Quantitative Analysis of Presynaptic Vesicle Luminal pH in Cultured Neurons
Quantitative Analysis of Presynaptic Vesicle Luminal pH in Cultured Neurons
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培养神经元突触前囊泡腔内 pH 值的定量分析
DOI：
10.1007/978-1-0716-1916-2_4
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Takamori Shigeo
中科院分区：
文献类型：
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作者：
Egashira Yoshihiro;Katsurabayashi Shutaro;Takamori Shigeo
新产生的突触小泡 (SV) 被液泡型 H+-ATP 酶的活性重新酸化。由于跨 SV 膜的 H+ 梯度驱动神经递质摄取进入 SV，因此稳态囊泡 pH 值和再酸化过程动态的精确测量将提供有关 H+ 驱动的神经递质摄取的重要信息。事实上，我们最近证明了培养的海马神经元中谷氨酸能囊泡和 GABA 能囊泡之间囊泡 pH 的稳态和动态的独特特征。在本文中，我们重点关注确定活体神经元中 SV 稳态管腔 pH 值所需的实验方案和设置。该方案包括在培养神经元的 SV 腔中有效表达 pH 敏感荧光蛋白，并在给定 pH 下局部应用酸性缓冲液和含离子载体溶液期间，在传统荧光显微镜下记录其荧光变化。这里描述的方法可以很容易地应用于测量培养细胞制剂中不同类型的分泌细胞器和其他酸性细胞器（例如溶酶体和内体）的腔内 pH 值。
Newly generated synaptic vesicles (SVs) are re-acidified by the activity of the vacuolar-type H+-ATPases. Since H+gradient across SV membrane drives neurotransmitter uptake into SVs, precise measurements of steady-state vesicular pH and dynamics of re-acidification process will provide important information concerning the H+-driven neurotransmitter uptake. Indeed, we recently demonstrated distinct features of steady state and dynamics of vesicular pH between glutamatergic vesicles and GABAergic vesicles in cultured hippocampal neurons. In this article, we focus on an experimental protocol and setup required to determine steady-state luminal pH of SVs in living neurons. This protocol is composed of efficient expression of a pH-sensitive fluorescent protein in the lumen of SVs in cultured neurons, and recordings of its fluorescence changes under a conventional fluorescent microscope during local applications of acidic buffer and ionophores-containing solution at a given pH. The method described here can be easily applied for measuring luminal pH of different types of secretory organelles and other acidic organelles such as lysosomes and endosomes in cultured cell preparations.