Histochemical and Molecular Genetical Studies on the Development of Cholinergic Neurons

胆碱乙酰转移酶（ChAT）是乙酰胆碱的生物合成酶，是目前胆碱能神经元最特异的标志物。从大鼠、小鼠和人的脊髓中提取ChAT的cDNA，并将分子生物学技术引入到解剖学研究中，以组织化学方法研究胆碱能神经元的发育。一种方法涉及从引入大肠杆菌的大鼠ChAT的cDNA表达ChAT蛋白并产生针对该蛋白的抗血清。使用获得的抗血清，建立免疫组织化学（IHC）。第二种方法涉及原位杂交（ISH）组织化学的发展，这使得精确定位和识别特定基因转录的单个细胞成为可能。我们成功地检测了大鼠和人的ChAT mRNA，并利用免疫组化和原位杂交技术，阐明了大鼠中枢胆碱能神经元的生后发育。在本研究过程中，我们发现在出生后第2天至第13天，在新皮质第VI层中出现了ChAT免疫反应阳性神经元，这一现象可能是由于这些神经元表型的改变，而不是由于神经元的死亡或迁移。我们还成功地在人胚胎脊髓运动神经元中检测到ChAT mRNA，另一方面，ChAT基因的分子遗传学分析表明，胚胎和成年小鼠脊髓中的mRNA结构不同，成年小鼠脊髓中存在多种形式的差异剪接产生的mRNA。此外，我们还成功地克隆了人ChAT的cDNA，并证明它比大鼠和小鼠的大得多。

Choline acetyltransterase (ChAT), the biosynthetic enzyme for ACh, is currently the most specific marker for the cholinergic neuron. cDNAs for ChAT were isolated from spinal cords of rat, mouse and human.To investigate the development of the cholinergic neuron histochemically, we introduced molecular biological techniques into our anatomical studies. One method involves expression of ChAT protein from cDNA for rat ChAT introduced into Echerichia coli and the production of antisera against the protein. Using an antiserum obtained, immunohistochemistry (IHC) was established. The second method involves the development of an in situ hybridization (ISH) histochemistry, which enables the precise localization and indentification of individual cells in which a particular gene is transcribed. We have succeeded in detecting mRNA for ChAT of rat and human.Using IHC and ISH, we clarified the postnatal development of the cholinergic neuron in the rat CNS. During the course of this study, we found a transient appearance of ChAT-immunoreactive neurons in the layer VI of the neocortex between postnatal day 2 and day 13.This phenomenon is suggested to be due to the change of phenotype in these neurons rather than the death or migration of the neurons.We have also succeeded in detecting mRNA for ChAT in motoneurons in the spinal cord of the human embryo, and this study will be continued.On the other hand, molecular genetical analyses of ChAT gene revealed that structures of mRNA differ between the fetal and adult mouse spinal cords and there are several forms of mRNAs generated by a differential splicing in the adult mouse spinal cord. In addition, we have succeeded in cloning cDNAfor human ChAT and demonstrated that it is far larger than those for rat and mouse ones.