Mechanisms and function of neurotransmitter corelease at an auditory synapse

虽然抑制性神经递质甘氨酸和GABA在许多听觉脑干突触中共同释放，但这些递质是如何共同释放的，以及这种共同传递的生理功能仍然不明确。在耳蜗背核（DCN），抑制甘氨酸/GABA共释放中间神经元称为车轮细胞严格控制是否响应于多感觉输入的目标神经元火灾。因此，缺乏知识的突触囊泡中的抑制性递质的量的细胞机制，严重限制了我们对DCN电路功能的理解。这个建议的目的是确定的机制，调节差异包装的甘氨酸和GABA在突触前囊泡，并建立如何共同释放支配的时间动力学突触后抑制。目的1将采用双全细胞记录在连接对车轮细胞，以确定在何种程度上膜甘氨酸转运蛋白GlyT 2调节甘氨酸/GABA的比例在突触囊泡。目的2将实验操纵突触前甘氨酸/GABA的比率在车轮细胞对测试是否共同释放功能，以提高失活动力学的突触后受体。目标3将使用双光子Na+成像来确定GlyT 2表达在来自同一细胞的多个终扣之间是否在功能上是同质的，或者甘氨酸转运蛋白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性共传递的基本机制，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.