Steinbusch H W, Wouterlood F G, de Vente J, Bol J G, Berkenbosch F
Department of Pharmacology, Faculty of Medicine, Free University of Amsterdam, The Netherlands.
Acta Histochem Suppl. 1988;35:86-106.
We have described immunocytochemical methods for the direct visualization of monoamine- and cGMP-containing neurons, using antibodies to the primary or secondary messengers themselves. The specificity and affinity of these antibodies were determined using quantitative immunofluorescence of gelatin models, as non-biological models, to which the native substances and their possibly cross-reacting compounds were incorporated. The use of retro- and anterograde tracers both in conjunction with immunohistochemical procedures provides the possibility to characterize transmitter specifically afferent and efferent pathways. With the presented double-label immunocytochemical procedures it is feasible to visualize and trace a projection between two brain areas, and in addition to specify the transmitters which are involved in their afferents and efferents and their target neurons and neurons of origin. Using the SCG, as a biological model, we could demonstrate that cGMP levels in individual cells could be elevated by cholinergic agonists. Moreover, using quantitative immunofluorescence of cGMP as a post- or presynaptic marker we have the possibility to measure the post-/presynaptic effects of monoamines or neuropeptides in individual neurons.
我们已经描述了免疫细胞化学方法，用于直接可视化含单胺和cGMP的神经元，该方法使用针对一级或二级信使本身的抗体。这些抗体的特异性和亲和力是通过使用明胶模型（作为非生物模型）的定量免疫荧光来确定的，在明胶模型中掺入了天然物质及其可能发生交叉反应的化合物。逆行和顺行示踪剂与免疫组织化学程序结合使用，为特异性表征递质传入和传出途径提供了可能性。通过本文介绍的双标记免疫细胞化学程序，可以可视化并追踪两个脑区之间的投射，此外还能确定参与其传入和传出的递质以及它们的靶神经元和起源神经元。以颈上神经节（SCG）作为生物模型，我们能够证明胆碱能激动剂可提高单个细胞中的cGMP水平。此外，使用cGMP的定量免疫荧光作为突触后或突触前标记，我们有可能测量单个神经元中单胺或神经肽的突触后/突触前效应。
