Cholinergic Modulation by Activation of Muscarinic Acetylcholine Receptor Subtypes Modulates Inspiratory Bursting in Hypoglossal Motoneurons in Mice in Vitro

Abstract

During wakefulness, tonic and respiratory input provides drive to upper airway dilatory muscles, and in particular genioglossus, the main tongue protruder, which stiffens the airway and keeps airway resistance low. These respiratory muscles experience decrements in activity during rapid eye movement sleep. Disruption in motor output and insufficient muscle tone of the tongue due to inhibition, or loss of excitation, of hypoglossal motoneurons (which innervate tongue muscles) contributes to sleep-disordered breathing. Recent data from adult rats in vivo suggest that muscarinic inhibition of hypoglossal motoneurons may be a major mechanism leading to loss of tongue muscle tone during sleep. However, available data indicate that there are contrasting effects of muscarinic modulation: in neonatal rhythmic slice preparations, muscarine applied within the hypoglossal nucleus has an excitatory effect on inspiratory burst amplitude. Muscarinic receptor subtypes can be broadly divided into those that are coupled to excitatory G-protein coupled receptors (M1, M3, M5) and those that are coupled to inhibitory G-protein coupled receptors (M2, M4). Hypoglossal motoneurons express M1, M2, M5, and possibly M3 receptor subtypes. Many modulatory systems undergo postnatal maturation, which we hypothesize underlies the switch in muscarinic modulation from net excitation to net inhibition of inspiratory bursting behavior at hypoglossal motoneurons. As an initial test of our hypothesis, our goal was to determine the contribution of excitatory and inhibitory muscarine receptor subtypes to the muscarinic modulation of inspiratory burst output from hypoglossal motoneurons in neonatal mice. Using the in vitro rhythmic slice preparation, we first investigated inspiratory behavior by observing the net effects of muscarine locally applied into the hypoglossal nucleus (100 mM, 30 s) on inspiratory burst amplitude in neonatal CD-1 mice (postnatal day, P0-P5). Our data confirm that in mice ages P0-5, local application of muscarine increased inspiratory burst amplitude (range: 53-180% potentiation, n = 9) compared to control inspiratory burst activity. Preliminary results suggest that the local application of M2 antagonist methoctramine in addition to muscarine (range: 20%-135% potentiation, n=4) had little effect on inspiratory behavior compared to baseline effects of muscarine (range: 4%-143% potentiation). Next, we tested the contribution of M3 receptors to muscarinic modulation on inspiratory burst amplitude. At baseline, locally applied muscarine increased inspiratory burst amplitude (range: 20%-63% potentiation); the muscarinic potentiation was attenuated during bath application of the M3 receptor-preferring antagonist 4-DAMP (range: 17%-25% potentiation) and recovered upon washout (range: 13%-117% potentiation, n=3). These preliminary results indicate muscarinic excitation is mediated in part by the M3-receptor subtype. Future experiments will determine how the M2 and M3 receptor contributions change with postnatal maturation.