Synaptic interactions of retrogradely labeled genioglossus motoneurons with substance P-like immunoreactive terminals in the cat: a double labeling immunoelectron microscopic study

The genioglossus muscle (GM) is the principal protruder of the tongue and comprises a major portion of the caudal body of the tongue. The position of the GM is a major determinant of upper airway resistance. Our laboratory is interested in identifying the neurochemical microcircuitry of functionally identified hypoglossal motoneurons. We have found that substance P (SP) makes synaptic contacts with hypoglossal motoneurons that innervate intrinsic tongue muscles [1] in the cat. The purpose of the present study was to investigate whether there are synaptic interactions between motoneurons that innervate the GM and SP-immunoreactive nerve terminals utilizing double labeling immunoelectron microscopy. Cholera toxinB conjugated to horseradish peroxidase (CTB-HRP) was injected into the right GM of three cats. Two days later, the animals were deeply anesthetized and perfused with an acrolein/paraformaldehyde mixture. Cells containing CTB-HRP were labeled with a histochemical reaction utilizing tetramethylbenzidine (TMB) as the chromogen. TMB forms a crystalline reaction product that is very distinct at the electron microscopic level. The tissues were then processed for immunocytochemistry using an antiserum against SP. The chromogen used in this case, diaminobenzidine, yields an amorphous reaction product. At the light microscopic level, retrogradely labeled cells were observed primarily ipsilaterally to the injection site. These neurons were located predominantly in the ventral and ventrolateral portions of the nucleus primarily at the level of the area postrema. At the electron microscopic level, numerous SP-labeled terminals were observed some of which made synaptic contacts with retrogradely labeled GM's. SP terminals formed synapses with retrogradely labeled dendrites and perikarya. SP-labeled terminals also synapsed on unlabeled dendrites. These are the first ultrastructural studies demonstrating synaptic interactions between hypoglossal motoneurons that innervate an extrinsic tongue muscle and SP nerve terminals. These studies demonstrate that motoneurons that innervate the GM are modulated by SP and that SP appears to play a role in the control of tongue movement and GM activity.