Electrophysiological characterization of enteric neurons isolated from the immortomouse

• Main Author: Hawkins, Edward G.
• Format: Others
• Published: VCU Scholars Compass 2011
• Subjects: enteric neurons; cell line; Medical Pharmacology; Medical Sciences; Medicine and Health Sciences
• Online Access: http://scholarscompass.vcu.edu/etd/2367; http://scholarscompass.vcu.edu/cgi/viewcontent.cgi?article=3366&context=etd

The availability of murine genetic models is extremely advantageous to studying gastrointestinal function, but the benefits afforded by studying enteric neurons in mice has been hindered by their accessibility. Fetal (E13) and 2 day post-natal (P2) enteric neuron cell lines (IM-FEN and IM-PEN, respectively) were recently developed from the H-2Kb-tsA58 immortomouse. Our goal was to identify the electrophysiological properties of these cell lines and clarify their utility as a model of enteric neurons. IM-PEN cells stained positively for the neuron specific markers βIII-tubulin and PGP9.5 and were negative for the glial cell marker S100. Detection of mRNA for TRPA1, TRPV1, ClCa1, KCa3.1, NaV1.3 and NaV1.9 were present while CaV2.2 and TASK1 were very faint. No significant difference was observed in the passive membrane properties of IM-FEN and IM-PEN. The cells had depolarized resting membrane potentials -29.8 ± 0.9mV (n=30) and high input resistances ranging from 552 ± 104MΩ (IM-FEN, n=6) to 728 ± 128MΩ (IM-PEN, n=20). xiv In current clamp, hyperpolarizing current was given to obtain a holding potential of -60mV or -80mV, yet neither IM-FEN (n=6) nor the IM-PEN cells (n=20) were able to generate action potentials in response to depolarizing pulses. In whole cell voltage clamp depolarization induced an inward current which was identified as an L-type Ca2+ channel. Niflumic acid inhibited the outward current as well as the tail currents indicating a ClCa current supporting the mRNA data. A volume sensitive chloride channel was also identified that was DCPIB sensitive (n=7) and removed when chloride was replaced with gluconate (n=4), displaying characteristics of ICl,swell. As a result IM-PEN cells had a high chloride conductance resulting in a depolarized membrane potential, which is a characteristic of immature neurons. The transcription factor MASH1 has been found to be required for enteric neuron differentiation. Transfection of MASH1 after 4 and 8 days did not alter the electrophysiological characteristics of IM-PEN (n=6). We conclude that IM-PEN may represent immature enteric neurons and are a useful model to examine the effect of factors required for the development of enteric neurons.