In vivo visualization of CaMKII activity in ocular dominance plasticity

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Brain and Cognitive Sciences, 2009.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Includes bibliographical references.Alterations in sensory experience can persistently modify the responses of cortical neurons. Ocular dominance (OD) plasticity, a process in which alternation of visual input induces a shift in cortical responsiveness, is an extensively studied model of such experience-dependent plasticity. However, the synaptic mechanisms underlying OD plasticity are not well understood. Recent studies revealed that both Hebbian and homeostatic mechanisms play a role in OD plasticity. Therefore, we were interested in monitoring the process of rapid plasticity at individual synapses in vivo to gain insight into the interplay of these two mechanisms. Ca2+/calmodulin dependent protein kinase II (CaMKII) is a major component of the postsynaptic density. Activation of CaMKII is necessary and sufficient for LTP induction, is required for OD plasticity, and its expression pattern coincides with the site of rapid plasticity in the supragranular layers II/III of the visual cortex. Moreover, CaMKII can convert transient Ca2+ influx into a prolonged biochemical process via autophosphorylation that renders CaMKII activity Ca2+ independent. Hence, CaMKII is well suited as a reporter of synaptic activity. We previously engineered a probe, Camui, which utilizes the optical phenomenon of fluorescence resonance energy transfer (FRET), to monitor CaMKII activation. This thesis embodies the work done to improve Camui to be a better tool for in vivo reporting of CaMKII activity, as well as the use of this improved probe for in vivo detection of CaMKII activity in single spines before and after 4 hrs of monocular deprivation (MD) in the ferret visual cortex.(cont.) We found that after only 4 hrs of MD, the overall CaMKII activity in spines, and adjacent dendritic regions, of neurons in the deprived eye domain increased significantly. This increase was also seen in the binocular eye domain. In the open eye domain, however, this overall increase in CaMKII activity was absent. These observations were specific to MD as control experiments did not show such changes. Moreover, detailed analysis revealed that spines that were eliminated after 4 hrs of MD had a low level of basal CaMKII activity. Our results lend support to the model that both Hebbian, as well as homeostatic compensatory mechanism can subserve OD plasticity.by Show Ming Kwok.Ph.D

Application of FRET probes in the analysis of neuronal plasticity

Breakthroughs in imaging techniques and optical probes in recent years have revolutionized the field of life sciences in ways that traditional methods could never match. The spatial and temporal regulation of molecular events can now be studied with great precision. There have been several key discoveries that have made this possible. Since green fluorescent protein (GFP) was cloned in 1992, it has become the dominant tracer of proteins in living cells. Then the evolution of color variants of GFP opened the door to the application of Förster resonance energy transfer (FRET), which is now widely recognized as a powerful tool to study complicated signal transduction events and interactions between molecules. Employment of fluorescent lifetime imaging microscopy (FLIM) allows the precise detection of FRET in small subcellular structures such as dendritic spines. In this review, we provide an overview of the basic and practical aspects of FRET imaging and discuss how different FRET probes have revealed insights into the molecular mechanisms of synaptic plasticity and enabled visualization of neuronal network activity both in vitro and in vivo.National Science Foundation (U.S.) (Grant R01DA17310

Global Transcriptional and Translational Repression in Human-Embryonic-Stem-Cell-Derived Rett Syndrome Neurons

Rett syndrome (RTT) is caused by mutations of MECP2, a methyl CpG binding protein thought to act as a global transcriptional repressor. Here we show, using an isogenic human embryonic stem cell model of RTT, that MECP2 mutant neurons display key molecular and cellular features of this disorder. Unbiased global gene expression analyses demonstrate that MECP2 functions as a global activator in neurons but not in neural precursors. Decreased transcription in neurons was coupled with a significant reduction in nascent protein synthesis and lack of MECP2 was manifested as a severe defect in the activity of the AKT/mTOR pathway. Lack of MECP2 also leads to impaired mitochondrial function in mutant neurons. Activation of AKT/mTOR signaling by exogenous growth factors or by depletion of PTEN boosted protein synthesis and ameliorated disease phenotypes in mutant neurons. Our findings indicate a vital function for MECP2 in maintaining active gene transcription in human neuronal cells.National Institutes of Health (U.S.) (Grant R01-HG002668)National Cancer Institute (U.S.) (P30-CA14051)David H. Koch Institute for Integrative Cancer Research at MITKathy and Curt Marble Cancer Research FundNational Institutes of Health (U.S.) (Grant HD 045022)National Institutes of Health (U.S.) (Grant R37-CA084198)Simons FoundationEuropean Leukodystrophy Associatio