Intracellular dialysis disrupts Zn2+ dynamics and enables selective detection of Zn2+ influx in brain slice preparations.
Intracellular dialysis disrupts Zn2+ dynamics and enables selective detection of Zn2+ influx in brain slice preparations.
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DOI：
10.1111/jnc.12246
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发表时间：
2013-06
影响因子：
4.7
通讯作者：
Shuttleworth CW
中科院分区：
文献类型：
--
作者：
Aiba I;West AK;Sheline CT;Shuttleworth CW
我们研究了细胞内透析对神经元细胞内 Zn2+ 积累荧光检测的影响。通过标准全细胞钳对两种透析条件（标准；20 分钟，简短；2 分钟）进行比较，结果显示细胞内 Zn2+ 缓冲液对细胞内透析非常脆弱。因此，低浓度的吡啶硫酮锌在标准透析的神经元中产生强烈的反应，但在短期透析的神经元中信号较小。与短暂透析的神经元反应相比，标准透析减少了氧化敏感 Zn2+ 库的释放。通过在透析溶液中加入重组金属硫蛋白-3，可以部分逆转透析效果。这些发现表明，可以利用广泛的透析来选择性检测跨膜 Zn2+ 流入。然后使用不同的透析条件来探测对突触刺激的反应。在标准透析条件下，突触刺激在野生型 (WT) 制剂中产生显着的 FluoZin-3 信号，但在缺乏囊泡 Zn2+ (ZnT3-KO) 的制剂中几乎不产生反应。相反，在短暂的透析条件下，WT 和 ZnT3-KO 制剂中的细胞内 Zn2+ 瞬态非常相似。这表明细胞内释放和跨膜通量都有助于突触刺激后细胞内 Zn2+ 的积累。这些结果证明了细胞内透析的显着混淆和潜在用途来研究细胞内 Zn2+ 积累机制。
We examined the impact of intracellular dialysis on fluorescence detection of neuronal intracellular Zn2+ accumulation. Comparison between two dialysis conditions (standard; 20minutes, brief; 2minutes) by standard whole-cell clamp revealed a high vulnerability of intracellular Zn2+ buffers to intracellular dialysis. Thus low concentrations of zinc-pyrithione generated robust responses in neurons with standard dialysis, but signals were smaller in neurons with short dialysis. Release from oxidation-sensitive Zn2+ pools were reduced by standard dialysis, when compared with responses in neurons with brief dialysis. The dialysis effects were partly reversed by inclusion of recombinant metallothionein-3 in the dialysis solution. These findings suggested that extensive dialysis could be exploited for selective detection of transmembrane Zn2+ influx. Different dialysis conditions were then used to probe responses to synaptic stimulation. Under standard dialysis conditions, synaptic stimuli generated significant FluoZin-3 signals in wild-type (WT) preparations, but responses were almost absent in preparations lacking vesicular Zn2+ (ZnT3-KO). In contrast, under brief dialysis conditions, intracellular Zn2+ transients were very similar in WT and ZnT3-KO preparations. This suggests that both intracellular release and transmembrane flux can contribute to intracellular Zn2+ accumulation after synaptic stimulation. These results demonstrate significant confounds and potential use of intracellular dialysis to investigate intracellular Zn2+ accumulation mechanisms.
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影响因子：
3.3
作者：
Vander Jagt TA;Connor JA;Weiss JH;Shuttleworth CW
通讯作者：
Shuttleworth CW
DOI：
10.1523/jneurosci.0026-12.2012
发表时间：
2012-06-27
期刊：
The Journal of neuroscience : the official journal of the Society for Neuroscience
影响因子：
--
作者：
Hall CN;Klein-Flügge MC;Howarth C;Attwell D
通讯作者：
Attwell D
影响因子：
16.2
作者：
Huang, Yang Z.;Pan, Enhui;McNamara, James O.
通讯作者：
McNamara, James O.
影响因子：
4.7
作者：
Kiedrowski, Lech
通讯作者：
Kiedrowski, Lech
影响因子：
6.2
作者：
Malaiyandi, LM;Dineley, KE;Reynolds, IJ
通讯作者：
Reynolds, IJ