Neuroimaging of human myenteric neurons v1

This protocol is to investigate the sensitivity of human colonic myenteric neurons to chemical and electrical stimulation using ultrafast neuroimaging technique with the voltage sensitive dye Di-8-ANEPPS. We compared tissue samples from different colonic sub regions. Tissue samples were taken during surgery (Medical school of Hannover, Germany), placed in ice-cold oxygenated Krebs solution for preparation and immediately transferred to our laboratory. Tissues were dissected in ice-cold oxygenated Krebs solution for preparation to obtain whole-mount myenteric plexus preparations. Samples were used for ultrafast neuroimaging technique combined with a voltage sensitive dye (VSD). In order to stain the ganglia, VSD Di-8-ANEPPS (20-40 μM) was applied directly into the ganglion at moderate pressure (≤ 0.5 bar). Ganglia were then stimulated by applying 100 μM nicotine or 1,000 serotonin on the surface of a ganglion. Electrical stimulation was performed with a self-designed platinum wire electrode with a tip diameter of 75 μm, placed onto interganglionic neuronal fiber tracts, applying 180 μA single-pulses for 0.5 ms. The preparations were examined with an inverted microscope equipped with an appropriate filter set. Pictures were acquired with a CMOS camera connected to a computer and controlled by Turbo SM 64 Software.