Single cell laser dissection with molecular beacon polymerase chain reaction identifies 2A as the predominant serotonin receptor subtype in hypoglossal motoneurons.

We hypothesize that sleep state-dependent withdrawal of serotonin (5-hydroxytryptamine, 5-HT) at upper airway (UAW) dilator motoneurons contributes significantly to sleep-related suppression of dilator muscle activity in obstructive sleep apnea. Identification of 5-HT receptor subtypes involved in postsynaptic facilitation of UAW motoneuron activity may provide pharmacotherapies for this prevalent disorder. We have adapted two assays to provide semi-quantitative measurements of mRNA copy numbers for 5-HT receptor subtypes in single UAW motoneurons. Specifically, soma of 111 hypoglossal (XII) motoneurons in 10 adult male rats were captured using a laser dissection microscope, and then used individually in single round molecular beacon polymerase chain reaction (PCR) for real-time quantitation of 5-HT(2A), 5-HT(2C), 5-HT(3), 5-HT(4), 5-HT(5A), 5-HT(5B), 5-HT(6) or 5-HT(7) receptor. Receptor mRNA copy numbers from single XII motoneurons were compared to control samples from within the XII nucleus and lateral medulla. All 20 motoneuronal soma assayed for the 5-HT(2A) receptor had measurable copy numbers (7028+/-2656 copies/cell). In contrast, copy numbers for the 5-HT(2A) receptor in XII non-motoneuronal (n=17) and lateral medulla (n=15) samples were 81+/-51 copies and 83+/-35 copies, respectively, P<0.05. Seven of 13 XII motoneurons assayed had measurable 5-HT(2C) receptor copy numbers of mRNA (287+/-112 copies/cell). XII soma had minimal 5-HT(3), 5-HT(4), 5-HT(5A), 5-HT(5B), 5-HT(6) or 5-HT(7) receptor mRNA. 5-HT(2A) receptor mRNA presence within XII motoneurons was confirmed with digoxigenin-labeled in situ hybridization. In summary, combined use of laser dissection and molecular beacon PCR revealed 5-HT(2A) receptor as the predominant 5-HT receptor mRNA in XII motoneurons, and identified small quantities of 5-HT(2C) receptor. This information will allow a more complete understanding of serotonergic control of respiratory activity.