Endogenous calcium buffering in motoneurones of the nucleus hypoglossus from mouse
Endogenous calcium buffering in motoneurones of the nucleus hypoglossus from mouse
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DOI：
10.1111/j.1469-7793.1998.105bi.x
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发表时间：
1998-08-15
影响因子：
5.5
通讯作者：
Keller, BU
中科院分区：
文献类型：
--
作者：
Lips, MB;Keller, BU
1.对2~6日龄小鼠脑干舌下核片中65个运动神经元进行同步膜片钳和快速微量钙测量。在体外切片分离过程中，舌下运动神经元特别容易受到机械或代谢应激的影响。因此，根据舌下神经(XII)的自发节律性活动判断，实验条件对功能完整性进行了优化。在去极化电压阶跃过程中，细胞胞体内的钙浓度以毫秒时间域的时间分辨率进行监测。用快速单色仪(开关10ms)、光电倍增管和钙敏感染料Fura-2和Mag-Fura-5.4进行比率荧光测量。研究了细胞内钙指示剂浓度对体细胞钙瞬变动力学的影响。钙瞬变的衰减近似为单一指数分量，衰减时间常数与染料浓度呈线性关系。在没有指示剂染料的情况下，外推的衰变时间为0.7+/-0.2 S，这表明生理条件下体细胞钙的快速动力学。通过反向外推，即“添加缓冲液”法，得到钙结合比为41+/-12(9个细胞)，表明98%进入舌下运动神经元的钙离子是由内源性缓冲液结合的。舌下运动神经元的内源性钙结合率与其他大小或形状相似的神经元相比较小。因此，我们的测量表明，舌下运动神经元对钙相关的兴奋性毒性的选择性易感性可能部分是由于这些细胞中低浓度的钙缓冲液所致。
1. Simultaneous patch clamp and rapid microfluorometric calcium measurements were performed on sixty-five motoneurones in slices of the nucleus hypoglossus in the brainstem of 2- to 6-day-old mice.2. Hypoglossal motoneurones were particularly vulnerable to mechanical or metabolic stress during isolation of in vitro slice preparations. Therefore, experimental conditions were optimized for functional integrity, as judged by spontaneous rhythmic activity of hypoglossal nerves (XII).3. Calcium concentrations in the cell soma were monitored with a temporal resolution in the millisecond time domain during depolarizing voltage steps. Ratiometric fluorescence measurements were made using a rapid monochromator (switching tau < 10 ms), a photomultiplier tube and the calcium sensitive dyes fura-2 and mag-fura-5.4. Dynamics of somatic calcium transients were investigated as a function of the concentration of calcium indicator dye in the cell. Decays of calcium transients were approximated to a single exponential component and decay time constants showed a linear dependence on dye concentration. The extrapolated decay time in the absence of indicator dye was 0.7 +/- 0.2 s, suggesting rapid somatic calcium dynamics under physiological conditions5. By a process of back-extrapolation, the 'added buffer' method, a calcium binding ratio of 41 +/- 12 (9 cells) was obtained indicating that 98% of the calcium ions entering a hypoglossal motoneurone were bound by endogenous buffers.6. Endogenous calcium binding ratios in hypoglossal motoneurones were small compared with those of other neurones with comparable size or geometry. Accordingly, our measurements suggest that the selective vulnerability of hypoglossal motoneurones to calcium-related excitotoxicity might partially result from low concentrations of calcium buffers in these cells.