The formation of memory in the hippocampus by long term potentiation (LTP) requires both fresh transcription of mRNAs encoding the proteins that enable synaptic transmission and localized translation of these mRNAs at the activated synapses. These mRNAs travel from the soma to the synapses as translationally repressed cargo on RNA granules that are actively transported through the dendrites. An understanding of the mechanisms by which activated synapses draw the granules to their domains, and then activate the mRNA cargo for translation, awaits the development of methods which will permit the imaging of mRNA dynamics in live neurons simultaneously with neuronal activation. Molecular beacon probes can fulfil this role, because they only fluoresce when they are hybridized to a specific mRNA sequence. Using multiple molecular beacons for each mRNA species, we will image the dendritic traffic of a select set of mRNAs that are believed to play significant roles in the establishment of LTP. The same probes will be used to determine the stoichiometry of these mRNAs in the granules. We will also image the assembly of the mRNAs into the granules in the soma and their eventual release for translation at the synapse. The single-molecule sensitivity necessary for this task will be obtained by inserting a tandemly repeated probe binding site into a non-translated region of each mRNA species. When multiple molecular beacons bind to these sites, each mRNA molecule is rendered so intensely fluorescent that it is visible as a diffraction-limited spot. These modified mRNAs will be expressed in cultured neurons and tracked with single-molecule sensitivity as they assemble into granules, travel through the dendrites, dock at the synapses, and are released from the granules by the activation of the synapse. The simultaneous use of different tandemly repeated probe binding sequences for different mRNA species will enable two different mRNA species to be distinctively labeled with differently colored molecular beacons. Moreover, the granules themselves can be labeled in a different color by the co-expression of a stable GFP-tagged protein constituent of the granules. The development of this system to study mRNA dynamics in live neurons with single-molecule sensitivity will enable an exploration of the molecular mechanisms that distinguish activated synapses from naive synapses.
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