Yang C C, Alvarez R B, Engel W K, Haun C K, Askanas V
USC Neuromuscular Center, Department of Neurology, Los Angeles, California 90017-1912, USA.
Exp Neurol. 1997 Nov;148(1):34-44. doi: 10.1006/exnr.1997.6663.
Neuronal (n) and inducible (i) nitric oxide synthase (NOS) were immunolocalized at the postsynaptic domain of human and rat neuromuscular junctions (NMJs) by light and electron microscopy. We applied polyclonal and monoclonal antibodies for colocalization with three other synaptic proteins, utilizing double and triple fluorescence labeling, and gold and peroxidase for immunoelectron microscopy. By light microscopy, nNOS and iNOS colocalized with desmin and dystrophin, known postsynaptic components, but not with neurofilament protein, a presynaptic component. By electronmicroscopy, nNOS, but not iNOS, colocalized postsynaptically on the same structures as desmin; iNOS was also postsynaptic, but did not colocalize with desmin immunoreactivity. At the NMJs of Duchenne muscular dystrophy patients, both nNOS and iNOS were strongly immunoreactive. At the NMJs of a patient with myasthenia gravis, nNOS was weaker than in controls. Total denervation of rat sciatic nerve did not cause any decrease of nNOS or iNOS immunoreactivity 7 days thereafter. At 15 days after denervation, there was a gradual decrease of immunoreactivity, and immunoreactivity disappeared 30 days after denervation, corresponding to the ultrastructurally detectable disorganization of the postsynaptic region. This seems to be the first combined light and electron microscopic description of the postsynaptic localization of nNOS and iNOS at human and rat NMJs.
通过光学显微镜和电子显微镜，对神经元型（n）和诱导型（i）一氧化氮合酶（NOS）在人和大鼠神经肌肉接头（NMJ）的突触后区域进行了免疫定位。我们应用多克隆和单克隆抗体，通过双重和三重荧光标记以及免疫电子显微镜中使用的金标和过氧化物酶，使其与其他三种突触蛋白进行共定位。通过光学显微镜观察，nNOS和iNOS与已知的突触后成分结蛋白和抗肌萎缩蛋白共定位，但不与突触前成分神经丝蛋白共定位。通过电子显微镜观察，nNOS而非iNOS在突触后与结蛋白位于相同结构上；iNOS也位于突触后，但不与结蛋白免疫反应性共定位。在杜兴氏肌营养不良患者的神经肌肉接头处，nNOS和iNOS均具有强烈的免疫反应性。在重症肌无力患者的神经肌肉接头处，nNOS比对照组弱。大鼠坐骨神经完全去神经支配7天后，nNOS或iNOS的免疫反应性没有任何降低。去神经支配15天后，免疫反应性逐渐降低，去神经支配30天后免疫反应性消失，这与突触后区域超微结构上可检测到的紊乱相对应。这似乎是首次对人和大鼠神经肌肉接头处nNOS和iNOS的突触后定位进行的光学显微镜和电子显微镜联合描述。
