A fluorescence technique to measure intracellular pH of single neurons in brainstem slices.
A fluorescence technique to measure intracellular pH of single neurons in brainstem slices.
复制标题
一种测量脑干切片中单个神经元细胞内 pH 值的荧光技术。
DOI：
10.1016/0165-0270(96)00051-9
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发表时间：
1996
影响因子：
3
通讯作者：
Putnam,RW
中科院分区：
文献类型：
--
作者：
Ritucci,NA;Erlichman,JS;Dean,JB;Putnam,RW
我们已经开发了一种技术，以测量单个神经元在脑干切片使用荧光成像系统的pH值。将切片加载pH敏感性荧光染料BCECF，并通过在500和440 nm处交替激发切片来观察荧光。在530 nm处发射的荧光被引导通过MTI GenIISys图像增强器和MTI CCD 72照相机。图像经Image-1/FL软件处理。测量在500和440 nm激发波长下发射的荧光的比率，并通过使用pH值范围为5.8至8.6的高K+/尼日利亚菌素溶液构建校准曲线来转换为pH。加载BCECF的切片显示出强烈荧光和弥散背景荧光的不同球体。切片标记的神经元特异性抗体，神经元特异性烯醇化酶，显示染色与球的强烈荧光BCECF加载的细胞。用胶质细胞特异性抗体（胶质细胞酸性蛋白）标记的切片显示弥漫性背景染色。用罗丹明珠逆行标记的神经元发出大球体的荧光，这与BCECF负载细胞的荧光完全相关。此外，大的荧光球具有约-60 mV的膜电位并产生动作电位。这些发现表明大的荧光球是神经元。在背侧和腹侧延髓化学敏感区的这些大球体（神经元）中测量pH，分别为7.32 ± 0.02（n = 110）和7.38 ± 0.02（n = 85）。
We have developed a technique to measure the pHiof single neurons in brainstem slices using a fluorescence imaging system. Slices were loaded with the pH-sensitive fluorescent dye BCECF and fluorescence was visualized by exciting the slices alternately at 500 and 440 nm. The emitted fluorescence at 530 nm was directed through an MTI GenIISys image intensifier and MTI CCD72 camera. The images were processed by Image-1/FL software. The ratio of emitted fluorescence at excitation wavelengths of 500 and 440 nm was measured and converted to pH by constructing a calibration curve using high K+/nigericin solutions at pH values ranging from 5.8 to 8.6. BCECF-loaded slices showed distinct spheres of intense fluorescence and diffuse background fluorescence. Slices labeled with a neuron-specific antibody, neuron-specific enolase, showed staining that correlated with the spheres of intense fluorescence of BCECF-loaded cells. Slices labeled with a glial-specific antibody, glial fibrillary acidic protein, showed a diffuse, background staining. Neurons that were retrograde-labeled with rhodamine beads fluoresced as large spheres that exactly correlated with the fluorescence from BCECF-loaded cells. Further, large fluorescent spheres had membrane potentials of about −60 mV and generated action potentials. These findings indicate that the large fluorescent spheres are neurons. pHiwas measured in these large spheres (neurons) in the dorsal and ventral medullary chemosensitive regions, and was 7.32 ± 0.02 (n = 110) and 7.38 ± 0.02 (n = 85), respectively.