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.

Public Health Relevance

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.

National Institute of Health (NIH)
National Institute of Mental Health (NIMH)
Research Project (R01)
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Special Emphasis Panel (ZRG1-ETTN-G (52))
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Freund, Michelle
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University of Pennsylvania
Schools of Medicine
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Mao, Yu-Ting; Zhu, Julia X; Hanamura, Kenji et al. (2018) Filopodia Conduct Target Selection in Cortical Neurons Using Differences in Signal Kinetics of a Single Kinase. Neuron 98:767-782.e8
Hruska, Martin; Henderson, Nathan T; Xia, Nan L et al. (2015) Anchoring and synaptic stability of PSD-95 is driven by ephrin-B3. Nat Neurosci 18:1594-605
Shahidullah, Mohammad; Le Marchand, Sylvain J; Fei, Hong et al. (2013) Defects in synapse structure and function precede motor neuron degeneration in Drosophila models of FUS-related ALS. J Neurosci 33:19590-8
Hruska, Martin; Dalva, Matthew B (2012) Ephrin regulation of synapse formation, function and plasticity. Mol Cell Neurosci 50:35-44
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
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

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