PROJECT SUMMARY Down syndrome (DS), or Trisomy 21, is the most frequent chromosomal abnormality in humans, resulting from a complete or partial extra copy of chromosome 21. Clinically, DS is identified after birth through the recognition of specific physical characteristics such as flat nasal bridge and midface, decreased muscle tone, clinodactyly, and short neck with excess skin at the back. Additionally, children and adults with DS have a higher likelihood of gastrointestinal dysfunction that markedly affects quality of life. While our understanding of the underlying cognitive and other central effects in DS have been studied for many years, our understanding of the gastrointestinal effects in these patients in still quite rudimentary. Gut motility is controlled by the interdependent actions of enteric neurons (ENS), interstitial cells of Cajal, and smooth muscle cells. Investigation of the enteric nervous system in the Ts65Dn mouse model of DS (a well-established mouse model of Trisomy 21) demonstrates that these mice have ENS neurons throughout the bowel, including in distal colon, and that longitudinal migration of enteric neural crest–derived cells during development is normal. However, adult Ts65Dn mice demonstrate reduced colonic motility. Our overarching hypothesis is that downregulation of ionic conductances, specifically, smooth muscle voltage-gated calcium channels by increased oxidative stress results in reduced contractility. In this proposal, we will test this hypothesis in the adolescent (PND 25) and young adult (PND65) Ts65Dn and their disomic littermates. In the first specific aim, we will conduct isometric tension recordings of distal smooth muscle strips in response to neuronal stimulation, acetylcholine induced contractions and calcium influx. In the second specific aim, we will test the hypothesis that trisomy 21 results in altered voltage-gated calcium currents (VGCCs) in smooth muscle in the distal colon, leading to altered smooth muscle motility. We propose to test all aspects of this hypothesis at levels spanning from in vitro gastrointestinal motility assays, to VGCCs functional alterations on individual smooth muscle cells in adolescence and in adults. This comprehensive approach will enhance validation and interpretation of findings through comparisons across these systems, enhancing scientific rigor. Our closely integrated, multidisciplinary, research plan is intended to establish for the first time a generalized framework to understand the mechanisms underlying karyotype-induced changes of bowel smooth muscle function in adolescence and in adulthood using a well-established mouse model of DS. Because these mechanisms are potentially shared across other gastrointestinal abnormalities (e.g. Hirschsprung’s disease), these studies may define a common thread spanning multiple diseases. These newly- defined mechanisms could then be targeted for therapeutic intervention by, e.g., manipulating VGCCs directly or through inte...