Overexpression of and RNA Interference with the CCAAT Enhancer-Binding Protein on Long-Term Facilitation ofAplysiaSensory to Motor Synapses

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Abstract
In the marine mollusk Aplysia, the CCAAT/enhancer-binding protein, ApC/EBP, serves as an immediate early gene in the consolidation of long-term facilitation in the synaptic connection between the sensory and motor neurons of the gill-withdrawal reflex. To further examine the role of ApC/EBP as a molecular switch of a stable form of long-term memory, we cloned the full-length coding regions of two alternatively spliced forms, the short and long form of ApC/EBP. Overexpression of each isoform by DNA microinjection resulted in a l6-fold increase in the expression of the coinjected luciferase reporter gene driven by an ERE promoter. In addition, when we overexpressed ApC/EBP in Aplysia sensory neurons, we found that the application of a single pulse of 5-HT that normally induced only short-term facilitation now induced long-term facilitation. Conversely, when we attempted to block the synthesis of native ApC/EBP by microinjecting double-strand RNA or antisense RNA, we blocked long-term facilitation in a sequence-specific manner. These data support the idea that ApC/EBP is both necessary and sufficient to consolidate short-term memory into long-term memory. Furthermore, our results suggest that this double-strand RNA interference provides a powerful tool in the study of the genes functioning in learning and memory in Aplysia by specifically inhibiting both the constitutive and induced expression of the genes.