Evaluating changes in hypoglossal motoneuron muscarinic acetylcholine receptor expression during mouse postnatal maturation

The decrease of upper airway patency observed during rapid eye movement sleep is in part due to loss of tongue muscle tone. This transient atonia can be attributed partially to inhibitory muscarinic activity at hypoglossal motoneurons (XII MNs). There are multiple muscarinic acetylcholine receptor (mAChR) subtypes found at XII MNs: excitatory subtypes M1, M3, M5 and inhibitory subtype M2. While mAChR activation at XII MNs in adult rats (in vivo) has a net inhibitory effect, mAChR activation has a net excitatory effect on inspiratory burst amplitude at XII MNs in neonatal mice (medullary slice preparations). One potential explanation for the dichotomous effect of mAChRs on XII MNs across postnatal maturation is developmental changes in the relative expressions of mAChR subtypes. Thus, the objective of this study was to identify and quantify any changes in mAChR expression as well as to determine when those changes are happening during postnatal maturation. We hypothesized that across maturation there is an increase in inhibitory M2 and decrease in excitatory M1, M3, M5 receptor subtypes. Immunofluorescence was employed on fixed medullary brainstem slices (transverse, 20μm) under identical conditions targeting mAChRs M1, M3, M5, and M2. Sample groups included mice at 6 ages: postnatal day (P)1, P4/5, P9, P12/13, P17, and Adulthood (AD) (n = 3). Fluorescence images were obtained on a confocal microscope. Using ImageJ software, the corrected total cell fluorescence was determined both at the whole XII nucleus and at 10 single XII MNs for each slice. All data were normalized. Data indicate that M1 expression at individual XII MNs exhibits a transient yet significant increase from P1 to P4/5, followed by a net decrease over the remainder of postnatal development (P1= 0.81 ± 0.15, P4/5= 1.43 ± 0.14, P9= 0.96 ± 0.01, P12/13= 1.1 ± 0.12, P17= 1.12 ± 0.11, adult= 0.76 ± 0.14, p= 0.01). M3 expression at individual XII MNs exhibits a transient yet significant increase at P17 with no clear pattern preceding it (P1= 0.74 ± 0.28, P4/5= 0.86 ± 0.18, P9= 0.70 ± 0.26, P12/13= 1.09 ± 0.21, P17= 1.78 ± 0.38, adult= 0.53 ± 0.25, p= 0.04). M5 expression at individual XII MNs exhibits no significant pattern (P1= 1.10 ± 0.26, P4/5= 1.23 ± 0.39, P9= 1.04 ± 0.27, P12/13= 1.15 ± 0.49, P17= 0.95 ± 0.20, adult= 0.55 ± 0.19, p= 0.16). M2 expression at individual XII MNs exhibits a significant net increase over postnatal development (P1= 0.68 ± 0.16, P4/5= 1.04 ± 0.047, P9= 1.08 ± 0.36, P12/13= 0.10 ± 0.15, P17= 1.40 ± 0.29, adult= 0.86 ± 0.01, p= 0.03). These data support the hypothesis that there is a decrease in excitatory mAChR subtype expression and an increase in inhibitory mAChR subtype expression over postnatal maturation in mice. The change in expression provides a possible explanation for the contrary effects of muscarinic modulation at XII MNs between the neonatal and adult periods. NIH/NHLBI R25 HL126140 (Moreno, Garcia, Parthasarathy, subaward to Revill). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.