Heterogeneity of ganglia of the guinea pig myenteric plexus: an in vitro study of the origin of terminals within single ganglia using a covalently bound fluorescent retrograde tracer

Abstract

Experiments were done to test the hypothesis that individual ganglia of the myenteric plexus of the guinea pig small intestine are heterogeneous with respect to the location of the neurons that provide terminals to them. The myenteric plexus, attached to the longitudinal layer of smooth muscle, was maintained in vitro. Individual ganglia were injected with a variety of potential retrograde tracers by pressure microejection from the tip (20-micron diameter) of a glass micropipette. The fluorescent dye 4-acetoamido, 4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS) was found to be an effective tracer, labeling neuronal perikarya, evidently by retrograde transport. SITS has previously been shown not to cross plasma membranes, but to be covalently bound to the outer surface of that membrane, and to be taken up by nerve terminals to be retrogradely transported to label neuronal cell bodies. SITS fluorescence was found in about 12% of the neurons within the ganglion into which it was injected and also in approximately ten times more neurons in discretely located distant ganglia. No labeling of neurons was found when SITS was injected into the bath or into the smooth muscle below the myenteric plexus. Damage to neural connectives obstructed the labeling of neurons in ganglia distal to the injection site. Individual SITS-injected myenteric ganglia were found to vary greatly in the ratios of intraganglionic SITS-labeled neurons to the total number of neurons within the injected ganglion. The ratios of the number of intraganglionic SITS-labeled neurons to SITS-labeled neurons in distant ganglia projecting to the injected ganglion from elsewhere in the myenteric plexus also varied greatly. More strikingly, individual ganglia differed over a wide range with respect to whether the neurons in distant ganglia that provided them with terminals were situated in the oral, anal, or circumferential direction. Although the majority of projections were found to be from orally located ganglia, individual ganglia were observed that received predominantly or exclusively anal or oral projections. Others received mixtures of terminals from ganglia that were anal, oral, or circumferential. This anatomical heterogeneity in the location of afferent inputs to individual myenteric ganglia is probably reflected in a functional heterogeneity as well and will have to be taken into account in further studies of the physiology of the myenteric plexus. Individual ganglia of the plexus can no longer be taken as anatomically and functionally equivalent to one another.