Background: cAMP-response-element-binding protein (CREB) is a ubiquitously expressed transcription factor in the brain that regulates neuroplasticity by modulating gene expression. There are numerous signaling pathways by which information is transmitted from the cell membrane to the nucleus that in turn affects CREB binding to DNA. The influx of calcium through N-methyl-D-aspartate receptors (NMDARs) is a well-defined mechanism that leads to the increased expression of CREB-dependent genes, including brain derived neurotrophic factor (BDNF), microRNA-132 (miR132), and activity-regulated cytoskeleton-associated protein (Arc). We have previously demonstrated that serine racemase knockout (SR-/-) mice, which exhibit NMDAR hypofunction, also have reduced mRNA and protein levels of the aforementioned CREB-dependent genes in the hippocampus. In addition, these molecules are reduced in schizophrenia.
Methods: Wild-type (WT; n=5-6) and SR-/- (n=5) mice were killed and their hippocampi were flash frozen on dry ice. Tissue samples were dissociated and then fixed with 1.5% paraformaldehyde. Chromatin was digested using the SimpleChIP Plus Enzymatic Chromatin IP kit (Cell Signaling Technologies). The chromatin was incubated with a rabbit anti-CREB antibody (1:50; Cell Signaling Technologies) overnight at 4°C on a rotisserie. The crosslinks were reversed and the DNA was purified for analysis using qPCR (Sybr Green). To determine the levels of CREB bound at each gene of interest, PCR primers were directed near the CRE sequence of each promoter. All of the primers were validated in previous publications. Melting temperature analysis was performed after every PCR to ensure accuracy.
