The major objective of this research is to provide insights into the regulation of biochemical and molecular events that occur while organisms learn and form memories. We are examining the hypothesis that regulation of gene transcription and protein translation is involved in the formation of long-term memories. Moreover, a cascade of transcription events is hypothesized to affect the translation of a diverse array of proteins which control a broad spectrum of cellular functions. The specific framework of our studies is sensitization of the defensive tail-siphon withdrawal reflex in Aplysia, and mRNA and protein changes in large homogeneous clusters of bilaterally-symmetrical sensory neurons in the pleural ganglia. These sensory neurons are key components of the reflex and its plasticity. This proposal has four Specific Aims which are designed to extend our previous research in this area.
Specific Aim 1 is to discover proteins whose synthesis is regulated by treatments that mimic some effects of sensitization training and then characterize their temporal responses.
Specific Aim 2 is to obtain partial amino acid sequences and gene sequences of proteins or mRNAs that are affected by induction treatments.
Specific Aim 3 is to determine if behavioral training or 5-HT alters levels of specific mRNAs in sensory neurons and define the time domains in which changes occur.
Specific Aim 4 is to obtain antibodies to an Aplysia Tolloid-like protein that is similar to a previously-identified, developmentally-regulated protein. We will use the antibodies to localize the protein in neural tissue and to examine the relationship between changes in Tolloid mRNA and changes in the synthesis of the Tolloid protein. The research will use quantitative analysis of proteins separated by two dimensional gel electrophoresis, peptide sequencing, gene cloning and sequencing, and other techniques of molecular biology. Ribonuclease protection assays will be used to measure levels of mRNAs. Antibodies will be used in several types of studies including Western blotting and immunocytochemistry. This research will provide substantial new insights into the mechanisms for the induction, storage and read-out of a form of long-term memory. Moreover, the research will provide general information on the role of specific proteins and genes in cellular function and plasticity.
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