Tsuji_2002_Brain.Res.Brain.Res.Protoc_9_16

Fresh myenteric plexus-longitudinal muscle preparations were dissected by cleavage of the ileum of guinea pigs. The plasma membrane remained impermeable to lipophobic histochemical agents and the perineuronal surface (non-somatic) acetylcholinesterase (AChE) activity of the neural network was exclusively stained by a modified Karnovsky's histochemical medium. The cleaved face of the myenteric plexus was directly exposed to the exterior and could be clearly seen with scanning electron microscopy. A high contrast image of the neural network in three-dimensional space was observed by backscattered electron imaging (BSEI) of the histochemical precipitate which was produced on the perineuronal surface. The Cu and Fe of the histochemical precipitate (cupric ferrocyanide) were detected by energy dispersive X-ray analysis (EDXA). Elementary X-ray mapping of Cu and Fe on whole specimens provided images of the histochemical precipitate which were completely superposable to those of the BSEI, or even finer than those of the BSEI. The present results confirm the presence of Karnovsky's reaction on the perineuronal surface of the neural plexus, and suggest a useful method for studying this extraneuronal AChE in neural networks.