Carbachol inhibits the glutamatergic input from parahypoglossal neurons to genioglossus motoneurons (PH^Glut → GMNs). A, Schematic diagram of the experimental design used to map synaptic connectivity of the PH^Glut → GMN input. We injected VGlut2-IRES-Cre mice with fluorescent CTB (Alexa Fluor-488; green) into the tongue to retrogradely labeled GMNs and AAV-DIO-ChR2-mCherry into the PH region to express ChR2 restricted in neurons lateral to the MoXII (area postrema level). We recorded fluorescently labeled neurons in the MoXII retrograde labeled with green CTB. B, Heatmap of AAV-DIO-ChR2-mCherry injection sites of seven recorded mice showing overlapping regions of ChR2-mCherry expressing neurons. C, Confocal images of retrogradely labeled GMN with F-CTB from the genioglossus muscle (green) and PH glutamatergic somata and fibers expressing ChR2-mCherry (red). The low-magnification image (left) shows both MoXII and the PH region containing ChR2-expressing neurons. Scale bar, 200 μm. The high-magnification image (right) shows retrogradely labeled GMN and dense PH glutamatergic projections expressing ChR2-mCherry. Scale bar, 20 μm. D, E, Photostimulation produced photo-evoked EPSCs in GMNs and carbachol (15 μm) reduced the photo-evoked EPSC charge (mean ± SEM; n = 9; RM one-way ANOVA: F_(1,9) = 23.61, p = 0.0009; Tukey's multiple comparison, **p < 0.01).