Arc protein-protein interactions in long-term potentiation in the rat dentate gyrus in vivo
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Synaptic plasticity is the ability of neuronal synapses to change in strength over time, and is considered to be a foundation for learning and memory. Long-term potentiation (LTP) is a widely studied form of synaptic plasticity in the mammalian brain. In LTP, brief high-frequency stimulation (HFS) of afferent fibers results in a long-lasting (hours to days) increase is excitatory synaptic transmission. HFS-evoked expression of the immediate early gene Arc, (activity-regulated cytoskeleton-associated protein) is required for stabilization LTP. However, the mechanisms by which Arc acts to generate and stabilize LTP are unknown. The identification of proteins that bind to interact with Arc during LTP is the key to understanding how Arc works. To this end, we evalutated possible interaction of Arc with two candidate interacting proteins, Dynamin 2 and Syntaxin 4 (Stx4). In vivo electrophysiological recording was done on anesthetized rats to determine LTP induction due to HFS and later compared with another group of rats, received baseline test stimuli but not any HFS. Differences between HFS-treated dentate gyrus and the contralateral control dentate gyrus in terms of Arc protein expression was measured by western blot method. HFS-treated and the contralateral control dentate gyrus were analyzed for Dynamin 2 and Stx4 co-immunoprecipitation (Co- IP) by Arc protein Immunoprecipitation (IP) at various time points. Our experimental proof indicated stable LTP induction after HFS and this percentage was higher to 40-50 % of increase to baseline. We also found that Arc is rapidly synthesized after tetanic stimulation. From our Co-IP experiments we found that Dynamin 2 and Stx4 are the prime binding partners of Arc protein during LTP. Arc-Dynamin 2 interaction may be highest during 60 minutes after HFS. Stx4 might be a candidate binding partner for Arc protein during LTP. Stx4 IP showed Dynamin 2 as a binding partner of Stx4. From this study we concluded that Arc is rapidly synthesized after HFS. During LTP Dynamin 2 and Stx4 are binding partners of Arc. Stx4 and Dynamin 2 are present in the sample protein complex.