了我们对DCN电路功能的理解。这项建议的目的是确定调节突触前囊泡中甘氨酸和GABA差异包装的机制，并确定共释放如何决定突触后抑制的时间动力学。目的1将在连接的两对车轮细胞中使用双全细胞记录来确定膜甘氨酸转运体GlyT2在多大程度上调节突触小泡中甘氨酸/GABA的比例。目的2将在实验中操纵侧翻细胞对突触前甘氨酸/GABA的比例，以测试共释放是否具有加剧突触后受体失活的作用。AIM 3将使用双光子Na+成像来确定GlyT2在同一细胞的多个启动子中的表达是否在功能上是均一的，或者甘氨酸转运体的亚细胞分布是否存在显著的变异性。这些实验将阐明调节抑制共传递的基本机制，并确定这如何塑造DCN中局部抑制的时间特性。

While the inhibitory neurotransmitters glycine and GABA are co-released at many auditory brainstem synapses, exactly how these transmitters are co-released, and the physiological function of this co-transmission remain ambiguous. In the dorsal cochlear nucleus (DCN), inhibition from glycine/GABA co-releasing interneurons termed cartwheel cells tightly controls whether target neurons fire in response to multi-sensory inputs. Thus, a lack of knowledge as to the cellular mechanisms governing the amount of inhibitory transmitters in synaptic vesicles severely limits our understanding of DCN circuit function. The objective of this proposal is to determine the mechanisms that regulate the differential packaging of glycine and GABA in pre-synaptic vesicles, and establish how co-release dictates the temporal kinetics of post-synaptic inhibition. Aim 1 will employ dual whole-cell recordings in connected pairs of cartwheel cells to determine the extent to which the membrane glycine transporter GlyT2 regulates the ratio of glycine/GABA in synaptic vesicles. Aim 2 will experimentally manipulate pre-synaptic glycine/GABA ratios in cartwheel cell pairs to test whether co-release functions to sharpen the deactivation kinetics of post-synaptic receptors. Aim 3 will use 2-photon Na+ imaging to determine whether GlyT2 expression is functionally homogenous across multiple boutons from the same cell, or if there exists significant variability in the sub-cellular distribution of glycine transporters. These experiments will shed light on the fundamental mechanisms that regulate inhibitory co-transmission, and establish how this shapes the temporal properties of local inhibition in the DCN.