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

Publication Date
DOI: 10.1016/s0306-4522(02)00137-9
  • Single Cell
  • Motor Neurons
  • Sleep Apnea
  • Upper Airway
  • Real-Time Pcr
  • Taqman
  • Laser Capture Microdissection
  • Digoxigenin-Labeled Andin Situhybridization
  • Medicine
  • Pharmacology


Abstract 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.

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