Distribution of muscarinic acetylcholine receptors in hypoglossal motoneurons across postnatal maturation
Authors: Kelley Kori , Koziol Sophia , White Monica , Banayat Tatum , Buhlman Lori , Revill Ann 
Published: May 2024

Upper airway patency is decreased during rapid eye movement (REM) sleep due to loss of genioglossus (primary tongue protruder) tongue muscle tone. During REM sleep, hypoglossal motoneurons (XII MNs), which innervate genioglossus, receive less excitatory noradrenergic drive and may be inhibited by activation of muscarinic acetylcholine receptors. Data from intact adult rats during natural sleep implicates an inhibitory effect of muscarinic acetylcholine receptors on XII MNs. However, data using the rhythmic slice preparation from neonatal mice indicate that muscarine has a net excitatory effect on inspiratory burst amplitude at XII MNs. Our project examines the changes to distribution of muscarinic acetylcholine receptors (mAChRs) at XII MNs across postnatal maturation. We hypothesize there is an increase in inhibitory (M2) and decrease in excitatory (M1, M3, M5) mAChRs at XII MNs with postnatal maturation. We performed double-labeled immunofluorescence experiments on perfused transverse brainstem slices (20μm) under identical conditions across six postnatal age groups: P0-P2, P3-P5, P6-P10, P11-P13, P14-P17, and adult against mAChR targets M1, M2, M3, M5. Slices were co-labeled with choline acetyltransferase (ChAT) and 4’,6-diamidino-2-phenylindole (DAPI). Images were collected using a confocal microscope. 10 individual XII MNs were identified in each slice using positive ChAT labeling as well as anatomical location. ImageJ was used to determine the average XII MN mAChR subtype intensity. Preliminary data (n = 3) indicate that M1 receptors showed a transitory increase in labeling intensity in XII MNs followed by a decrease across development (P1=89±11%, P4-5=100%, P9=76±18%, P12-13=67±8%, P17=67±4%, adult=56±7%, p = 0.13). M3 (n = 3) showed a trend for a transient increase in labeling intensity at P17 (P1=78±41%, P4-5=87±3%, P9=61±13%, P12-13=86±34%, P17=100%, adult=80±23, p = 0.60). M5 (n = 3) expression intensity decreased consistently over maturation (P1=100%, P4-5=92±34%, P9=75±23%, P12-13=70±10%, P17=66±14%, adult=39±12%, p = 0.0257). In contrast, M2 (n = 3) receptor expression intensity remained relatively consistent across maturation (P1 = 88±23%, P4-5 = 100%, P9=88±10%, P12-13 = 75±15%, P17 = 86±18%, adult = 75±10%). These data partly support our hypothesis of a decrease in expression intensity of excitatory M1, M3, and M5 receptor subtypes into adulthood. Contrary to our hypothesis, we observed minimal change in the expression intensity of inhibitory M2 receptors across maturation. The decrease in labeling intensity of excitatory muscarinic receptor subtypes may support the observed shift in muscarinic modulation from excitation to inhibition with postnatal maturation. Arizona Alzheimer's Consortium, NIH. This is the full abstract presented at the American Physiology Summit 2024 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.