Atypical localization of myenteric neurons in the opossum lower esophageal sphincter

This study investigated sphincter-body differences in neuronal density and morphometry between the esophageal sphincter and body with a view to determining whether previously reported differences are authentic. The anatomical limits of the opossum lower esophageal sphincter were correlated with its physiological behavior by manometric demarcation. Following this, peeled whole mounts and paraffin and cryosections were used to study the morphology and morphometry of the esophageal myenteric plexus. Thirty animals were used and seven quantitated. The plexus of the esophageal body was located as usual in a plane between the longitudinal and circular muscle, which coincided with the plane of cleavage when these muscle layers were peeled apart for studying the plexus in whole mounts. In contrast, the plexus was located in several planes in the lower esophageal sphincter, which had no cleavage plane. Therefore, peeling the sphincter removes neurons and yields falsely low counts, making peel preparations of this region unsuitable for neuronal quantitation. In paraffin sections, the neuron density in the esophageal body 7 cm above the sphincter was 6,353 +/- 850/cm2, but decreased significantly to 2,254 +/- 353/cm2 at the 1-cm segment. In the lower esophageal sphincter, the neuronal count increased again to 8,530 +/- 1,606/cm2. Flash-frozen cryosections, which produced neuronal morphology similar to the in vivo condition, showed that there was no difference in neuronal size between esophageal body and sphincter. These studies show that atypical myenteric plexus localization causes spuriously low neuronal counts reported in the lower esophageal sphincter and that reported neuronal size differences are technique-dependent.