Using pharmacological approaches, several recent studies suggest that local protein synthesis is required for synaptic plasticity. Convincing demonstrations of bona fide dendritic protein synthesis in mammalian neurons are rare, however. We developed a protein synthesis reporter in which the coding sequence of green fluorescent protein is flanked by the 5' and 3' untranslated regions from CAMKII-alpha, conferring both dendritic mRNA localization and translational regulation. In cultured hippocampal neurons, we show that BDNF, a growth factor involved in synaptic plasticity, stimulates protein synthesis of the reporter in intact, mechanically, or "optically" isolated dendrites. The stimulation of protein synthesis is blocked by anisomycin and not observed in untreated neurons. In addition, dendrites appear to possess translational hot spots, regions near synapses where protein synthesis consistently occurs over time.
Methods
Cell Culture and MicroscopyDissociated postnatal (P1-2) rat hippocampal neuron cultures, plated at a density of 230-460/ mm 2 , were prepared as previously described (1). In all experiments, neurons were used following 14-23
The regulation of protein synthesis is vital for a host of cell biological processes, but investigating roles for protein synthesis have been hindered by the inability to selectively interfere with it. To inhibit protein synthesis with spatial and temporal control, we have developed a photo-releasable anisomycin compound, N-([6-bromo-7-hydroxycoumarin-4-yl]methyloxycarbonyl)anisomycin (Bhc-Aniso), that can be removed through exposure to UV light. The area of protein synthesis inhibition can be restricted to a small light-exposed region or, potentially, the volume of two-photon excitation if a pulsed IR laser is the light source. We have tested the compound's effectiveness with an in vitro protein-translation system, CHO cells, HEK293 cells, and neurons. The photo-released anisomycin can inhibit protein synthesis in a spatially restricted manner, which will enable the specific inhibition of protein synthesis in subsets of cells with temporal and spatial precision.
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