GABAergic transmission and chloride equilibrium potential are not modulated by pyruvate in the developing optic tectum of Xenopus laevis tadpoles.
GABAergic transmission and chloride equilibrium potential are not modulated by pyruvate in the developing optic tectum of Xenopus laevis tadpoles.
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DOI：
10.1371/journal.pone.0034446
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Aizenman CD
中科院分区：
文献类型：
--
作者：
Khakhalin AS;Aizenman CD
在发育中的哺乳动物脑中，由于未成熟神经元细胞内高水平的Cl-−，γ-氨基丁酸被认为起到兴奋而不是抑制的作用。然而，这一想法受到了最近的研究的质疑，这些研究表明，基于葡萄糖的人工脑脊液(ACSF)可能不足以用于对未成熟和发育中的大脑进行实验。这些研究表明，未成熟的神经元可能需要替代能源，如乳酸或丙酮酸。缺乏这些其他能源被认为是导致细胞内ClGABA浓度人为升高的原因，从而导致更具去极化的−受体(GABA)逆转电位。由于不同物种之间的葡萄糖代谢差异很大，因此测试这些替代能源对不同实验制剂的影响是很重要的。我们通过记录顶盖神经元对视神经刺激的反应性，并通过测量Granicidin穿孔贴片结构中局部应用GABA引起的电流，来测试丙酮酸是否影响体外发育的野生型非洲爪哇蝌蚪大脑中的GABA能传递。我们发现，与先前报道的结果相反，GABAR介导的电流的逆转潜力在发育阶段45和49之间没有显著变化。丙酮酸部分替代葡萄糖对45和49期顶盖神经元的GABA翻转电位和反应性的影响都很小。完全耗尽ACSF的能量来源不会影响神经反应。我们还报道了GABA逆转电位的强烈空间梯度，靠近外侧和尾部增殖区的未成熟细胞具有更多的正逆转电位。我们的结论是，在本实验制备中，以葡萄糖为基础的ACSF是一种适合于体外实验的细胞外介质。
In the developing mammalian brain, gamma-aminobutyric acid (GABA) is thought to play an excitatory rather than an inhibitory role due to high levels of intracellular Cl− in immature neurons. This idea, however, has been questioned by recent studies which suggest that glucose-based artificial cerebrospinal fluid (ACSF) may be inadequate for experiments on immature and developing brains. These studies suggest that immature neurons may require alternative energy sources, such as lactate or pyruvate. Lack of these other energy sources is thought to result in artificially high intracellular Cl− concentrations, and therefore a more depolarized GABA receptor (GABAR) reversal potential. Since glucose metabolism can vary widely among different species, it is important to test the effects of these alternative energy sources on different experimental preparations. We tested whether pyruvate affects GABAergic transmission in isolated brains of developing wild type Xenopus tadpoles in vitro by recording the responsiveness of tectal neurons to optic nerve stimulation, and by measuring currents evoked by local GABA application in a gramicidin perforated patch configuration. We found that, in contrast with previously reported results, the reversal potential for GABAR-mediated currents does not change significantly between developmental stages 45 and 49. Partial substitution of glucose by pyruvate had only minor effects on both the GABA reversal potential, and the responsiveness of tectal neurons at stages 45 and 49. Total depletion of energy sources from the ACSF did not affect neural responsiveness. We also report a strong spatial gradient in GABA reversal potential, with immature cells adjacent to the lateral and caudal proliferative zones having more positive reversal potentials. We conclude that in this experimental preparation standard glucose-based ACSF is an appropriate extracellular media for in vitro experiments.
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