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首页> 外文期刊>The European Journal of Neuroscience >Glutamate-induced elevations in intracellular chloride concentration in hippocampal cell cultures derived from EYFP-expressing mice.
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Glutamate-induced elevations in intracellular chloride concentration in hippocampal cell cultures derived from EYFP-expressing mice.

机译：谷氨酸诱导的表达EYFP的海马细胞培养物中细胞内氯浓度的升高。

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Abstract The homeostasis of intracellular Cl(-) concentration ([Cl(-)](i)) is critical for neuronal function, including gamma-aminobutyric acid (GABA)ergic synaptic transmission. Here, we investigated activity-dependent changes in [Cl(-)](i) using a transgenetically expressed Cl(-)-sensitive enhanced yellow-fluorescent protein (EYFP) in cultures of mouse hippocampal neurons. Application of glutamate (100 micro m for 3 min) in a bath perfusion to cell cultures of various days in vitro (DIV) revealed a decrease in EYFP fluorescence. The EYFP signal increased in amplitude with increasing DIV, reaching a maximal response after 7 DIV. Glutamate application resulted in a slight neuronal acidification. Although EYFP fluorescence is sensitive to pH, EYFP signals were virtually abolished in Cl(-)-free solution, demonstrating that the EYFP signal represented an increase in [Cl(-)](i). Similar to glutamate, a rise in [Cl(-)](i) was also induced by specific ionotropic glutamate receptor agonists and by increasing extracellular [K(+)], indicating that an increase in driving force for Cl(-) suffices to increase [Cl(-)](i). To elucidate the membrane mechanisms mediating the Cl(-) influx, a series of blockers of ion channels and transporters were tested. The glutamate-induced increase in [Cl(-)](i) was resistant to furosemide, bumetanide and 4,4'-diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS), was reduced by bicuculline to about 80% of control responses, and was antagonized by niflumic acid (NFA) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). We conclude that membrane depolarization increases [Cl(-)](i) via several pathways involving NFA- and NPPB-sensitive anion channels and GABA(A) receptors, but not through furosemide-, bumetanide- or DIDS-sensitive Cl(-) transporters. The present study highlights the vulnerability of [Cl(-)](i) homeostasis after membrane depolarization in neurons.
机译：摘要细胞内Cl（-）浓度（[Cl（-）]（i）的稳态对神经元功能至关重要，包括γ-氨基丁酸（GABA）能量突触传递。在这里，我们调查了在小鼠海马神经元的文化中使用转基因表达的Cl（-）敏感的增强型黄色荧光蛋白（EYFP）在[Cl（-）]（i）中的活动依赖性变化。浴灌注中谷氨酸（100微米，3分钟）在体外不同天数的细胞培养中的应用（DIV）表明EYFP荧光减少。 EYFP信号的幅度随DIV的增加而增加，在7 DIV之后达到最大响应。谷氨酸的应用导致轻微的神经元酸化。尽管EYFP荧光对pH敏感，但是在不含Cl（-）的溶液中EYFP信号实际上被消除了，这表明EYFP信号代表[Cl（-）]（i）的增加。与谷氨酸相似，[Cl（-）]（i）的升高也由特定的离子型谷氨酸受体激动剂和细胞外[K（+）]诱导，表明Cl（-）驱动力的增加足以满足增加[Cl（-）]（i）。为了阐明介导Cl（-）流入的膜机制，测试了一系列离子通道和转运蛋白的阻滞剂。谷氨酸诱导的[Cl（-）]（i）的增加对速尿，布美他尼和4,4'-二异硫氰基-二苯乙烯-2,2'-二磺酸（DIDS）有抵抗力，双小分子降低至约80％的控制反应，并被烟酰胺酸（NFA）和5-硝基-2-（3-苯基丙基氨基）苯甲酸（NPPB）拮抗。我们得出结论，膜去极化通过涉及NFA-和NPPB敏感阴离子通道和GABA（A）受体的几种途径增加[Cl（-）]（i），但不通过呋塞米，布美他尼或DIDS敏感Cl（-）增加运输者。本研究突出了神经元的膜去极化后[Cl（-）]（i）稳态的脆弱性。

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