The long term goal of the proposed project is to understand how Ca2+ and lipid second messenger signals are used by neurons and astrocytes to regulate signaling responses. The lipid second messengers phosphatidylinositol-4,5-bisphosphate (PIP2), phosphatidylinositol-3,4,5-triphosphate (PIP3), diacyiglycerol (DAG) and phosphatidic acid (PA) as well as Ca2+ signals are connected by cross-talk and feedback loops and can be considered as key components of a complex """"""""signaling network."""""""" The understanding of how different Ca2+ and lipid second messenger signaling systems work in neurons and astrocytes will likely lead to the identification of new classes of drug targets for different brain diseases. Our laboratory has developed a fluorescent microscopy strategy for monitoring local Ca2+ and lipid second messenger signals and we are now in a good position to ask questions on how and where positive and negative feedback loops and cross-talk is operational within the two cell types. We will be using lipid binding domains conjugated with the GFP color variants CFP and YFP as translocation biosensors and total internal reflection fluorescence (TIRF) and confocal microscopy to simultaneously monitor local changes in Ca2+ and lipid second messenger concentration in astrocytes and in the dendrites of hippocampal neurons as a function of time. Using these strategies, we will 1. test the model that glutamate stimulated calcium waves and oscillations in astrocytes require DAG and investigatorP2 oscillations, 2. use a new wide-field TIRF system that we developed to test the hypothesis that crosstalk and positive and negative feedback processes between Ca2+ and lipid second messengers define an interconnected second messenger signaling network in astrocytes, 3. test the hypothesis of a """"""""geometric delay mechanism"""""""" and other spatial and temporal control mechanisms that are thought to regulate translocating signaling proteins in the dendrites of hippocampal neurons, and 4. investigate local PIP3 and other positive feedback mechanisms and determine whether localized second messenger signals occur at postsynaptic terminals. Furthermore, we will test if such local signals define where and when dendritic branches and synapses are formed. The overall goal of these aims is to obtain a model that describes the key features of the Ca2+ and lipid second messenger signaling networks of astrocytes and neurons and their regulation by different electrical and receptor stimuli.
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