Consolidation of synaptic plasticity seems to require transcription, but the way the nucleus is informed in this context remains to be unknown. not need NMDA receptors. These data show that synaptic stimulation induces many biochemical occasions linked to transcription and that NMDA receptors do not need to be straight involved. Strategies Slice planning and electric stimulation Hippocampal slices (350 m) had been prepared from 5C7 week older Sprague-Dawley rats. Slices had been lower on a vibratome at 4C in artificial cerebrospinal liquid (ACSF) that contains (in mM): KCl, 4; sucrose, 240; NaH2PO4, 1.25; NaHCO2, AG-014699 supplier 26; CaCl2, 1; MgCl2, 3; glucose, 10; bubbled with 95/5% O2/CO2. Mini-slices of CA1 had been microdissected in ice-cool cutting ACSF, and they were put into an interface documenting chamber and perfused with regular ACSF (NaCl, 124; KCl, 4; NaH2PO4, 1.25; NaHCO2, 26; CaCl2, 2.5; MgCl2, 1.5; and glucose, 10) at 34C for 2C3 hours ahead of stimulation. After program of 10 M bicuculline or bicuculline + 50 M APV for 20C40 mins, a concentric bipolar stimulating electrode (FHC) put into the stratum radiatum was AG-014699 supplier utilized to AG-014699 supplier stimulate the mini-slices with a theta-burst design (130 s duration, 100 A). This pattern of stimulation is known to induce LTP in CA1 pyramidal neurons [14] and induces action potentials resistant to the inhibitory effects of APV [7]. LTP was effectively blocked by this concentration of APV, even in the presence of bicuculline (n=3, data not shown). This stimulus intensity and duration was found to evoke population spikes AG-014699 supplier to the edge of the mini-slices, resulting in an estimated 60C80% of the cells being activated based on phospho-ERK staining [15]. Five (EMSA) or fifteen (qPCR) minutes after electrical stimulation, slices were removed from the chamber, snap-frozen on dry ice, and stored at ?80C. One control (non-stimulated) slice was removed and frozen for each stimulated slice to match for time after cutting and drug exposure. Electrical stimulation without bicuculline did not reliably induce 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 SERPINF1 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 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 AG-014699 supplier 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 NFB consensus sequence oligonucleotides when action potentials were maintained (Figure 1). The effect of NMDA receptor blockade on SRE binding trended toward, but did not reach, significance (p=0.04, =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. Open in a separate window 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 M D-APV), synaptic stimulation was delivered in a theta burst pattern of stimulation (TBS) with bicuculline [7]. Hippocampal CA1 mini-slices were sampled 5 minutes after stimulation. Nuclear protein extracts were assessed for transcription factor binding by electrophoretic mobility shift assays (EMSAs). Example gels are shown on the left, with arrows marking the bands representing specific binding. Plotted.