Vesicular glutamate transporter 3 is required for synaptic transmission in zebrafish hair cells
Vesicular glutamate transporter 3 is required for synaptic transmission in zebrafish hair cells
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DOI：
10.1523/jneurosci.5230-07.2008
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发表时间：
2008-02-27
影响因子：
5.3
通讯作者：
Nicolson, Teresa
中科院分区：
文献类型：
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作者：
Obholzer, Nikolaus;Wolfson, Sean;Nicolson, Teresa
毛细胞探测声音和运动，并通过特殊的带状突触传递这些信息。在此，我们报道了在乙基亚硝胺诱变斑马鱼幼体中发现的编码囊泡谷氨酸转运蛋白3(Vlut3)的基因。VGlu3外显子2的剪接位点突变导致预测的蛋白产物严重截断，针对vlug 3 ATG起始点或受影响的剪接连接的吗啉复制小行星的表型。原位杂交结果表明，vlut3仅在耳部和侧线器官的毛细胞中表达。在斑马鱼毛细胞中也表达了第二个转运蛋白基因vher1，但在没有vlut3的情况下，vlu1的mRNA水平不会增加。抗Vher3的抗体标记毛细胞的基底端，而在小行星/vlut3突变体中不存在标记。根据毛细胞中Vher3的定位，我们推测在小行星/vlut3突变体中缺乏前庭-眼睛和听觉惊厥反射是由于毛细胞中突触传递的缺陷。支持这一观点的是，突触后听觉外侧神经细胞的动作电流在小行星/vlut3突变体中不存在。在超微结构水平上，突变的小行星/vlut3毛细胞显示与突触相关的突触小泡数量减少，这表明Vlut3在突触小泡的生物发生和/或与突触小体的拴系中发挥作用。突变体中缺乏突触后动作电流表明，剩余的毛细胞突触小泡含有不足以在一级神经元中产生动作电位的谷氨酸水平。
Hair cells detect sound and movement and transmit this information via specialized ribbon synapses. Here we report that asteroid, a gene identified in an ethylnitrosourea mutagenesis screen of zebrafish larvae for auditory/vestibular mutants, encodes vesicular glutamate transporter 3 ( Vglut3). A splice site mutation in exon 2 of vglut3 results in a severe truncation of the predicted protein product and morpholinos directed against the vglut3 ATG start site or the affected splice junction replicate the asteroid phenotype. In situ hybridization shows that vglut3 is exclusively expressed in hair cells of the ear and lateral line organ. A second transporter gene, vglut1, is also expressed in zebrafish hair cells, but the level of vglut1 mRNA is not increased in the absence of Vglut3. Antibodies against Vglut3 label the basal end of hair cells and labeling is not present in asteroid/vglut3 mutants. Based on the localization of Vglut3 in hair cells, we suspected that the lack of vestibulo-ocular and acoustic startle reflexes in asteroid/vglut3 mutants was attributable to a defect in synaptic transmission in hair cells. In support of this notion, action currents in postsynaptic acousticolateralis neurons are absent in asteroid/vglut3 mutants. At the ultrastructural level, mutant asteroid/vglut3 hair cells show a decrease in the number of ribbon-associated synaptic vesicles, indicating a role for Vglut3 in synaptic vesicle biogenesis and/or tethering to the ribbon body. Lack of postsynaptic action currents in the mutants suggests that the remaining hair-cell synaptic vesicles contain insufficient levels of glutamate for generation of action potentials in first-order neurons.