Department of Biochemistry, Saitama Medical University, 38 Morohongo, Moroyama-machi, Iruma-gun, Saitama 350-0495, Japan.
Department of Biochemistry, Saitama Medical University, 38 Morohongo, Moroyama-machi, Iruma-gun, Saitama 350-0495, Japan.
Neurochem Int. 2018 Oct;119:140-150. doi: 10.1016/j.neuint.2017.08.012. Epub 2017 Aug 24.
Neuronal plasma membrane has been thought to retain a lot of lipid raft components which play important roles in the neural function. Although the biochemical analyses of lipid raft using brain tissues have been extensively carried out in the past 20 years, many of their experimental conditions do not coincide with those of standard neuroscience researches such as neurophysiology and neuropharmacology. Hence, the physiological methods for lipid raft analysis that can be compatible with general neuroscience have been required. Herein, we developed a system to physiologically analyze ganglioside GM1-enriched lipid rafts in brain tissues using the "Enzyme-Mediated Activation of Radical Sources (EMARS)" method that we reported (Kotani N. et al. Proc. Natl. Acad. Sci. U S A 105, 7405-7409 (2008)). The EMARS method was applied to acute brain slices prepared from mouse brains in aCSF solution using the EMARS probe, HRP-conjugated cholera toxin subunit B, which recognizes ganglioside GM1. The membrane molecules present in the GM1-enriched lipid rafts were then labeled with fluorescein under the physiological condition. The fluorescein-tagged lipid raft molecules called "EMARS products" distributed differentially among various parts of the brain. On the other hand, appreciable differences were not detected among segments along the longitudinal axis of the hippocampus. We further developed a device to label the lipid raft molecules in acute hippocampal slices under two different physiological conditions to detect dynamics of the lipid raft molecules during neural excitation. Using this device, several cell membrane molecules including Thy1, known as a lipid raft resident molecule in neurons, were confirmed by the EMARS method in living hippocampal slices.
神经细胞的质膜被认为保留了许多脂质筏成分，这些成分在神经功能中发挥着重要作用。尽管过去 20 年来已经广泛开展了使用脑组织进行脂质筏的生化分析，但它们的许多实验条件与神经生理学和神经药理学等标准神经科学研究并不相符。因此，需要开发与一般神经科学兼容的用于脂质筏分析的生理方法。在此，我们使用我们报道的“酶介导的自由基源激活（EMARS）”方法（Kotani N. 等人，Proc. Natl. Acad. Sci. U S A 105, 7405-7409 (2008)），开发了一种在生理条件下分析脑组织中神经节苷脂 GM1 富集的脂质筏的系统。该 EMARS 方法应用于使用 EMARS 探针（HRP 缀合霍乱毒素亚基 B，其识别神经节苷脂 GM1）在 aCSF 溶液中制备的来自小鼠大脑的急性脑切片。然后，在生理条件下，用荧光素标记存在于 GM1 富集的脂质筏中的膜分子。这种被称为“EMARS 产物”的荧光素标记的脂质筏分子在大脑的不同部位呈现出不同的分布。另一方面，在海马体的纵轴上的各个节段之间没有检测到明显的差异。我们进一步开发了一种设备，用于在两种不同的生理条件下标记急性海马切片中的脂质筏分子，以检测神经兴奋过程中脂质筏分子的动态变化。使用该设备，通过 EMARS 方法在活体海马切片中确认了几种细胞膜分子，包括 Thy1，它被认为是神经元中的脂质筏驻留分子。
