Imaging of sialidase activity in rat brain sections by a highly sensitive fluorescent histochemical method
Imaging of sialidase activity in rat brain sections by a highly sensitive fluorescent histochemical method
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DOI：
10.1016/j.neuroimage.2011.06.017
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发表时间：
2011-09-01
期刊：
影响因子：
5.7
通讯作者：
Suzuki, Takashi
中科院分区：
文献类型：
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作者：
Minami, Akira;Shimizu, Hirotaka;Suzuki, Takashi
唾液酸酶 (EC 3.2.1.18) 可去除唾液酸糖缀合物中的唾液酸。由于细胞外应用于大鼠海马的唾液酸酶影响许多神经功能，包括突触可塑性和谷氨酸能神经元的神经支配，细胞外膜表面上的内源性唾液酸酶活性也可能影响神经功能。然而，唾液酸酶活性在大脑中的分布仍然未知。为了可视化大鼠大脑膜表面上的细胞外唾液酸酶活性，将急性脑切片与 5-溴-4-氯吲哚-3-基-α-D-N-乙酰神经氨酸 (X-Neu5Ac) 和固红紫 LB (FRV LB) 在 pH 7.3 下孵育。 1小时后，大鼠大脑中髓磷脂丰富的区域显示出强烈的荧光。虽然大脑中的海马体显示出微弱的荧光，但海马体中的苔藓纤维末端却显示出相对较强的荧光。这些荧光强度被唾液酸酶特异性抑制剂 2,3-脱氢-2-脱氧-N-乙酰神经氨酸（DANA，1 mM）减弱。此外，X-Neu5Ac和FRV LB引起的荧光强度与用4-甲基伞形基-α-D-N-乙酰神经氨酸（4MU-Neu5Ac）测量的唾液酸酶活性相关，4MU-Neu5Ac是每个脑区唾液酸酶活性定量测量的经典底物。因此，X-Neu5Ac 和 FRV LB 染色对唾液酸酶具有特异性，可用于唾液酸酶活性的定量分析。结果表明，大鼠脑白质具有强烈的唾液酸酶活性。 (C) 2011 Elsevier Inc. 保留所有权利。
Sialidase (EC 3.2.1.18) removes sialic acid from sialoglycoconjugates. Since sialidase extracellularly applied to the rat hippocampus influences many neural functions, including synaptic plasticity and innervations of glutamatergic neurons, endogenous sialidase activities on the extracellular membrane surface could also affect neural functions. However, the distribution of sialidase activity in the brain remains unknown. To visualize extracellular sialidase activity on the membrane surface in the rat brain, acute brain slices were incubated with 5-bromo-4-chloroindol-3-yl-alpha-D-N-acetylneuraminic acid (X-Neu5Ac) and Fast Red Violet LB (FRV LB) at pH 7.3. After 1 h, myelin-abundant regions showed intense fluorescence in the rat brain. Although the hippocampus showed weak fluorescence in the brain, mossy fiber terminals in the hippocampus showed relatively intense fluorescence. These fluorescence intensities were attenuated with a sialidase-specific inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA, 1 mM). Additionally, the fluorescence intensities caused by X-Neu5Ac and FRV LB were correlated with the sialidase activity measured with 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid (4MU-Neu5Ac), a classical substrate for quantitative measurement of sialidase activity, in each brain region. Therefore, staining with X-Neu5Ac and FRV LB is specific for sialidase and useful for quantitative analysis of sialidase activities. The results suggest that white matter of the rat brain has intense sialidase activity. (C) 2011 Elsevier Inc. All rights reserved.