Microarray analysis of gene expression profiles with or without manipulations of neuronal activity

The process of synapse turnover is regulated by specific signaling mechanisms. Various molecules that promote formation and differentiation of synapses have been identified. Some of these molecules were classified as “synapse organizers,” as they were shown to initiate differentiation of hemi-synapses when they were expressed in non-neuronal cells and co-cultured with neurons.On the other hand, little is known about the mechanisms underlying destabilization and elimination of unnecessary synapses. We used microarray profiling of dissociated hippocampal culture to identify genes in response to the down-regulation of neuronal activity in hippocampal neurons. Cultured hippocampal neurons were prepared from embryonic day 16.5 ICR mouse embryos. Hippocampi were dissected, trypsinized, and dissociated. Cells were plated onto 35 mm-diameter culture dishes (BD Falcon) . Cells were cultured in Minimum Essential Medium with B18 supplement and 5.5% Fetal Bovine Serum (Equitech-Bio). RNA from primary cultured neurons at 12 DIV with or without pharmacological treatments (2 μM TTX for 2days ) was purified using SV Total RNA Isolation System (Promega) according to the manufacturer's instructions. After confirming the absence of RNA degradation, RNA samples were hybridized to Agilent 8X60K Whole Mouse Genome microarrays. Array data were analyzed by quantile normalization, determination of differential gene expression, and classification of different gene expression by ontology-based methods.