Yasuhara Osamu, Aimi Yoshinari, Shibano Akira, Matsuo Akinori, Bellier Jean-Pierre, Park Masami, Tooyama Ikuo, Kimura Hiroshi
Molecular Neuroscience Research Center, Shiga University of Medical Science, Otsu 520-2192, Japan.
J Comp Neurol. 2004 Apr 26;472(2):232-45. doi: 10.1002/cne.20077.
We have recently discovered a splice variant of choline acetyltransferase (ChAT) mRNA and designated the variant protein pChAT because of its preferential expression in peripheral neuronal structures. In this study, the presence of pChAT in rat iris was examined by immunohistochemistry and Western blot using a pChAT antiserum, in combination with RT-PCR analysis and ChAT enzyme assay. For comparison, the conventional ChAT (cChAT) was studied in parallel. By pChAT immunohistochemistry, intense labeling was found to occur in nerve fibers of the iris and in neurons of the ciliary and trigeminal ganglia. Denervation studies, analyzed by semiquantitative morphometry, indicated that these iridial pChAT fibers originated about half from the ciliary ganglion and the other half from the trigeminal ganglion. The presence of pChAT protein in the iris and trigeminal ganglion was confirmed by Western blot. The expression of pChAT mRNA in the ciliary and trigeminal ganglia was proved by RT-PCR. Although cChAT protein and mRNA were detected in the ciliary ganglion, neither was detectable in the trigeminal ganglion. The contributions of the ciliary and trigeminal ganglia to the iridial ChAT enzyme activity were verified by the present ChAT assay. Here, we provide evidence that iridial pChAT nerves are composed of postganglionic parasympathetic efferents from the ciliary ganglion and, more interestingly, somatic sensory afferents of the trigeminal ophthalmic nerve.
我们最近发现了胆碱乙酰转移酶（ChAT）mRNA的一种剪接变体，并将该变体蛋白命名为pChAT，因为它在外周神经结构中优先表达。在本研究中，我们使用pChAT抗血清，通过免疫组织化学和蛋白质印迹法检测大鼠虹膜中pChAT的存在，并结合逆转录聚合酶链反应（RT-PCR）分析和ChAT酶活性测定。为作比较，同时对传统的ChAT（cChAT）进行了研究。通过pChAT免疫组织化学发现，虹膜神经纤维以及睫状神经节和三叉神经节的神经元中出现了强烈的标记。通过半定量形态学分析的去神经研究表明，这些虹膜pChAT纤维约一半起源于睫状神经节，另一半起源于三叉神经节。蛋白质印迹法证实了虹膜和三叉神经节中存在pChAT蛋白。RT-PCR证明了睫状神经节和三叉神经节中pChAT mRNA的表达。虽然在睫状神经节中检测到了cChAT蛋白和mRNA，但在三叉神经节中均未检测到。本ChAT活性测定验证了睫状神经节和三叉神经节对虹膜ChAT酶活性的贡献。在此，我们提供证据表明，虹膜pChAT神经由来自睫状神经节的节后副交感传出神经组成，更有趣的是，还由三叉神经眼支的躯体感觉传入神经组成。
