This is a competitive revision of the parent award R01-NS012969-34, and is being submitted in response to the Notice Number (NOT-OD-09-058) and Notice Title: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. The enteric nervous system (ENS), which lacks the protection of bone, is highly exposed and thus potentially threatened, not only because of its location, but also because it must deal with the vagaries of ingestion and the large microbial population that is harbored within the bowel. Because of its evident vulnerability, it would seem likely that means of protection and regeneration that are utilized elsewhere in the nervous system should also function in the ENS. We have recently demonstrated that neurogenesis occurs in the adult mouse gut. Surprisingly, the new neurons were found to be generated, not within enteric ganglia, but in extraganglionic """"""""germinal niches"""""""", which appear to represent a special environment in which stem cells are maintained. Enteric neurogenesis, furthermore, was not demonstrable until it was provoked. Two neurogenic stimuli that were found to be effective were administration of a 5-HT4 agonist and by induction of intestinal mucosal inflammation. Following their generation, the post-mitotic enteric neuroblasts slowly (1.25 5m/month) migrated into myenteric ganglia. The developmental origin of the stem cells that produce new neurons in the adult bowel has not yet been determined. One possibility is that subsets of the neural crest-derived cells that colonize the fetal bowel and give rise to the ENS survive to adulthood in """"""""germinal niches"""""""" where they can be mobilized by appropriate stimuli to generate new neurons. Alternatively, adult stem cells may have a different origin, such as bone marrow. We will thus first test the hypothesis that the neurogenic cells in """"""""germinal niches"""""""" are crest-derived progenitors. The Wnt1 promoter will be used to drive expression of Cre recombinase in crest-derived cells in Rosa-floxed-stop-YFP mice. Stable expression of YFP will thus mark cells in the bowel that are of neural crest origin. Neurogenesis will be elicited in animals given BrdU, which is incorporated into newly generated neuroblasts during their terminal mitosis. """"""""Germinal niches"""""""" can then be recognized as clusters of cells that are doubly labeled with BrdU and a neuronal marker such as HuC/D. The neural crest origin of the newly generated neuroblasts will be made evident by determining whether or not they co-express YFP. We will also test the hypothesis, suggested by the neurogenic activity of 5-HT4 agonists, that innervation of """"""""germinal niches"""""""" by myenteric neurons contributes to the regulation of adult neurogenesis. To do so, we will use LM and EM immunocytochemistry to visualize nerve fibers, synaptic proteins, and receptors for putative neurotransmitters. Retrograde tracing will be employed with immunocytochemistry to identify the chemical coding of neurons that project to """"""""germinal niches"""""""". That identification will, in turn, permit the putative neuronal control of neurogenesis to be tested physiologically.
The current application is a Competitive Revision submitted in response to the program NOT-OD-09- 058, """"""""NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications"""""""". This application is intended to enhance and accelerate progress of the parent award NS12969-34, """"""""Neural Control of Gastrointestinal Activity"""""""". The major discovery made during the prior funding period of NS12969 that new neurons could be stimulated to form in vivo in the adult gut. The current competitive revision seeks to identify the origin, neural crest or bone marrow, of the stem cell-derived precursors that give rise to newly generated neurons in the adult gut. Information derived from the studies outlined in the current competitive revision is intended to facilitate future efforts to repair nerve damage or congenital defects of the bowel with a minimum of surgical intervention.
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