Histological staining of pre- and postsynaptic components of amphibian neuromuscular junctions

We have developed a combined staining technique whereby pre- and postsynaptic structures of the neuromuscular junction can be simultaneously visualized in the light microscope. The general approach was first to stain presynaptic nerve terminals using nitroblue tetrazolium (NBT), which when reduced to its diformazan state coloured the entire nerve terminal arborization blue. When the NBT stain was combined with the Karnovsky acetylcholinesterase (AChE) procedure the blue-coloured nerve terminal processes were vividly outlined by the brown copper ferrocyanide-AChE reaction product. Experiments were performed to ensure that the NBT AChE method stained nerve reliably and that even extremely small neural processes were stained and visualized in the light microscope. This method stained cholinergic neuromuscular junctions and unmyelinated axons in a variety of preparations. Aldehyde pre-and poststaining fixation markedly affected the quality of NBT nerve stain. In addition, glutaraldehyde had a direct role in the staining process. The quality of the nerve terminal staining was affected by the pH and the constituents of the staining solution. The powerful experimental advantage of the NBT-AChE stain for neuromuscular junctions resulted from the sharp colour contrast which made possible accurate determinations of the relationship between the presynaptic nerve terminal arborization and the postsynaptic junctional AChE activity.