(A) Schematic of single-cell mRNA sequencing paradigm. Postnatal day 0 (P0) medullary brainstem slices containing the preBötC bilaterally (circled in red) were dissected and isolated for sequencing. (B), Single cell transcriptome profiling of cells isolated from P0 preBötC. Of 1817 cells isolated, only 267 were presumed preBötC neurons of which only 21 expressed Oprm1 mRNA. (C), Heatmap of scaled transcript abundance for Oprm1 and markers of glutamatergic, gabaergic, and glycinergic preBötC neurons. Oprm1 expressing cells are both excitatory and inhibitory. (D), Schematic of extracellular recordings of the preBötC rhythm in P0-4 medullary brainstem slices. The preBötC has input to the hypoglossal motor neurons which form the CN12 rootlet, relaying an inspiratory motor command to the tongue in intact animals. Due to its input from the preBötC, extracellular recording from this rootlet display autonomous rhythmic activity corresponding to in vitro respiration (Mansour et al., 1994). (E), Representative recording of bursting activity after application of 50 nM DAMGO and 100 nM Naloxone. Top: in control (Oprm1f/f) slices bath application of 50 nM DAMGO quickly slowed and decreased the amplitude from baseline bursting. After rhythm cessation, bath application of Naloxone restored the rhythm. Bottom: in Slc17a6-cre;Oprm1f/f mice 50 nM DAMGO did not stop, or even slow rhythmic activity. (F), Percent of slices from each genotype with rhythm cessation after 50 nM DAMGO application. Control (n = 11), Gad2-Cre;Oprm1f/f (n = 6), Slc32a1-Cre;Oprm1f/f (n = 2), Slc6a5-Cre;Oprm1f/f (n = 4), and Slc17a6-Cre;Oprm1f/f (n = 3). (G), Schematic showing three subpopulations of glutamatergic lineages delineated by transcription factors Dbx1 and Foxp2. Foxp2 neurons represents a smaller, and overlapping, population of Dbx1 neurons. (H,) Identification of molecular subtypes of Oprm1 preBötC excitatory neurons. Sagittal section of the preBötC from a P0 Oprm1-mCherry;Dbx1-Cre;Rosa-LSL-YFP mouse immunostained for mCherry (OPRM1 fused to mCherry, red), YFP (Green) and FOXP2 (Blue). About ~50% of Oprm1 preBötC neurons are glutamatergic/Dbx1 derived (arrowhead) and of those,~35% express FOXP2 (asterisk). Scale bar, 50 µM. (I,) Quantification of the number of preBötC for each molecular subtype identified in H. J), Dose response curve for the bursting rate and amplitude after bath application of 0, 50, 100, and 500 nM DAMGO applied to Slc17a6-Cre;Oprm1f/f (gray, n = 3), Dbx1-Cre;Oprm1f/f (green, n = 3), Foxp2-Cre;Oprm1f/f (blue, n = 4) and control (black, n = 11) P0-4 preBötC slices. Rate and amplitude for each slice are normalized to baseline. (K), Schematic summary showing that the key node for opioids to suppress breathing is the preBötC and within this site, elimination of Oprm1 from just a small subset of those neurons,~70–140 excitatory neurons, prevents opioid respiratory suppression.