Understanding neuronal function and synaptic physiology and plasticity is key to understanding the response to drugs of abuse and addiction. It is well demonstrated that local protein translation impacts both short- and long-term responses of synapses, including long-term potentiation (LTP) and long-term depression (LTD). Here, we propose to develop an innovative method for probing biological processes at subcellular resolution and to apply it to selective transcriptome profiling of individual cell type and to profiling of locally translated mRNAs. This technology, which we term Laser Tag, is based on the development of caged substrates for the CLIP and SNAP epitope tags. Uncaging of these substrates using single or two-photon illumination will allow us to activate the compounds in any region of interest, either intracellularly or extracellularly, and enable selectiv tagging of proteins with a high degree of spatial resolution that is designated solely by the light beam. By combining Laser Tag with a previously validated methodology to recover actively translating messages (Ribotag), we can profile mRNAs selectively in discrete cell types or subcellular compartments. In this case, we will examine profiles of locally translated mRNAs in dendrites and measure changes in these populations in response to a variety of chemical and genetic manipulations. The overall outcome of these studies will not only be a deeper understanding of local translation and how it is regulated but also a broad platform technology that can be used to investigate a wide range of biological processes.

Public Health Relevance

Understanding neuronal function is key to understanding the biology of addiction. We propose to develop a new methodology to enable the study of biological processes with a high degree of spatial precision. We will apply this to the study of local protein translation in neurons, a process, which is critical for the function of synapses and in turn for learning and memory.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21DA035612-02
Application #
8623127
Study Section
Special Emphasis Panel (ZDA1-SXC-E (09))
Program Officer
Satterlee, John S
Project Start
2013-04-01
Project End
2015-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
2
Fiscal Year
2014
Total Cost
$220,956
Indirect Cost
$41,445
Name
Cold Spring Harbor Laboratory
Department
Type
DUNS #
065968786
City
Cold Spring Harbor
State
NY
Country
United States
Zip Code
11724
Amatrudo, Joseph M; Olson, Jeremy P; Agarwal, Hitesh K et al. (2015) Caged compounds for multichromic optical interrogation of neural systems. Eur J Neurosci 41:16-May
Yagishita, Sho; Hayashi-Takagi, Akiko; Ellis-Davies, Graham C R et al. (2014) A critical time window for dopamine actions on the structural plasticity of dendritic spines. Science 345:1616-20