Dendritic Protein Synthesis in Hippocampal Neurons

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DESCRIPTION (provided by applicant): Many studies of behavioral and synaptic plasticity have demonstrated that long-lasting changes in synaptic transmission and behavior require both gene transcription and mRNA translation. To understand long-term information storage, the problem is to determine how the potentiated synapses of a single neuron become selectively modified during the long-lasting phases of synaptic plasticity. That is, how do the products of transcription and/or translation reach the modified synaptic sites without affecting the unmodified sites within the same neuron? We hypothesize that proteins synthesized locally, in dendrites, may allow for this specificity. In order to visualize dendritic protein synthesis directly, we have developed a series of reporter GFP constructs that possess a dendritic localization domain in their mRNA. Thus, the GFP message is routed to the dendrite and can be translated locally. Using this fluorescent reporter and time-lapse confocal microscopy, we have shown that BDNF, a growth factor implicated in synaptic plasticity, can stimulate dendritic protein synthesis in cultured hippocampal neurons. To extend these initial findings, we will ask whether long-term potentiation (LTP) or metabotropic-receptor-dependent long-term depression (LTD) results in dendritic protein synthesis in hippocampal slices. We will re-examine the protein synthesis dependence of LTP by examining whether protein synthesis inhibitors applied after LTP induction can affect the level of synaptic potentiation. We will also attempt to spatially restrict the area of protein synthesis inhibitor to the dendrite using a caged protein synthesis inhibitor. To determine the specificity of synthesis and destination of dendritically synthesized proteins, we will determine the spatial domain over which dendritic protein synthesis occurs when it is stimulated in a local, restricted area. We will examine this by local application of agonists in cultured neurons coupled with time-lapse imaging of the entire dendrite. In hippocampal slices we will selectively stimulate and induce plasticity in spatially distinct axon populations that make synapses with CA1 neurons. Lastly, we will determine the cellular mechanisms underlying dendritic protein synthesis.