Activity-dependent genes such as arc
are quickly transcribed after LTP-inducing stimulation, and so the transcription factors (TFs) regulating those genes can be studied using electrophoretic mobility shift assays (EMSAs). In previous work from our lab, we used transcription factor arrays to identify TFs of interest from rat hippocampal slices that had been electrically stimulated to induce LTP (Hudgins and Dudek, 2002 Society for Neuroscience abstracts). Using oligonucleotides with the consensus sequences to TF binding sites identified in the arrays and others known to be in the arc
promoter region [16
], we performed EMSAs on similar nuclear extracts to test for the role of the NMDA receptors. To test whether or not LTP-inducing stimulation could continue to activate TFs when action potentials were maintained, extracts were made from slices that had been electrically stimulated either in the presence or absence of APV, an NMDA receptor antagonist. This treatment consisted of eliminating fast synaptic inhibition with bicuculline, a GABA-A receptor antagonist that preserved action potentials during the continued NMDA receptor blockade.
Five minutes post-stimulation we found that NMDA receptors were not required for the increase in TF binding to AP-1, CBF, CREB, or NFκB consensus sequence oligonucleotides when action potentials were maintained (). The effect of NMDA receptor blockade on SRE binding trended toward, but did not reach, significance (p=0.04, α=0.01). Thus, synaptic stimulation induces rapid transcription factor binding to at least several of the consensus sequences related to plasticity-regulated genes, and the binding is independent of NMDA receptor activation.
Fig. 1 Stimulation-induced transcription factor binding does not require NMDA receptors (or LTP) if action potentials are maintained. To insure that action potentials were maintained during NMDA receptor antagonist exposure (50 μM D-APV), synaptic stimulation (more ...)
Based on this data, we sought to determine whether a similar response could be seen with a gene whose induction had been previously shown to be NMDA receptor-dependent [5
]. Using real-time quantitative PCR analysis, we tested whether arc
, an immediate early gene, could be induced in the absence of NMDA receptor activation while maintaining action potentials with bicuculline. Like transcription factor binding, we found that LTP-inducing stimulation in the presence of bicuculline led to an increase in arc
transcription, and it was unaffected by the NMDA receptor blockade (). L-type voltage sensitive calcium channels were likely contributing, at least in part, to the calcium levels in the postsynaptic neurons: nifedipine, a blocker of voltage-gated calcium channels, significantly reduced the stimulation-induced increase in arc
in APV and bicuculline (APV/bicuculline: 1.53±0.12; APV/bicuculline/10 μM nifedipine: 1.25±0.11; p<0.018). We conclude that NMDA receptors are not necessary for stimulation-induced changes in arc
transcription when action potentials are preserved.
Fig. 2 Stimulation-induced arc expression does not require NMDA receptors if action potentials are maintained. To insure that action potentials were maintained during NMDA receptor antagonist exposure (50 μM D-APV), synaptic stimulation was delivered (more ...)