Morphological, immunocytochemical, and functional characterization of esophageal enteric neurons in primary culture

The enteric nervous system of the esophagus plays an important role in its sensory and motor functions. Although the esophagus contains enteric neurons, they have never been isolated and characterized in primary culture. We isolated and cultured enteric neurons of the rat esophagus and determined their morphological appearance, chemical coding for neurotransmitters, and functional characteristics. After primary culture for 2 wk, dendrites and axons appeared in the enteric neurons, which usually have one axon and several dendrites. Although the size of neuronal bodies varied from Dogiel type I to type II, their average size was 39 ± 1.8 μm in length and 23 ± 1.4 μm in width. Immmunocytochemical studies revealed that over 95% of these cells were positively stained for two general neuronal markers, PGP 9.5 or Milli-Mark Fluoro. Chemical coding showed that the neurons were positively stained for choline acetyltransferease (53 ± 6%) or nNOS (66 ± 13%). In functional studies, membrane depolarization and stimulation of several G protein-coupled receptors (GPCRs) induced Ca²⁺ signaling in the esophageal enteric neurons. The GPCR stimulation was found to induce both intracellular Ca²⁺ release and extracellular Ca²⁺ entry. The functional expressions of Ca²⁺ channels (voltage-gated Ca²⁺ channels and store-operated channels) and Ca²⁺ pump (sarcoplasmic reticulum Ca²⁺-ATPase) were also demonstrated on these neurons. We have grown, for the first time, esophageal enteric neurons in primary culture, and these contain excitatory and inhibitory neurotransmitters. The functional integrity of GPCRs, Ca²⁺ channels, and Ca²⁺ pump in these neurons makes them a useful cell model for further studies.