Characterizing the Contribution of Muscarinic Receptor Subtypes to Inspiratory Bursting Activity at Hypoglossal Motor Neurons of CD1 Mice.

Obstructive sleep apnea (OSA) is characterized by cessation of breathing during sleep, due to repetitive episodic collapse of the airway or increased airway resistance. This loss of airway patency during sleeping conditions is controlled in part by the tongue, innervated by the hypoglossal (XII) motor nucleus. Decreased excitability of XII motor neurons (MNs) that innervate the tongue may occur due to activation of muscarinic acetylcholine receptors acting through G-protein coupled signal transduction pathways. The five types of muscarinic acetylcholine receptors can be broadly categorized as excitatory or inhibitory. M1, M3, and M5 receptors are excitatory receptors coupled to Gq, whereas M2 and M4 receptors are inhibitory receptors coupled to Gi. XII MNs express M1, M2, M3, and M5 subtypes of muscarinic receptors, but the specific mechanisms of how cholinergic signaling modulates excitability of XII MNs are not well understood. The objective of this study was to investigate how the muscarinic receptor subtypes individually contribute to inspiratory activity at XII MNs. We tested the hypothesis that activation of M1 and M5 receptors potentiate, whereas activation of M2 receptors inhibit inspiratory bursting in XII MNs. Using medullary rhythmic slice preparations from neonatal CD1 mice (postnatal day P0-5) local application of muscarine (30s, 100 μM) to the XII motor nucleus increased inspiratory burst amplitude to 180 ± 7% [n = 23]. Blocking M1 receptors locally with pirenzepine (10 μM & 100 μM) decreased the muscarine-mediated potentiation of inspiratory bursting to 94 ± 15% and 72 ± 14% of control muscarine response [n = 9]. Activating M1 receptors locally with cevimeline (1 mM) for 30s and 60s increased burst amplitude to 116 ± 7% and 117 ± 7% of control muscarine response [n = 6]. Blocking M2 receptors locally with AF-DX 116 (1 μM & 10 μM), the muscarine-mediated effect was 101 ± 10% [n = 3] and 95 ± 10% [n = 3] of control muscarine-mediated excitation. M2 receptor blockade was also tested by application of the M2 receptor antagonist, methoctramine (MTH). Local application of MTH (2 μM) had no effect on muscarine-mediated potentiation of inspiratory bursting (171 ± 15% compared to control muscarine response of 177 ± 14% [n = 10]). Similarly, blocking M2 receptors with bath application of MTH (200 nM) had no effect on muscarine-mediated potentiation of inspiratory bursting (166 ± 20% compared to 161 ± 14% control muscarine response) or on inspiratory burst period (10 ± 0.3% compared to 10 ± 0.4% control muscarine response) [n = 5]. Modulating M5 receptors locally with VU 0238429 (20 μM and 1000 μM) increased burst amplitude to 116% ± 5% and 119 ± 4% of control muscarine-mediated excitation [n = 3]. Our data partially support a role of M1 receptors contributing to the muscarinic potentiation of inspiratory bursting in XII MNs. Future research will evaluate other muscarinic receptor agonists and antagonists, as well as determine how the muscarinic modulation of inspiratory bursting changes with postnatal maturation.