During the past decade stunning advances have been made in imaging, molecular biology and biochemistry that enable the visualization of the behavior of single proteins in vivo. Here, I propose to develop and visualize the temporal and spatial dynamics of intracellular signaling within living neurons. Much as early work in calcium imaging redefined our understanding of the importance of calcium influx by defining its spatial and temporal characteristics, I believe visualizing the spatial and temporal dynamics of intracellular signaling will have similar benefits to our understanding of the nervous system. Initially we have developed indicators that enable visualization of one of the key first steps in many intracellular signaling cascades: tyrosine phosphorylation. During the past several years, we have developed a system that relies on ratiometric imaging of changes in a genetically encoded fluorescent indicator of phosphorylation. We now propose three specific aims to develop these tools into a system for monitoring signaling during neuronal plasticity and development. We propose to: 1) Develop a library of indicators targeted to report activity of specific kinases;2) Develop indicators that localize to specific cellular compartments;3) Develop indicators to report activity of multiple signaling molecules simultaneously. Using our indicators, workers will be able to elucidate the dynamics of signals that underlie synaptic plasticity. Thus, our tools will enable novel insights into essential mechanisms that underlie neuronal plasticity.
Project Narrative Neuronal plasticity underlies many fundamental functions within the brain, while abnormal neuronal plasticity is associated with disease. Excessive plasticity may underlie diseases like epilepsy and addiction, while defects in plasticity could play important roles in epilepsy, neurodegenerative, and autism spectrum disorders. Our research will have broad impacts across all these levels by developing new tools to visualize dynamic neuronal signaling with subcellular resolution.
|Sheffler-Collins, Sean I; Dalva, Matthew B (2012) EphBs: an integral link between synaptic function and synaptopathies. Trends Neurosci 35:293-304|
|Kayser, Matthew S; Lee, Anderson C; Hruska, Martin et al. (2011) Preferential control of basal dendritic protrusions by EphB2. PLoS One 6:e17417|
|Nolt, Mark J; Lin, Ying; Hruska, Martin et al. (2011) EphB controls NMDA receptor function and synaptic targeting in a subunit-specific manner. J Neurosci 31:5353-64|
|Antion, Marcia D; Christie, Louisa A; Bond, Allison M et al. (2010) Ephrin-B3 regulates glutamate receptor signaling at hippocampal synapses. Mol Cell Neurosci 45:378-88|
|McClelland, Andrew C; Hruska, Martin; Coenen, Andrew J et al. (2010) Trans-synaptic EphB2-ephrin-B3 interaction regulates excitatory synapse density by inhibition of postsynaptic MAPK signaling. Proc Natl Acad Sci U S A 107:8830-5|
|Dalva, Matthew B (2010) Remodeling of inhibitory synaptic connections in developing ferret visual cortex. Neural Dev 5:5|
|Dalva, Matthew B (2010) Ephecting excitatory synapse development. Cell 143:341-2|
|McClelland, Andrew C; Sheffler-Collins, Sean I; Kayser, Matthew S et al. (2009) Ephrin-B1 and ephrin-B2 mediate EphB-dependent presynaptic development via syntenin-1. Proc Natl Acad Sci U S A 106:20487-92|