Ye Xiaoying, Xie Fang, Romanova Elena V, Rubakhin Stanislav S, Sweedler Jonathan V
Department of Chemistry and the Beckman Institute, University of Illinois, Urbana, Illinois 61801.
ACS Chem Neurosci. 2010 Mar 17;1(3):182-193. doi: 10.1021/cn900016z.
Nitric oxide (NO), an intercellular signaling molecule, helps coordinate neuronal network activity. Here we examine NO generation in the Aplysia central nervous system using 4,5-diaminofluorescein diacetate (DAF-2 DA), a fluorescent reagent that forms 4,5-diaminofluorescein triazole (DAF-2T) upon reaction with NO. Recognizing that other fluorescence products are formed within the biochemically complex intracellular environment, we validate the observed fluorescence as being from DAF-2T; using both capillary electrophoresis and mass spectrometry we confirm that DAF-2T is formed from tissues and cells exposed to DAF-2 DA. We observe three distinct subcellular distributions of fluorescence in neurons exposed to DAF-2 DA. The first shows uniform fluorescence inside the cell, with these cells being among previously confirmed NOS-positive regions in the Aplysia cerebral ganglion. The second, seen inside buccal neurons, exhibits point sources of fluorescence, 1.5 ± 0.7 µm in diameter. Interestingly, the number of fluorescence puncta increases when the tissue is preincubated with the NOS substrate L-arginine, and they disappear when cells are preexposed to the NOS inhibitor L-NAME, demonstrating that the fluorescence is connected to NOS-dependent NO production. The third distribution type, seen in the R2 neuron, also exhibits fluorescent puncta, but only on the cell surface. Fluorescence is also observed in the terminals of cultured bag cell neurons loaded with DAF-2 DA. Surprisingly, fluorescence at the R2 surface and bag cell neuron terminals is not modulated by L-arginine or L-NAME, suggesting it has a source distinct from the buccal and cerebral ganglion DAF 2T-positive tissues.
一氧化氮（NO）作为一种细胞间信号分子，有助于协调神经网络活动。在此，我们使用4,5-二氨基荧光素二乙酸酯（DAF-2 DA）来检测海兔中枢神经系统中NO的生成，DAF-2 DA是一种荧光试剂，与NO反应后会形成4,5-二氨基荧光素三唑（DAF-2T）。鉴于在生物化学复杂的细胞内环境中会形成其他荧光产物，我们验证了观察到的荧光来自DAF-2T；通过毛细管电泳和质谱分析，我们证实DAF-2T是由暴露于DAF-2 DA的组织和细胞形成的。我们在暴露于DAF-2 DA的神经元中观察到三种不同的亚细胞荧光分布。第一种在细胞内显示均匀荧光，这些细胞位于先前已确认的海兔脑神经节中一氧化氮合酶（NOS）阳性区域。第二种出现在颊神经元内，呈现直径为1.5±0.7 µm的荧光点源。有趣的是，当组织用NOS底物L-精氨酸预孵育时，荧光点的数量会增加，而当细胞预先暴露于NOS抑制剂L-硝基精氨酸甲酯（L-NAME）时，荧光点会消失，这表明荧光与NOS依赖的NO生成有关。第三种分布类型见于R2神经元，也呈现荧光点，但仅在细胞表面。在加载了DAF-2 DA的培养袋状细胞神经元的终末也观察到了荧光。令人惊讶的是，R2表面和袋状细胞神经元终末的荧光不受L-精氨酸或L-NAME的调节，这表明其来源与颊神经节和脑神经节中DAF-2T阳性组织不同。
