The experiments of this proposal continue our studies of how neurons construct postsynaptic sites, especially during reinnervation. The experiments are the outgrowth of our discovery of synapse-associated polyribosomes, and the fact that this machinery is particularly prominent during periods of synapse growth. The central hypotheses that guide our current work are: I) that the polyribosomes synthesize key protein constituents of the synaptic junction; 2) that this local synthesis is critical for the construction of the synaptic site; 3) that the synthetic activity of synapse-associated polyribosomes is regulated by the presence and/or activity of the presynaptic element; and 4) that changes in synapse-associated polyribosomes during reinnervation are part of the molecular process that makes postsynaptic cells receptive to reinnervating fibers. We will test these hypotheses in the experiments of the present project. The experiments of Specific Aim I seek to define the signals that cause polyribosomes to dock beneath synaptic sites We will use time-lapse video microscopy techniques to follow the development of individual synapses on neurons in culture beginning at the stage at which an axon first contacts a dendrite. We will then evaluate the individual synapses electron microscopically at different times after contact has been made so as to define how the postsynaptic cytoplasm becomes organized as the synapse develops. The experiments of Specific Aim 2 seek to identify the mRNAs that are present in dendrites. We will isolate mRNA from subcellular fractions enriched in dendrites (""""""""synaptodendrosomes"""""""") and from dendrites that have been isolated using a special two-phase tissue culture technique. We will clone and sequence the dendritic mRNAs and then compare the sequences with known sequences in the data bank. The experiments of Specific Aim 3 use biochemical techniques to identify the proteins that are synthesized within dendrites. Pulse-labeling and subcellular fractionation techniques will be used in conjunction to selectively label the proteins that are synthesized in dendrites. 2-D PAGE fluorography will be used to identify and characterize the proteins that are locally synthesized. The experiments of Specific Aim 4 will evaluate whether different proteins are synthesized within the dendrites of different types of neurons (from cortex, cerebellum, hippocampus, and brainstem). The experiments of Specific Aim 5 test the hypothesis that the synthesis of particular proteins beneath synaptic sites is regulated by synaptic activity. We will use 2-D PAGE fluorography to determine whether the local synthesis of particular synaptic junctional proteins synaptodendrosomes is selectively modified by neurotransmitter activation. These experiments will lead to new insights into how neurons build and modify postsynaptic sites, and may lead to strategies that could promote the re-establishment of particular types of connections by fibers that are induced to regenerate following CNS injury.
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