Immunohistochemical and electrophysiological characterization of submucous neurons from the guinea-pig small intestine in organ culture

Immunohistochemical and electrophysiological properties of submucous neurons were investigated in organ cultures of the guinea-pig small intestine. Preparations of submucosa, with or without the myenteric plexus attached, were maintained in vitro for 3 to 5 days. Immunohistochemical labelling for peptides revealed that the cultured submucous plexus remained substantially intact and the immunoreactivity of cell bodies was well preserved. Substantial sprouting of nerve fibers immunoreactive for vasoactive intestinal peptide (VIP) or neuropeptide Y (NPY) was evident in submucous ganglia after 5 days in organ culture. Nerve fibers immunoreactive for substance P, somatostatin, 5-hydroxytryptamine or tyrosine hydroxylase were substantially depleted in submucous ganglia or perivascular nerves at 3 days and had virtually disappeared after 5 days in cultures of isolated submucosa. During intracellular recording from submucous neurons, action potentials were initiated by depolarizing current pulses in all neurons cultured with or without the myenteric plexus and muscle layers. Electrical stimulation of internodal strands evoked fast excitatory synaptic potentials (fast EPSPs) in nearly all neurons whether or not the myenteric plexus was present during the culture period up to 5 days. The removal of myenteric plexus and extrinsic nerves did not abolish fast EPSPs from submucous neurons, suggesting that some fast EPSPs may originate from neurons in the submucous plexus, although the possibility that new synapses formed by sprouting, or surviving axons severed from myenteric or sympathetic ganglia may have been functional, cannot be entirely excluded. This work demonstrates that the immunohistochemical and electrophysiological characteristics of submucous neurons are largely maintained in organ cultures of the submucosa.