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Integrative Neural Behavior in a Mammalian Nervous System Revealed Using Ca2+ Imaging
AuthorBayguinov, Peter O.
AdvisorSmith, Terence K.
Biochemistry and Molecular Biology
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Historically, research on nervous systems has been hindered by the complexity of such systems, and the inability to record activity in multiple neurons at the same time. As such, our current understanding of the way a nervous system functions has been the result of electrophysiological studies performed on single, or small groups of neurons, as well as research on relatively simple invertebrate models. In the work presented in this dissertation, we have used imaging techniques to explore the functions of several classes of neurons and auxiliary cells in the enteric nervous system of the murine large bowel during a compound motor behavior, the colonic migrating motor complex (CMMC).Using low-powered Ca<super>2+</super> imaging in whole tissue preparations, we explored the relationship between the longitudinal and circular muscle layers of the colon, showing a high degree of synchronization between the two layers during the CMMC. This synchronization is further shown to be a function of enteric innervation, as addition of nicotinic and muscarinic antagonists disrupted coordinated Ca<super>2+</super> waves in the two muscle layers. Using a mouse model for Hirschsprung's disease (aganglionosis), we have shown that CMMCs do not occur in mice that lack enteric innervation, and Ca<super>2+</super> waves that persist are unsynchronized, and do not propagate sufficient distances to generate pellet propulsion.The myenteric plexus of the colon contains 13 different classes of enteric neurons, including sensory neurons, ascending and descending interneurons, and both excitatory and inhibitory motor neurons. The use of Ca<super>2+</super> imaging as a technique to study the activity of enteric neurons provided little information regarding the class of neurons being observed. This dissertation details a number of techniques which we have developed to facilitate the identification of these enteric neurons using post hoc staining, and have subsequently used to identify inhibitory motor neurons, sensory neurons, types of interneurons and different classes of interstitial cells in the murine colon.Our work has identified AH/Dogiel Type II neurons, which stained intensely with the mitochondrial marker, Mitotracker, as the first responders in the generation of the CMMC in response to mucosal stimulations. Responses in these cells were abolished in the presence of 5- HT<sub>3</sub> receptor antagonists, and when the mucosa was removed, suggesting that 5-HT release from enterochromaffin cells in the mucosa activates AH/Dogiel Type II neurons to generate a CMMC. Furthermore, we have shown that during the CMMC, there is an increase in Ca<super>2+</super> transient frequency in neurons that tested negative for NOS, and were presumably excitatory motor neurons, as well as a marked decrease in the activity of NOS+ve neurons, which were likely inhibitory motor neurons.Recent studies have identified a colonic occult reflex, which is activated by colonic elongation, leading to the activation of mechanosensitive descending inhibitory interneurons, which inhibit the activation of the peristaltic reflex. This occult reflex is thought to underlie slow transit constipation, and as the CMMC in the murine large bowel is responsible for fecal pellet propulsion, we sought to establish the effects of colonic elongation on the CMMC. Our findings show that colonic elongation led to a nitric oxide-mediated reduction in the amplitude of migrating complexes. During such elongation, enteric neurons, including AH/Dogiel Type II neurons exhibited reduced amplitude and frequency of spontaneous Ca<super>2+</super> transients.Ca<super>2+</super> imaging experiments of the myenteric plexus region during the CMMC revealed an increase in the Ca<super>2+</super> transient frequency of ICC-MY, which, in the colon, are believed to underlie synchronization of the longitudinal and circular muscle layers. Using high-power imaging, we observed both excitatory and inhibitory nerve varicosities in close apposition to ICC-MY, which activated or inactivated the cells, respectively. Pharmacological studies revealed that ICC-MY have both muscarinic and NK1 receptors, which are likely the targets of ACh and TK release from excitatory motor neurons.Collectively these findings offer insight into the neuronal mechanism that underlies the generation and propagation of the colonic migrating motor complex in the murine large intestine. From a larger perspective, studies described in this dissertation represent an important step in our understanding of how neural networks in a mammalian nervous system functions to generate a complex rhythmic motor behavior.