Simultaneous demonstration of catecholamines and acetylcholinesterase in peripheral autonomic nerves

Certain primary catecholamines remain well localized during brief fixation with cold, buffered formalin. In the present study, the diffusibility of catecholamines was further explored, using guinea‐pigs and cats pretreated with Nialamide and norepinephrine. Preliminary experiments showed that catecholamines were well localized after perfusion fixation with buffered formalin. Furthermore, catecholamines remained in situ in tissues immersed in cold fixative or phosphate buffer for as long as six hours. It seemed, therefore, that catecholamines might remain localized in fixed tissue, during incubation for acetylcholinesterase. To test this hypothesis, formaldehyde‐fixed, cryostat sections were incubated in a cold thiocholine medium for two to four hours to demonstrate acetylcholinesterase activity. They were then exposed to formaldehyde gas to demonstrate catecholamines. Acetylcholinesterase‐positive and catecholaminecontaining nerve fibers were identified simultaneously in the same section under mixed ultraviolet and red light. When a dark‐field condenser was used, adrenergic nerves appeared green and acetylcholinesterase‐positive nerves red on a black background. We were able to demonstrate adrenergic presynaptic terminals on cholinergic myenteric ganglion cells and acetylcholinesterase‐positive terminals close to cardiac chromaffin cells. We also have observed contiguities between peripheral adrenergic and cholinesterase‐positive nerves.