Genioglossal hypoglossal muscle motoneurons are contacted by nerve terminals containing delta opioid receptor but not mu opioid receptor-like immunoreactivity in the cat: a dual labeling electron microscopic study

Abstract

This study has investigated (1) the distribution of delta opioid receptor (DOR) or mu opioid receptor (MOR) containing elements in the hypoglossal nucleus of the adult cat; and (2) the association of these processes with retrogradely labeled genioglossus muscle motoneurons. Cholera toxin B conjugated to horseradish peroxidase (CTB-HRP) was injected into the genioglossus muscle on the right side of four isoflurane-anesthetized cats. Forty-four to 52 h later, the animals were sacrificed. Motoneurons containing HRP were labeled with a histochemical reaction utilizing tetramethylbenzidine (TMB) as the chromogen. The tissues were then processed for immunocytochemistry, using an antiserum raised against DOR or MOR using diaminobenzidine (DAB) as the chromogen. At the light microscopic level, retrogradely labeled cells were observed primarily ipsilaterally in ventral and ventrolateral subdivisions of the hypoglossal nucleus. The majority of these labeled cells were observed immediately caudal to obex. DOR-like immunoreactive processes were apparent at the light microscopic level in the hypoglossal nucleus, but MOR-like immunoreactive processes were not. Both DOR and MOR-like immunoreactive processes were observed in other brainstem areas such as the spinal trigeminal nucleus. At the electron microscopic level, DOR-like immunoreactive nerve terminals formed synaptic contacts with retrogradely labeled genioglossus muscle motoneuronal dendrites and perikarya in the hypoglossal nucleus. Nineteen (19) percent of the DOR terminals contacted retrogradely labeled genioglossus muscle motoneurons. DOR-immunoreactive terminals also synapsed on unlabeled dendrites and somata. Few MOR-like immunoreactive terminals were found at the EM level in the hypoglossal nucleus, and none of these terminals contacted retrogradely labeled neuronal profiles from the GG muscle. These are the first ultrastructural studies demonstrating synaptic interactions between functionally identified hypoglossal motoneurons and DOR terminals, and that enkephalins most likely act presynaptically to modulate the release of other neurotransmitters that affect GG motoneuron activity. These studies demonstrate that hypoglossal motoneurons which innervate the major protruder muscle of the tongue, the genioglossus muscle, are modulated by terminals containing DOR, and that enkephalins acting on DOR but not MOR in the hypoglossal nucleus may play a role in the control of tongue protrusion.

Introduction

Tongue muscles are under the direct control of the hypoglossal nerve (the twelfth cranial nerve). The hypoglossal nerve originates from the hypoglossal nucleus. It has been shown in several species that hypoglossal motoneurons are myotopically arranged [5], [12], [14], [18], [19], [21].

Nerve terminals containing enkephalins have been described in the hypoglossal nucleus [1], [9]. All regions of the hypoglossal nucleus contained both leu- and met-enkephalin; however, density was greatest in the ventral subcompartments where protrusor muscle motoneurons are located. It is not known which opiate receptor subtype(s) the enkephalins act upon in the hypoglossal nucleus, nor is it known whether the enkephalins act pre- or postsynaptically. There are five major subtypes of opiate receptors: the mu receptor, delta receptor, kappa receptor, epsilon receptor, and sigma receptor. Opiates have been long known as respiratory depressants in most species. Systemic administration of opiates such as morphine produces a profound decrease in hypoglossal nerve activity in the cat [6]. In the present study, we have used a modified double-labeling electron microscopic method [13] to retrogradely label hypoglossal motoneurons that selectively innervate the genioglossus muscle while simultaneously utilizing immunocytochemistry to visualize central nervous system afferents containing either the delta opioid receptor (DOR) or the mu opioid receptor (MOR). With this technique, one could also ascertain whether the receptors are found in postsynaptic elements as well. The focus of the present study is to determine the location of DOR and MOR within the hypoglossal nucleus and determine the relationship between these processes with genioglossus muscle motoneurons. A preliminary report of these findings has been previously published [16].