Identification of synaptic activity-dependent genes by exposure of cultured cortical neurons to tetrodotoxin followed by its withdrawal.
Activity-dependent gene expression is one of the key mechanisms of synaptic plasticity that form the basis of higher order functions such as learning and memory. In the present study, we surveyed for activity-dependent genes by analyzing gene expression changes accompanying reversible inhibition of synaptic activity by tetrodotoxin (TTX) using two types of DNA microarrays; our focused oligo DNA microarray "Synaptoarray" and the commercially available high-density array. Cerebral cortical cells from E18 rat embryos were cultured for 14 days to ensure synaptogenesis, then treated with 1 muM TTX for 48 hr without detectable effect on cell viability. Synaptic density estimated by the amount of Synapsin I and Synaptotagmin I was decreased 21-24% by TTX treatment, but recovered to the control level 48 hr after TTX withdrawal. Comparison of gene expression profiles by competitive hybridization of fluorescently labeled cRNA from TTX-treated and control cells showed an overall downregulation of the genes on the Synaptoarray by TTX-treatment with different recovery rates after TTX withdrawal. With 16 representative genes, microarray data were validated by real-time PCR analysis. Genes most severely downregulated by TTX and upregulated above the control level at 5 hr after TTX withdrawal were munc13-1 (involved in docking and priming of synaptic vesicles) and Shank2 (involved in the postsynaptic scaffold). In addition, comprehensive screening at 5 hr after TTX withdrawal using high density arrays resulted in additional identification of Rgs2, a regulator of trimeric G-protein signaling, as an activity-dependent gene. These three genes are thus likely to be key factors in the regulation of synaptic plasticity. (c) 2007 Wiley-Liss, Inc.
Kitamura
Chikara
C
Department of Chemistry and Biological Science, School of Science and Engineering, Aoyama-Gakuin University, Sagamihara, Kanagawa, Japan.
Takahashi
Masaki
M
Kondoh
Yasumitsu
Y
Tashiro
Hideo
H
Tashiro
Tomoko
T
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000187:drug effects / Q000502:physiology; D015870:Gene Expression / Q000187:drug effects*; D020869:Gene Expression Profiling; D007150:Immunohistochemistry; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000187:drug effects; D009473:Neuronal Plasticity / Q000187:drug effects / Q000235:genetics; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D020411:Oligonucleotide Array Sequence Analysis; D011042:Poisons / Q000008:administration & dosage*; D020710:RGS Proteins / Q000096:biosynthesis / Q000187:drug effects; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013569:Synapses / Q000187:drug effects / Q000235:genetics*; D013779:Tetrodotoxin / Q000008:administration & dosage*; D013997:Time Factors
D009419:Nerve Tissue Proteins; D011042:Poisons; D020710:RGS Proteins; C514563:RGS2 protein, rat; C485686:Shank2 protein, rat; C103912:Unc13a protein, rat; D013779:Tetrodotoxin