A quantitative assessment of glutamate uptake into hippocampal synaptic terminals and astrocytes: new insights into a neuronal role for excitatory amino acid transporter 2 (EAAT2).
A quantitative assessment of glutamate uptake into hippocampal synaptic terminals and astrocytes: new insights into a neuronal role for excitatory amino acid transporter 2 (EAAT2).
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DOI：
10.1016/j.neuroscience.2008.08.043
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发表时间：
2008-11-11
期刊：
影响因子：
3.3
通讯作者：
Danbolt NC
中科院分区：
文献类型：
--
作者：
Furness DN;Dehnes Y;Akhtar AQ;Rossi DJ;Hamann M;Grutle NJ;Gundersen V;Holmseth S;Lehre KP;Ullensvang K;Wojewodzic M;Zhou Y;Attwell D;Danbolt NC
兴奋性氨基酸转运体2(EAAT2)在突触终末和星形胶质细胞之间的相对分布，以及EAAT2在终末摄取中的重要性仍未得到解决。在这里，我们使用了戊二醛固定的D-天冬氨酸的抗体来在电子显微镜下识别D-天冬氨酸在海马片中积累的位置。辐射层CA1中约3/4的终末通过一种主动的二氢海氨酸敏感机制积累D-天冬氨酸免疫反应，而EAAT2谷氨酸转运体基因敲除小鼠则不存在。这些终末负责脑片中所有D-天冬氨酸摄取的一半以上。这是意想不到的，因为在完整的脑组织中观察到的EAAT2免疫反应主要与星形胶质细胞有关。然而，当检查细胞外间隙大于灌流固定组织的突触体和切片制备时，证实大多数EAAT2位于星形胶质细胞(约80%)。树突棘中未检测到D-天冬氨酸摄取和EAAT2蛋白的表达。约6%的EAAT2免疫反应阳性物质位于突触终末的质膜上(突触间隙内外)。大部分残留免疫反应(8%)分布在轴突内，分布在比星形胶质细胞大几倍的质膜表面积内。这解释了为什么尽管CA3锥体细胞中有高水平的mRNA，但神经元EAAT2的密度很低，但不能解释为什么海马片制备的EAAT2摄取外源性底物的一半以上。这一点以及完整大脑中终末摄取与神经胶质摄取的相对量仍有待发现。
The relative distribution of the excitatory amino acid transporter 2 (EAAT2) between synaptic terminals and astroglia, and the importance of EAAT2 for the uptake into terminals is still unresolved. Here we have used antibodies to glutaraldehyde-fixed D-aspartate to identify electron microscopically the sites of D-aspartate accumulation in hippocampal slices. About 3/4 of all terminals in the stratum radiatum CA1 accumulated D-aspartate-immunoreactivity by an active dihydrokainate-sensitive mechanism which was absent in EAAT2 glutamate transporter knockout mice. These terminals were responsible for more than half of all D-aspartate uptake of external substrate in the slices. This is unexpected as EAAT2-immunoreactivity observed in intact brain tissue is mainly associated with astroglia. However, when examining synaptosomes and slice preparations where the extracellular space is larger than in perfusion fixed tissue, it was confirmed that most EAAT2 is in astroglia (about 80%). Neither D-aspartate uptake nor EAAT2 protein was detected in dendritic spines. About 6% of the EAAT2-immunoreactivity was detected in the plasma membrane of synaptic terminals (both within and outside of the synaptic cleft). Most of the remaining immunoreactivity (8%) was found in axons where it was distributed in a plasma membrane surface area several times larger than that of astroglia. This explains why the densities of neuronal EAAT2 are low despite high levels of mRNA in CA3 pyramidal cell bodies, but not why EAAT2 in terminals account for more than half of the uptake of exogenous substrate by hippocampal slice preparations. This and the relative amount of terminal versus glial uptake in the intact brain remain to be discovered.
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