Immunohistochemical localization of monoamines and cyclic nucleotides. Their application in quantitative immunofluorescence studies and tracing monoaminergic neuronal connections

Abstract

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.