The experiments of this proposal seek to define some of the cellular and molecular mechanisms of synapse regeneration following denervation and synapse formation during development. The central hypothesis is based on our discovery of protein synthetic machinery (comprised of polyribosomes and associated membranes) beneath synapses on CNS neurons; this machinery is particularly prominent during the initial formation of synaptic connections during normal development and increasing under synaptic sites during reinnervation. We believe that the polyribosomes produce proteins that are required for the construction or consolidation of the synaptic junction and that the local synthesis of protein at the postsynaptic site plays a role in regulating innervation. This 5 year project has 5 Specific Aims: 1) We will determine at what stage in the mutation of the synaptic junction the polyribosomes are most prominent and evaluate whether the positioning of the polyribosomes at postsynaptic sites is determined by a program of differentiation within the postsynaptic cell, by the presence or activity of the presynaptic terminal, by an interaction between pre- and postsynaptic elements during synaptogenesis, or by factors that are extrinsic to the pre- and postsynaptic neurons. Quantitative electron microscopic techniques will be used to evaluate polyribosome distribution on neurons in vivo and in vitro while varying the extent of afferent innervation and the rate of synaptogenesis. 2) We will evaluate whether synapse replacement following lesions depends on local synthesis of protein at the postsynaptic site; we will evaluate whether synaptogenesis is blocked when synthesis in dendritic laminae is inhibited during the period of synapse construction. 3) We will evaluate whether afferent activity affects protein synthesis in dendrites. 4) We will use pulse-labeling techniques in vitro in conjunction with subcellular fractionation to determine whether some of the proteins that are produced at the postsynaptic site are constituents of the synaptic junctional complex. 5) We will determine what types of mRNA are present at the postsynaptic site by isolating mRNA from dendrites and evaluating whether the message is capable of directing the synthesis of functional neurotransmitter receptors or channels dendrite specific cytoskeletal proteins or constituents of the psd. 6) We will define how the arborization pattern of individual axons of the crossed temporodentate pathway changes during sprouting using the PHA- L method.
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