.MU.-Opioid and N-Methyl-D-aspartate Receptors Are Localized at Laryngeal Motoneurons of Guinea Pigs

Abstract

Behavior of coughing is produced by combination of a series of laryngeal and respiratory muscle contraction. Movements of laryngeal muscles are governed by laryngeal motoneurons via two main efferent nerves.1) The superior laryngeal nerve (SLN) innervates the cricothyroid muscle, and the recurrent laryngeal nerve innervates the posterior cricoarytenoid, thyroarytenoid, lateral cricoarytenoid and interarytenoid muscles. The cricothyroid muscle contracts in concert with the posterior cricoarytenoid muscle and can facilitate laryngeal opening during coughing. 1,2)It has been reported that opioids including morphine, codeine, endomolphin-1 and 2 inhibit occurrence of cough reflex in cats, 3) guinea pigs 4,5) and mice. 6) Moreover, blockade of the N-methyl-D-aspartate (NMDA) receptors effectively suppresses cough reflex in rats, 7) rabbits 8,9) and cats. 10)These results suggest that m-opioid and NMDA receptors play important roles in generation of cough reflex, by which these mechanisms act in the central regions such as the nucleus tractus solitarius (NTS) and the cough/respiratory neuron network. 5,10,11) However, whether such mechanisms are directly involved in cough-related efferent signals is not clarified yet. The present study focused on modulation of the activity of laryngeal motoneurons (LMNs) providing efferent fibers to the SLN by m-opioid and NMDA mechanisms. The LMNs were identified immunohistochemically by application of the fluorescent wheat-germ agglutinin (WGA) conjugate, and then double staining was undertaken with antibodies to either m-opioid or NMDA receptors. MATERIALS AND METHODS Preparation of SlicesThis study was conducted in accordance with Guiding Principles for the Care and Use of Laboratory Animals approved by The Japanese Pharmacological Society. Eight guinea pigs (Hartley strain, 400-700 g body weight) were anesthetized with ketamine and xylazine (80 and 8 mg/kg, intramuscularly). The right SLN was dissected free from the surrounding tissue and the proximal central cut end of the nerve was soaked in 3 ml of 5% WGA-Fl (WGA-Alexa Fluor 488 conjugate, Molecular Probes, Eugene, OR, U.S.A.) in saline for 90 min. After 24 to 48 h, the animals were anesthetized deeply with ketamine and xylazine, and perfused transcardially with 200 ml of physiological saline containing heparin (5 U/ml), followed by 150 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brainstem was dissected, post-fixed for 60 min, and placed in 10 mM phosphate buffered saline (PBS, pH 7.4) at 4°C. Serial coronal slices (50 mm thickness) were cut with a vibratome.Immunoreactivity of m m-Opioid and NMDA Receptors Free floating sections were incubated in PBS containing 0.2% Tween 20 for 20 min at room temperature, washed three times with PBS for 5 min and incubated in a blocking buffer containing 10% of goat normal serum diluted with PBS solution for 10 min. Tissue sections were then incubated at 4°C for 18 h with following a primary antibody: either rabbit polyclonal antibody raised against the 3rd extracellula...

The pattern and extent of retrograde transsynaptic transport of WGA-Alexa 488 in the phrenic motor system is dependent upon the site of application

“…WGA-Alexa tracers are well established (Panchuk-Voloshina et al, 1999), are exceptionally bright and are excellent for tracing axonal pathways in the central nervous system (Reeber et al, 2011; Yamazaki et al, 2009; Panchuk-Voloshina et al, 1999). Both anterograde (Reeber et al, 2011) and retrograde (Yamazaki et al, 2009) track tracing studies have been conducted with WGA-Alexa 488.…”

“…Both anterograde (Reeber et al, 2011) and retrograde (Yamazaki et al, 2009) track tracing studies have been conducted with WGA-Alexa 488. Transneuronal/transsynaptic transport of WGA-Alexa 488, however, has never been demonstrated in vivo .…”