Cameron Morven, Kékesi Orsolya, Morley John W, Tapson Jonathan, Breen Paul P, van Schaik André, Buskila Yossi
Biomedical Engineering and Neuroscience group, The MARCS Institute, Western Sydney University, Penrith, NSW, Australia.
School of Medicine, Western Sydney University, Penrith, NSW, Australia.
PLoS One. 2016 May 16;11(5):e0155468. doi: 10.1371/journal.pone.0155468. eCollection 2016.
Calcium-imaging is a sensitive method for monitoring calcium dynamics during neuronal activity. As intracellular calcium concentration is correlated to physiological and pathophysiological activity of neurons, calcium imaging with fluorescent indicators is one of the most commonly used techniques in neuroscience today. Current methodologies for loading calcium dyes into the tissue require prolonged incubation time (45-150 min), in addition to dissection and recovery time after the slicing procedure. This prolonged incubation curtails experimental time, as tissue is typically maintained for 6-8 hours after slicing. Using a recently introduced recovery chamber that extends the viability of acute brain slices to more than 24 hours, we tested the effectiveness of calcium AM staining following long incubation periods post cell loading and its impact on the functional properties of calcium signals in acute brain slices and wholemount retinae. We show that calcium dyes remain within cells and are fully functional >24 hours after loading. Moreover, the calcium dynamics recorded >24 hrs were similar to the calcium signals recorded in fresh tissue that was incubated for <4 hrs. These results indicate that long exposure of calcium AM dyes to the intracellular cytoplasm did not alter the intracellular calcium concentration, the functional range of the dye or viability of the neurons. This data extends our previous work showing that a custom recovery chamber can extend the viability of neuronal tissue, and reliable data for both electrophysiology and imaging can be obtained >24hrs after dissection. These methods will not only extend experimental time for those using acute neuronal tissue, but also may reduce the number of animals required to complete experimental goals.
钙成像技术是监测神经元活动期间钙动力学变化的一种灵敏方法。由于细胞内钙浓度与神经元的生理和病理生理活动相关，因此利用荧光指示剂进行钙成像已成为当今神经科学中最常用的技术之一。目前，将钙染料加载到组织中的方法，除了切片后的解剖和恢复时间外，还需要较长的孵育时间（45-150分钟）。这种较长的孵育时间限制了实验时间，因为切片后的组织通常只能维持6-8小时。我们使用最近推出的一种恢复室，它能将急性脑片的存活时间延长至24小时以上，测试了细胞加载后长时间孵育钙黄绿素乙酰甲酯（Calcium AM）染色的有效性，以及它对急性脑片和视网膜整体标本中钙信号功能特性的影响。我们发现，钙染料在加载后24小时以上仍能保留在细胞内并保持其全部功能。此外，在24小时后记录的钙动力学与在新鲜组织（孵育时间<4小时）中记录的钙信号相似。这些结果表明，钙黄绿素乙酰甲酯染料长时间暴露于细胞内细胞质中，并不会改变细胞内钙浓度、染料的功能范围或神经元的活力。这些数据扩展了我们之前的研究成果，即定制的恢复室可以延长神经元组织的存活时间，并且在解剖后24小时以上仍可获得可靠的电生理和成像数据。这些方法不仅将延长使用急性神经元组织的实验时间，而且还可能减少完成实验目标所需的动物数量。
