Effects of postnatal maturation on muscarinic acetylcholine receptor distribution in hypoglossal motorneurons

Abstract

Upper airway patency is decreased during rapid eye movement (REM) sleep due to loss of genioglossus (primary tongue protruder) tongue muscle tone. Hypoglossal motoneurons (XII MNs), which innervate the genioglossus muscle, receive less excitatory noradrenergic drive, and may be inhibited by activation of muscarinic acetylcholine receptors, during REM sleep. Indeed, preliminary data from intact adult rats during natural sleep implicated an inhibitory effect of muscarinic acetylcholine receptors on hypoglossal motoneurons. However, data using the rhythmic slice preparation from neonatal mice indicate that muscarine has a net excitatory effect on inspiratory burst amplitude at hypoglossal motoneurons. Our project examines the distribution of muscarinic acetylcholine receptors at XII MNs, and how that changes across postnatal maturation. We hypothesizes there would be an increase in inhibitory (M2) and decrease in excitatory (M1, M3, M5) muscarinic receptors in XII MNs with postnatal maturation. We performed double‐labeled immunofluorescence experiments on perfused 20μm transverse brainstem slices across six postnatal age groups under identical conditions: P0‐P2, P3‐P5, P6‐P10, P11‐P13, P14‐P17, and adult against muscarinic targets M1, M2, M3, M5. Slices were co‐labeled with choline acetyltransferase (ChAT) and 4’,6‐diamidino‐2‐phenylindole (DAPI). Images of the hypoglossal motor nucleus (HMN) were collected using a confocal microscope. ImageJ was used to determine the average HMN muscarine receptor subtype intensity across postnatal maturation. Preliminary data (n = 1) indicate that M1 receptors showed a consistent decrease in labeling intensity in the HMN across development (P5=100%, P13=80%, P17=67%, adult = 40%), whereas M3 (n = 1) (P4= 90%, P9= 73%, P13=89%, P17= 100%, adult= 41%) showed a transitory increases in expression before a final decrease into adulthood. M5 (n = 1 or 2) expression shows a bimodal distribution of labeling intensity (P0‐2= 100%, P4‐6= 58‐99%, P7‐10 = 52‐70%, P11‐13= 35‐58 %, P14‐17 = 27 % adult= 17‐37 %). In contrast, M2 receptor expression intensity remained relatively high with small fluctuations into adulthood (n=2), (P0‐2 = 92‐94 %, P4‐6 = 100%, P7‐10 = 92‐95 %, P11‐13 = 78‐107 %, P14‐17 = 88‐107 %, adult = 73‐77 %). These data partly support our hypothesis that there would be 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. We speculate that the decrease in labeling intensity of excitatory muscarinic receptor subtypes may support the observed shift of muscarinic modulation from excitation to inhibition with postnatal maturation.