A double-labeling method for AChE and fluorescent retrograde tracers

Abstract

Staining for the degradative enzyme acetylcholinesterase (AChE) is an important tool in studying central cholinergic/cholinoceptive systems. AChE staining has also been useful in identifying the projections of AChE-containing neurons and codistribution of AChE with other neurotransmitters. The intensity and opacity of conventional AChE histochemical reaction products, however, pose problems for such double-labeling studies. Here, we have successfully combined a modified version (37) of the Koelle-Friedenwald AChE reaction with retrograde transport of the fluorescent tracer, Fluoro-Gold (FG). By omitting the final intensification steps of the Koelle-Friedenwald reaction, a translucent, light-stable reaction product is created. Viewed under darkfield illumination, this precipitate is of similar intensity and sensitivity to that produced by conventional AChE histochemical processing. Prior administration of an AChE-inhibitor yields preferential staining of AChE-positive neuronal somata. This nonintensified darkfield AChE (NIDA) histochemical method was compatible with visualization of retrogradely transported FG in AChE-positive neurons, allowing unambiguous identification of the projections of AChE-containing neurons.