Intracellular Calcium Spikes in Rat Suprachiasmatic Nucleus Neurons Induced by BAPTA-Based Calcium Dyes
Intracellular Calcium Spikes in Rat Suprachiasmatic Nucleus Neurons Induced by BAPTA-Based Calcium Dyes
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DOI：
10.1371/journal.pone.0009634
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发表时间：
2010-03-10
期刊：
影响因子：
3.7
通讯作者：
Lee, Kyoung J.
中科院分区：
文献类型：
--
作者：
Hong, Jin Hee;Min, Cheol Hong;Lee, Kyoung J.
背景资料：据报道，位于下丘脑视交叉上核（SCN）内的哺乳动物昼夜节律起搏神经元的自发动作电位（AP）放电频率和胞浆游离钙浓度的昼夜节律。还报道了“Ca 2+尖峰”的存在（即，[Ca2+]（c）具有10，100秒带宽的瞬变），但尚不清楚这些SCN Ca 2+尖峰是否与缓慢的昼夜节律有关。方法学/主要发现：我们基于Ca 2+指示染料（fluo-4）和蛋白质Ca 2+传感器（黄色cameleon）解决了这个问题。用Fluo-4AM染料，我们在急性脑片中发现18%的大鼠SCN细胞内有自发性Ca ~（2+）峰，但Ca ~（2+）峰频率无昼夜变化。我们用大鼠（和小鼠）SCN切片培养物重复相同的实验，所述SCN切片培养物表达黄色骆驼基因用于许多不同的昼夜节律阶段，并且令人惊讶的是，几乎没有观察到自发的Ca 2+尖峰（< 3%）。然而，当除了表达cameleon的SCN培养物之外还加载fluo-4 AM或BAPTA-AM时，表现出Ca 2+尖峰的细胞数量增加到13个，接近14%。结论/意义：尽管我们进行了大量的实验，但在SCN的自发Ca 2+尖峰活动中没有发现昼夜节律的证据。此外，我们的研究强烈表明，自发的Ca 2+尖峰活性是由基于BAPTA的fluo-4染料的Ca 2+螯合作用引起的。因此，这种诱导的活性似乎与SCN神经元中[Ca 2 +]（c）的内在昼夜节律无关。基于BAPTA的染料的问题是众所周知的，我们的研究提供了一个明确的关注案例，特别是SCN Ca 2+尖峰。另一方面，我们的研究既没有使这些染料作为一个整体的使用无效，也没有破坏SCN Ca 2+尖峰在SCN功能中的潜在作用。
Background: Circadian rhythms in spontaneous action potential (AP) firing frequencies and in cytosolic free calcium concentrations have been reported for mammalian circadian pacemaker neurons located within the hypothalamic suprachiasmatic nucleus (SCN). Also reported is the existence of "Ca2+ spikes" (i.e., [Ca2+](c) transients having a bandwidth of 10,100 seconds) in SCN neurons, but it is unclear if these SCN Ca2+ spikes are related to the slow circadian rhythms.Methodology/Principal Findings: We addressed this issue based on a Ca2+ indicator dye (fluo-4) and a protein Ca2+ sensor (yellow cameleon). Using fluo-4 AM dye, we found spontaneous Ca2+ spikes in 18% of rat SCN cells in acute brain slices, but the Ca2+ spiking frequencies showed no day/night variation. We repeated the same experiments with rat (and mouse) SCN slice cultures that expressed yellow cameleon genes for a number of different circadian phases and, surprisingly, spontaneous Ca2+ spike was barely observed (< 3%). When fluo-4 AM or BAPTA-AM was loaded in addition to the cameleon-expressing SCN cultures, however, the number of cells exhibiting Ca2+ spikes was increased to 13 similar to 14%.Conclusions/Significance: Despite our extensive set of experiments, no evidence of a circadian rhythm was found in the spontaneous Ca2+ spiking activity of SCN. Furthermore, our study strongly suggests that the spontaneous Ca2+ spiking activity is caused by the Ca2+ chelating effect of the BAPTA-based fluo-4 dye. Therefore, this induced activity seems irrelevant to the intrinsic circadian rhythm of [Ca2+](c) in SCN neurons. The problems with BAPTA based dyes are widely known and our study provides a clear case for concern, in particular, for SCN Ca2+ spikes. On the other hand, our study neither invalidates the use of these dyes as a whole, nor undermines the potential role of SCN Ca2+ spikes in the function of SCN.