Neuronal muscarinic acetylcholine receptor subtype modulation of inspiratory bursting at hypoglossal motoneurons in developing neonatal mice in vitro
Authors: Dudley Sydney , Banayat Tatum , Kurup Asha , Jeong Kellie , Koziol Sophia , Buhlman Lori , Revill Ann 
Published: May 2023

Muscarinic acetylcholine receptor modulation contributes to changes in hypoglossal motoneuron excitability through pre- and post-synaptic effects, an important mechanism for regulation of airway tone during sleep. Previous data indicate a net excitatory effect of muscarinic modulation of inspiratory bursting in neonatal mouse brainstem slices, yet a net inhibitory effect on inspiratory bursting in adult rats. We tested the hypothesis that a developmental shift in muscarinic modulation impacts hypoglossal motoneurons, using neuroanatomical and electrophysiological methods. Immunofluorescence confirmed hypoglossal motoneurons express M1, M2, M3, and M5 muscarinic subtypes. We observed maturational decreases in M1 and M5 labeling intensity, M3 labeling intensity generally decreased with postnatal (P) age, although there was a transient increase in expression at P17. M2 labeling intensity did not change, although distribution of M2 labeling with postnatal maturation may change. Next, using the in-vitro rhythmic slice preparation from CD1 mice of either sex, local application of muscarine (100 μM) into the hypoglossal nucleus was tested in mice P0-14. The net effect of applying muscarine results in excitatory output after local application into the rhythmic slice because each data point is a positive percent change from control. The magnitude of this potentiation appears to lessen as the mice age toward P14. To evaluate the mechanisms through which the magnitude of muscarinic potentiation of inspiratory bursting changes with postnatal maturation, we next assessed the role of excitatory and inhibitory muscarinic receptor subtypes. Blocking M1 locally (pirenzepine, 100 μM) significantly decreased muscarine-mediated potentiation of inspiratory bursting (to 72±14% of control muscarine response, n=9, P0-5). Preliminary data indicate bath application of 4-DAMP (M3 antagonist) attenuated muscarinic potentiation of inspiratory bursting (range: to 7%-25% of control burst response, n=3, P0-5). Modulating M5 locally with a positive allosteric modulator VU 0238429 (1000 μM) had little effect on burst amplitude (119±4% of control burst response, n=3, P0-6). Blocking M2 locally (methoctramine, 2μM) showed no effect on inspiratory burst potentiation after applying muscarine (181±65% vs 187±69%, n=7, P0-5). Bath application of methoctramine did not have a significant effect on inspiratory burst potentiation by muscarine (179±65% vs control 182±90%, n=10) in P0-5 mice while preliminary analysis suggests an increasing effect of blocking M2 with postnatal maturation (147±56% vs control 134±30%, n=4, P9-14). These data support excitatory muscarinic receptor subtypes contributing to the excitatory response early in postnatal maturation. Based on our neuroanatomical data, we speculate the increasing inhibitory response to muscarinic modulation with postnatal maturation is due to a decreased contribution of excitatory muscarinic receptor subtypes. NIH R15HL148870 (Revill) and NIH/NHLBI R25 HL126140 (Moreno, Garcia, Parthasarathy) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.