The long range aim of this research project is to identify the molecular mechanisms underlying the formation of associations in the nervous system. The studies proposed in this grant renewal will provide insights into general principles underlying memory formation following one-trial and multi-trial Pavlovian conditioning, and contribute to the elucidation of events that are essential for the transformation of memory into an enduring form. To pursue these goals we are conducting a proteomic analysis of Pavlovian conditioning in the marine mollusk Hermissenda, a preparation that has been used extensively for over 30 years in biochemical, biophysical, and molecular studies of associative learning. The proposed research will use a combination of proteomic techniques and mass spectrometry to identify proteins whose abundance is regulated by conditioning followed by cloning full length cDNA's based upon identified peptide sequences. Four specific aims form the basis for this renewal application. The first will involve proteomic profiling to examine differences in protein abundance between conditioned groups and unpaired controls at different times following one-trial in vitro conditioning using difference gel electrophoresis (DIGE) and Cy Dye labeling of proteins in lysate samples. Mass spectrometry (MS) will be used to provide identification of proteins that exhibit statistically significant differences in protein abundance. We will also examine long-term changes in protein abundance supporting the maintenance of memory produced by multi-trial Pavlovian conditioning. The second aim will involve detecting changes in protein abundance regulated by one-trial in vitro conditioning in two types of identified neurons shown previously to express intrinsic cellular and synaptic plasticity. Isolated eyes and identified type I interneurons will receive in vitro conditioning followed by DIGE and gel image analysis to identify proteins regulated by one-trial conditioning. The proposed proteomic analysis of identified neuron types will be less likely to produce an overall average result that might mask subtle, localized changes since there will be a homogeneity of cell types studied following conditioning. The third aim will be addressed by the application of quantitative double-label autoradiography and phosphor imaging to identify proteins whose synthesis is regulated by one trial-conditioning at different time points post-conditioning. We will identify proteins in the 2D gel field of all 35S-labeled proteins that are phosphoproteins by incubating lysate samples of conditioned and control nervous systems with both 35S-methionine and 32P-orthophosphate, and imaging the same gel twice, before and after the 35S signal is blocked by a filter. The final aim will assess the role of candidate proteins in memory formation by blocking protein expression with RNAi techniques. Based upon pilot data we will also examine the role of tropomyosin and gelsolin in memory formation. These studies in conjunction with MS analysis will provide evidence for both de novo synthesis and post-translational modifications of identified proteins contributing to the induction and maintenance of long-term memory.