Department of Neuroscience, Cell Biology, and Physiology, Boonshoft School of Medicine, Wright State University, Dayton, OH 45435.
Department of Neuroscience, Cell Biology, and Physiology, Boonshoft School of Medicine, Wright State University, Dayton, OH 45435
eNeuro. 2019 Aug 1;6(4). doi: 10.1523/ENEURO.0349-18.2019. Print 2019 Jul/Aug.
We investigated the calcium dynamics of dorsal root ganglion (DRG) neurons using transgenic mice to target expression of the genetically encoded calcium indicator (GECI), GCaMP6s, to a subset of neurons containing parvalbumin (PV), a calcium-binding protein present in proprioceptors and low-threshold mechanoreceptors. This study provides the first analysis of GECI calcium transient parameters from large-diameter DRG neurons. Our approach generated calcium transients of consistent shape and time-course, with quantifiable characteristics. Four parameters of calcium transients were determined to vary independently from each other and thus are likely influenced by different calcium-regulating mechanisms: peak amplitude, rise time (RT), decay time, and recovery time. Pooled analysis of 188 neurons demonstrated unimodal distributions, providing evidence that PV+ DRG neurons regulate calcium similarly as a population despite their differences in size, electrical properties, and functional sensitivities. Calcium transients increased in size with elevated extracellular calcium, longer trains of action potentials, and higher stimulation frequencies. RT and decay time increased with the addition of the selective sarco/endoplasmic reticulum calcium ATPases (SERCA) blocker, thapsigargin (TG), while peak amplitude and recovery time remained the same. When elevating bath pH to 8.8 to block plasma-membrane calcium ATPases (PMCA), all measured parameters significantly increased. These results illustrate that GECI calcium transients provide sufficient resolution to detect changes in electrical activity and intracellular calcium concentration, as well as discern information about the activity of specific subclasses of calcium regulatory mechanisms.
我们使用转基因小鼠研究背根神经节 (DRG) 神经元的钙动力学，以将基因编码钙指示剂 (GECI)，GCaMP6s，靶向表达于包含钙结合蛋白肌球蛋白（PV）的神经元亚群中，肌球蛋白存在于本体感受器和低阈值机械感受器中。这项研究首次分析了大直径 DRG 神经元中 GECI 钙瞬变参数。我们的方法产生了具有一致形状和时间过程的钙瞬变，具有可量化的特征。四个钙瞬变参数被确定为彼此独立变化，因此可能受到不同的钙调节机制的影响：峰值幅度、上升时间 (RT)、衰减时间和恢复时间。对 188 个神经元的汇总分析表明，存在单峰分布，这为 PV+DRG 神经元作为一个群体调节钙的方式提供了证据，尽管它们在大小、电特性和功能敏感性方面存在差异。钙瞬变的大小随细胞外钙浓度的升高、动作电位的长串和刺激频率的增加而增大。RT 和衰减时间随选择性肌浆/内质网钙 ATP 酶 (SERCA) 阻滞剂他帕霉素 (TG) 的加入而增加，而峰值幅度和恢复时间保持不变。当将浴 pH 值升高至 8.8 以阻断质膜钙 ATP 酶 (PMCA) 时，所有测量的参数均显著增加。这些结果表明，GECI 钙瞬变提供了足够的分辨率来检测电活动和细胞内钙浓度的变化，并辨别特定钙调节机制亚类活动的信息。
