During development, neuronal activity plays an important role in shaping functionalsynaptic networks. Moreover, in adults, neuronal activity is thought to produce long-termchanges during learning and memory. For these changes to persist, however, neuronalactivity must engage signaling pathways that result in alterations in gene expression.Pathways that activate the transcription factor CREB (cAMP-response element-bindingprotein), which binds to CREs (cAMP response elements) in gene promoters, have beenimplicated in this long-term, activity-dependent plasticity. The focus of my research is toinvestigate the mechanisms of CRE-dependent gene expression induced by physiologicalsynaptic activity. Together with my supervisor, I developed a sensitive biosensor tomeasure CRE-dependent gene expression in neurons; this biosensor consists of apromoter-reporter construct derived from the major immediate-early promoter/enhancerof the human cytomegalovirus (hCMV promoter) coupled to Green Fluorescent Protein(GFP). Our studies show that this promoter acts as an activity-dependent switch inneurons. The hCMV promoter functions poorly in unstimulated neurons, but is stronglyinduced by neuronal activity or other stimuli that activate CREB; moreover, site-directedmutagenesis of the five CREs in the promoter eliminated this inducibility. To express thisbiosensor in neurons, we used adenoviral-mediated gene transfer which has an efficiencyof>95%, allowing one to monitor reporter gene expression in individual neurons of afunctional network for several days. Using this system we, discovered that nicotine, atlow-levels in the range that circulates in smokers, acts in a distributed manner throughout a neuronal network to alter CRE-dependent gene expression induced by network activity . Further, exploiting the sensitivity of this biosensor, we found that weak neuronal activity,including spontaneous release of neurotransmitter, is a sufficient stimulus to recruitsignaling pathways that resu
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.95597 |
| Date | January 2003 |
| Creators | Wheeler, Damian George |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Physiology) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | Theses scanned by McGill Library. |
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