We have previously shown that repetitive exposures to glutamate (100 μM, 3 min, three times at 24-hr intervals) induced a long-lasting synaptic enhancement accompanied by synaptogenesis in rat hippocampal slice cultures, a phenomenon termed RISE (for repetitive LTP-induced synaptic enhancement). To investigate the molecular mechanisms underlying RISE, we first analyzed the time course of gene expression changes between 4 hr and 12 days after repetitive stimulation using an original oligonucleotide microarray: "synaptoarray. " The results demonstrated that changes in the expression of synapse-related genes were induced in two time phases, an early phase of 24-96 hr and a late phase of 6-12 days after the third stimulation. Comprehensive screening at 48 hr after the third stimulation using commercially available high-density microarrays provided candidate genes responsible for RISE. From real-time PCR analysis of these and related genes, two categories of genes were identified, 1) genes previously reported to be induced by physiological as well as epileptic activity (bdnf, grm5, rgs2, syt4, ania4/carp/dclk) and 2) genes involved in cofilin-based regulation of actin filament dynamics (ywhaz, ssh1l, pak4, limk1, cfl). In the first category, synaptotagmin 4 showed a third stimulation-specific up-regulation also at the protein level. Five genes in the second category were coordinately upregulated by the second stimulation, resulting in a decrease in cofilin phosphorylation and an enhancement of actin filament dynamics. In contrast, after the third stimulation, they were differentially regulated to increase cofilin phosphorylation and enhance actin polymerization, which may be a key step leading to the establishment of RISE.
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