Appositions made by axons of descending interneurons in the guinea-pig small intestine, investigated by confocal microscopy

Abstract

There are four major classes of descending interneurons in the myenteric plexus of the guinea-pig small intestine. In this study, the connections made by two of these classes of descending interneurons with other interneurons and with inhibitory motor neurons have been investigated using confocal, conventional fluorescence and electron microscopy. The terminals of descending interneurons known to contain both bombesin (BN) and nitric oxide synthase (NOS) were identified by BN immunoreactivity (IR). Cholinergic interneurons known to contain somatostatin (SOM) were identified by SOM-IR. The connections of these two groups of interneurons with the following three classes of nerve cell bodies were examined: those with NOS-IR that also contain γ-aminobutyric acid (GABA) (inhibitory motor neurons), those with only NOS-IR (descending interneurons and inhibitory motor neurons) and those with only GABA-IR (motor neurons). The BN-IR and SOM-IR interneurons were found to form connections with each other, and both types of interneurons provided inputs to motor neurons. Most previous analyses of interconnections in the enteric plexuses have been by conventional fluorescence microscopy and electron microscopy. In the present work these are compared with confocal microscopy. BN-IR varicosities formed pericellular baskets around each class of nerve cell that were easily identifiable with all techniques. Using confocal microscopy, BN-IR varicosities that were in contact with NOS-IR and GABA-IR nerve cells were quantified. Confocal microscopy demonstrated over twice as many contacts as were shown by a previous electron microscopic study. In contrast, conventional fluorescence microscopy showed little indication that SOM-IR varicosities formed inputs to NOS-IR or GABA-IR nerve cells, despite the fact that confocal microscopy revealed direct appositions and electron microscopy revealed synapses. This study has shown that confocal analysis is a valuable adjunct to conventional fluorescence microscopy for determining neuronal circuitry. Moreover, it allows a more rapid collection of data than does electron microscopy. It is concluded that chains of BN-IR and SOM-IR interneurons form descending pathways in the small intestine and that both types of interneuron connect with muscle motor neurons.