Projections of submucosal neurons to the myenteric plexus of the guinea pig intestine: in vitro tracing of microcircuits by retrograde and anterograde transport

The enteric nervous system (ENS) can mediate reflex activity without input from the brain or spinal cord. The ENS thus contains intrinsic primary afferent neurons that link mucosal sensory receptors with motor neurons in the myenteric plexus. The intrinsic primary afferent neurons of the gut have not yet been identified. Although the submucosal plexus is known to innervate the mucosa, where enteric sensory receptors are located, no submucosal to myenteric projections have previously been found. In order to determine whether such projections exist, the submucosal plexus was examined following the microinjection of a retrograde tracer (Fluoro-Gold or 4-acetoamido, 4'-isothiocyanostilbene-2,2'-disulphonic acid [SITS]) into single myenteric ganglia. In addition, the myenteric plexus was studied following the iontophoretic injection of an anterograde tracer (Phaseolus vulgaris leucoagglutinin; [PHA-L]) into single submucosal ganglia. Ganglia were visualized by use of differential interference contrast optics and were injected from the beveled tip of a glass micropipette; 2.5-3.0 hours were allotted for retrograde and 20-24 hours (under culture conditions) for anterograde transport. In the myenteric plexus, a small number of the neurons of each injected ganglion were fluorescent and additional neurons in distant myenteric ganglia (predominantly orad) were also retrogradely labeled. About five to six submucosal neurons deep to but not directly underneath the injected myenteric ganglion were labeled by Fluoro-Gold or SITS and only rarely was there more than one labeled neuron in a submucosal ganglion. When control injections of Fluoro-Gold were placed into the muscle instead of a ganglion, some myenteric neurons near the injection site became labeled indicating an innervation of the circular muscle by myenteric neurons; however, there was no labeling of neurons in the submucosal plexus. Similarly, if connections between the myenteric and submucosal plexuses were severed before injecting Fluoro-Gold, no submucosal neurons were labeled. Following injection of PHA-L into a single submucosal ganglion, small-diameter axons were labeled in approximately 2 myenteric ganglia as well as in several distant submucosal ganglia (mainly anal and circumferential to the injection site). Additional labeled fibers traveled with blood vessels or surrounded mucosal crypts. It is concluded that submucosal neurons project to the myenteric plexus as well as to the mucosa and to one another. These observations are consistent with the hypothesis that at least some intrinsic enteric primary afferent neurons reside in the submucosal plexus.