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，但这些递质究竟是如何共同释放的，以及这种共同传递的生理功能尚不清楚。在耳蜗背核（DCN）中，被称为侧翻细胞的甘氨酸/GABA共释放中间神经元的抑制作用严格控制着靶神经元是否对多感觉输入作出反应。因此，缺乏对突触囊泡中抑制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。本提案的目的是确定调节突触前囊泡中甘氨酸和GABA的差异包装的机制，并确定共同释放如何决定突触后抑制的时间动力学。目的1将采用双全细胞记录连接成对的侧翻细胞，以确定膜甘氨酸转运体GlyT2调节突触囊泡中甘氨酸/GABA比例的程度。目的2将通过实验操纵侧翻细胞对突触前甘氨酸/GABA比率，以测试共释放功能是否会增强突触后受体的失活动力学。Aim 3将使用双光子Na+成像来确定GlyT2在同一细胞的多个钮孔中的表达是否在功能上是同质的，或者甘氨酸转运体的亚细胞分布是否存在显著的变异性。这些实验将揭示调控抑制共传递的基本机制，并确定这如何影响DCN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.