Intellectual Merit: Neuronal function requires the precise spatio-temporal control of mRNA translation in dendrites in response to electrical and chemical signaling events between cells. In addition, certain forms of synaptic plasticity, which may be molecular correlates of learning and memory, are mediated by protein-synthesis dependent changes within dendrites. It is known that various mRNAs encoding proteins critical to synapse function are transported into dendrites, where subsequent synapse activity regulates their translation, but the timing and location between synapse activity and these events remains unclear. This project aims to test the hypothesis that localized synaptic stimulation results in the subsequent capture and translation of mRNAs involved in the modification of activated synapses very rapidly, and that their translation rates will be exponentially increased for a brief but significant time after stimulation. Using molecular approaches including live imaging of single mRNA particle movements and translation events in living dendrites, these activities will be quantified in response to spatially restricted activation at synapses of hippocampal neurons. mRNAs known to be critical for changes in synaptic efficacy and localized in response to synaptic stimulation will be used as markers for these transport and translation activities. These approaches represent a significant departure from previous experiments that have used biochemical fractions of subcellular components to assess changes in dendritic mRNA abundance and translation rates since these are often not pure and do not give spatial information about these processes in vivo. Establishment of live cell assays for mRNA transport and translation in neurons will be an important advance for the assessment of gene expression studies in response to localized subcellular activation.
Broader Impact: This project is attractive and well suited to training of students interested in pursuing careers in the neurosciences. It is fundamentally intuitive since the experiments are based in live cell imaging and therefore, the highly complex processes of neuronal function can be visualized directly. In addition, the techniques are based in digital fluorescence microscopy, which represents a major new component to biological education at many levels of future curriculum development in the biological sciences. Hunter College is one of the largest public institutions in the United States that serves an underrepresented population of ethnic undergraduate students that originates from around the world. Students will be able to simultaneously take classes in related neurobiological topics while applying them in the laboratory, where they will learn basic molecular techniques coupled to modern cell biological approaches to complex neurobiological problems, such as the molecular regulation of learning and memory function.
