The overarching objective of this proposal is to understand alterations in the spatiotemporal gradients of nitric oxide (NO) within enteric ganglia and how these gradients change with age. The central hypothesis to be tested in the proposed research is that enteric neurotransmission is spatially and temporally altered with age. NO, a transmitter synthesized by a subpopulation of enteric neurons, plays a critical role in autonomic reflex pathways within the enteric nervous system (ENS). Within the ENS, NO has been shown to mediate relaxation of smooth muscle contraction as a transmitter of inhibitory motor neurons, and may also be involved in integration between sensory and motor activities as a transmitter of interneurons. The first specific aim concerning the neurochemical coding of enteric nitrergic neurons in the colon of animals of advanced age, will be accomplished using immunocytochemistry to characterize changes in the population of enteric-neuronal nitric oxide synthase (nNOS) immunoreactive neurons. It is critical to learn what subsets of neurotransmitters and neuromodulatory substances exist in enteric nitrergic neurons in old animals to better understand age-related changes in the nitrergic pathways of the ENS. Immunocytochemistry and quantitative morphometric analyses will likewise be used to determine age-related changes in the spatial relationships between NO-releasing and putative NO-receptive neurons. The population of colonic enteric neurons declines with age, and this decline may be an intrinsic factor in age-related alterations of colonic motility. Because of the critical role of NO in the ENS, it is crucial to address the question: What are the spatial and temporal characteristics of NO release within enteric ganglia of old animals? This second specific aim will be accomplished by using novel NO-sensing fluorescent molecules as probes to quantify the spatiotemporal parameters of NO gradients within the ENS of old vs young rat colon. Techniques previously used for electrophysiological studies of enteric neurons in vitro will be combined with state of the art imaging methods to record NO gradients in enteric ganglia. These experiments will reveal the extent to which age-related remodeling of enteric ganglia changes the NO-mediated signaling in the gut. To know how altered changes in neuronal phenotypes and NO release effect colonic function it is necessary to learn the answer to the third specific aim: Which enteric neurons respond to NO in the colon of aged animals? By combining digital fluorescence imaging with electrophysiological techniques enteric neurons that are responsive to NO will be morphologically and physiologically identified. These experiments will utilize a novel monolayer coated optical fiber to deliver brief, calibrated pulses of exogenous NO to preparations of enteric neurons in vitro. Ordered propulsive colonic motility is a necessity for continued good health, yet disordered colonic motility has been described as endemic in the elderly and adequate therapy is unavailable. Understanding the intrinsic age-related changes in NO signaling that occur in the wall of the bowel is a necessary first step in learning how novel medical therapies might improve the quality of life in the elderly.
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