Characterization of effector ion channels that mediate excitatory cholinergic modulation of XII motoneurons in neonatal mice in vitro

Abstract

Obstructive sleep apnea is a sleep disorder characterized by repeated stops and starts of breathing during sleep. During an apneic episode there is a decrease in upper airway muscle tone, particularly of the genioglossus muscle (primary tongue protruder). Recent data suggest that this sleep specific loss of muscle tone is due to muscarinic modulation of hypoglossal (XII) motoneurons that innervate genioglossus. In adult rats, during REM sleep, activation of muscarinic acetylcholine receptors depresses genioglossus inspiratory output. By contrast, in neonatal mouse brainstem slices, muscarine potentiates inspiratory output when locally applied to the XII motor nucleus. To determine whether these different effects of muscarine are due to postnatal maturation, we first need to determine the effector ion channels that mediate the excitatory response to muscarine in neonatal mice. In individual XII motoneurons recorded from neonatal brainstem slices, muscarinic receptor activation inhibited an apamin‐sensitive small conductance calcium dependent potassium current (SK) and potentiated a hyperpolarized‐activated mixed cation current (Ih). It was hypothesized that both effector ion channels contribute to the net potentiation of inspiratory drive by muscarine in neonatal mice. Therefore, we aimed to determine 1) the effect of inhibition of SK, and 2) the contribution of Ih modulation to the modulation of inspiratory drive by muscarinic acetylcholine receptor activation at XII motoneurons in neonatal mice. Using rhythmically medullary slice preparations from neonatal CD1 mice (postnatal day P0–5), we first investigated the effects of blocking SK on inspiratory burst output. Local application of apamin (30s) to the XII motor nucleus had a net effect of increasing inspiratory burst amplitude (n=3) to 134% of baseline (range 118–143%) or 132% (range 111–169%) of baseline (2 and 20 mM respectively). These data are important to establish the contributions of SK to inspiratory burst amplitude before testing how muscarine may modulate SK during inspiratory bursts. We then confirmed that muscarine had a net excitatory effect on inspiratory burst amplitude in neonatal mice (128 ± 6% of baseline, n = 9). Next, we aimed to investigate if muscarinic excitation is mediated by blocking SK channels. In one P3 preparation, local application of apamin attenuated the muscarinic‐mediated increase in XII inspiratory burst amplitude from 157% to 137%. Lastly, in one P2 brainstem slice preparation, contrary to our hypothesis, blocking Ih with ZD7288 caused the potentiation of inspiratory burst amplitude by muscarine to increase from 130% to 166%. Future experiments will be needed to confirm these results. Our next steps will focus on characterization of developmental changes in muscarinic modulation of XII motoneuron inspiratory burst output.