Enteric neurons of the adult mouse; successful isolation through immunoselection and immunocytochemical and electrophysiological characterization

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The enteric nervous system (ENS) is often referred to as the ‘second brain’ and plays an essential role in gastrointestinal peristalsis. However, isolation of individual neurons from adult mice has proven difficult. In this study we isolated mouse enteric neurons by immunoselection. Neurons from digested longitudinal muscle myenteric plexus preparations were labeled with an extracellular p75NTRantibody coupled to modified biotin and selected by streptavidin-coated magnetic beads. Cells were cultured for up to 2 weeks in modified Neurobasal A media. All neurons positively stained for the neuronal marker βIII-tubulin, and many grew in clusters. Immunocytochemistry revealed positive membrane staining for the μ opioid receptor and β-arrestin 2. Select neurons showed positive staining for choline acetyltransferase and weak nitric oxide synthase immunoreactivity. Electrophysiological studies of these neurons suggest at least two distinct neuronal populations. One population displays strong outward rectifying 4-aminopyridine-sensitive K+ currents. In a second set of neurons, current injection elicited action potentials. Robust Na+ currents are seen in tandem with these regenerative action potentials. We have developed a methodology for the successful isolation and study of viable adult mouse enteric neurons for systematic electrophysiological investigation. Supported by NIH DA024009.