Goedert M, Hunt S P
MRC Laboratory of Molecular Biology, Cambridge, U.K.
Neuroscience. 1987 Sep;22(3):983-92. doi: 10.1016/0306-4522(87)92974-5.
The cellular distribution of preprotachykinin A messenger RNA in the bovine nervous system was investigated by in situ hybridization and its tissue distribution by Northern and dot blotting. The latter results were compared with the levels of substance P-like immunoreactivity as determined by radio-immunoassay. The highest levels of preprotachykinin A messenger RNA were found in striatum and trigeminal ganglion, medium levels in retina and lower levels in hypothalamus, spinal cord, pituitary gland and adrenal medulla. The cellular localization of preprotachykinin A messenger RNA was obtained in striatum and trigeminal ganglion using either single-stranded DNA or complementary RNA probes labelled with 32P, 35S or 3H. Specific labelling of small trigeminal ganglion neurones and of medium-sized striatal nerve cells was observed with probes in the anti-messenger RNA sense orientation. Only background labelling was obtained with probes in the messenger RNA sense orientation. The technique was further validated by the demonstration that the same cells in the trigeminal ganglion were labelled by both in situ hybridization and immunohistochemistry. The present findings allow an unambiguous identification of the cellular sites of synthesis of preprotachykinin A messenger RNA; in situ hybridization should also prove a useful technique for investigating the regulation of neuropeptide biosynthesis at the cellular level.
通过原位杂交研究了前速激肽原A信使核糖核酸在牛神经系统中的细胞分布，并通过Northern印迹法和斑点印迹法研究了其组织分布。将后一种结果与放射免疫测定法测定的P物质样免疫反应水平进行了比较。在前速激肽原A信使核糖核酸水平方面，纹状体和三叉神经节中含量最高，视网膜中含量中等，下丘脑、脊髓、垂体和肾上腺髓质中含量较低。使用用32P、35S或3H标记的单链DNA或互补RNA探针，在前速激肽原A信使核糖核酸的细胞定位方面，获得了纹状体和三叉神经节中的定位结果。用反义信使核糖核酸方向的探针观察到了三叉神经节小神经元和中等大小纹状体神经细胞的特异性标记。用正义信使核糖核酸方向的探针仅获得了背景标记。通过证明原位杂交和免疫组织化学标记三叉神经节中相同的细胞，进一步验证了该技术。目前的研究结果能够明确鉴定前速激肽原A信使核糖核酸的细胞合成位点；原位杂交也应证明是一种在细胞水平上研究神经肽生物合成调控的有用技术。
