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• W2130270377 abstract "Activity-mediated gene expression is thought to play an important role in many forms of neuronal plasticities. We have used pentylenetetrazol-induced seizure that produces synchronous and sustained neuronal activity as a model to examine the mechanism(s) of gene activation. The transcription factor CREB (Ca2+/cAMP response element-binding protein) is thought to be necessary for long-term memory formation both in invertebrates and vertebrates. When phosphorylated on Ser133 either by cAMP-dependent protein kinase and/or Ca2+/calmodulin-dependent protein kinases, CREB increases transcription of genes containing the CRE (cAMP response element) sequence. Using an antibody that detects Ser133-phosphorylated CREB protein, we show that CREB phosphorylation is maximal between 3 and 8 min after the onset of seizure activity and declines slowly both in the hippocampus and the cortex. The total amount of CREB protein did not change at the time points examined. The increased phosphorylation of CREB protein is preceded by an increase in the amount of cAMP, suggestive of cAMP-dependent protein kinase activation, in the hippocampus and activation of Ca2+/calmodulin-dependent protein kinases in the cortex. Subsequent to CREB phosphorylation, the expression of the CRE-containing gene, c-fos, and the AP-1 complexes (heterodimers of Fos and Jun family members) is increased. These findings support the role of CREB-mediated gene expression in activity-dependent neuronal plasticities. Activity-mediated gene expression is thought to play an important role in many forms of neuronal plasticities. We have used pentylenetetrazol-induced seizure that produces synchronous and sustained neuronal activity as a model to examine the mechanism(s) of gene activation. The transcription factor CREB (Ca2+/cAMP response element-binding protein) is thought to be necessary for long-term memory formation both in invertebrates and vertebrates. When phosphorylated on Ser133 either by cAMP-dependent protein kinase and/or Ca2+/calmodulin-dependent protein kinases, CREB increases transcription of genes containing the CRE (cAMP response element) sequence. Using an antibody that detects Ser133-phosphorylated CREB protein, we show that CREB phosphorylation is maximal between 3 and 8 min after the onset of seizure activity and declines slowly both in the hippocampus and the cortex. The total amount of CREB protein did not change at the time points examined. The increased phosphorylation of CREB protein is preceded by an increase in the amount of cAMP, suggestive of cAMP-dependent protein kinase activation, in the hippocampus and activation of Ca2+/calmodulin-dependent protein kinases in the cortex. Subsequent to CREB phosphorylation, the expression of the CRE-containing gene, c-fos, and the AP-1 complexes (heterodimers of Fos and Jun family members) is increased. These findings support the role of CREB-mediated gene expression in activity-dependent neuronal plasticities. Neuronal activity plays a critical role in many forms of plasticities such as learning and memory (1Bear M.F. Kleinschmidt A. Gu Q. Singer W. J. Neurosci. 1990; 10: 909-925Crossref PubMed Google Scholar, 2McNaughton B.L. Siefert W. Neurobiology of the Hippocampus. Academy, New York1983: 233-252Google Scholar). Studies on the formation of long-term memory indicate that the induction of immediate-early genes is often associated with memory storage (3Rose S.P.R. Trends Neurosci. 1991; 14: 390-397Abstract Full Text PDF PubMed Scopus (255) Google Scholar, 4Mello C.V. Vicario D.S. Clayton D.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6818-6822Crossref PubMed Scopus (508) Google Scholar). The immediate-early gene products are thought to activate late-effector genes, which alter the structural and functional properties of nerve cells (5Goelet P. Castellucci V.F. Schacher S. Kandel E.R. Nature. 1986; 322: 419-422Crossref PubMed Scopus (802) Google Scholar, 6Cole A.J. Saffen D.W. Baraban J.M. Worley P.F. Nature. 1989; 340: 474-476Crossref PubMed Scopus (885) Google Scholar, 7Sheng M. Greenberg M.E. Neuron. 1990; 4: 477-485Abstract Full Text PDF PubMed Scopus (1938) Google Scholar, 8Morgan J.I. Curran T. Annu. Rev. Neurosci. 1991; 14: 421-451Crossref PubMed Scopus (2443) Google Scholar). How neuronal activity is coupled to alteration in gene expression is poorly understood. However, previous studies have shown that phosphorylation of the transcription factor CREB 1The abbreviations used are: CREBcAMP response element-binding proteinPBSphosphate-buffered salineTRE12-O-tetradecanoylphorbol-13-acetate response elementPMSFphenylmethylsulfonyl fluorideCaMKcalmodulin-dependent protein kinasePTZpentylenetetrazolPKAprotein kinase ACREcAMP response element. appeared to be important in mediating the expression of several immediate-early genes (9Yamamoto K.K. Gonzalez G.A. Biggs III, W.A. Montminy M.R. Nature. 1988; 334: 494-498Crossref PubMed Scopus (961) Google Scholar, 10Ginty D.D. Kornhauser J.M. Thompson M.A. Bading H. Mayo K.E. Takahashi J.S. Greenberg M.E. Science. 1993; 260: 238-241Crossref PubMed Scopus (731) Google Scholar, 11Konradi C. Cole R.L. Heckers S. Hyman S.E. J. Neurosci. 1994; 14: 5623-5634Crossref PubMed Google Scholar). Moreover, CREB protein has been implicated in the formation of long-term memory. For example, blockade of CREB by injection of CRE sequence containing oligonucleotides impedes long-term facilitation, a correlate of memory in the marine mollusc Aplysia californica (12Dash P.K. Hochner B. Kandel E.R. Nature. 1990; 345: 718-721Crossref PubMed Scopus (583) Google Scholar, 13Alberini C.M. Ghirardi M. Metz R. Kandel E.R. Cell. 1994; 76: 1099-1114Abstract Full Text PDF PubMed Scopus (489) Google Scholar). “Knock out” mice, with a deletion in the CREB gene, and Drosophila melanogaster, expressing a repressor form of CREB protein, show deficits in long-term memory without any effect on short-term memory (14Bourtchuladze R. Frenguelli B. Blendy J. Cioffi D. Schutz G. Silva A.J. Cell. 1994; 79: 59-68Abstract Full Text PDF PubMed Scopus (1547) Google Scholar, 15Yin J.C.P. Wallach J.S. Vecchio M. Wilder E.L. Zhou H. Quinn W.G. Tully T. Cell. 1994; 79: 49-58Abstract Full Text PDF PubMed Scopus (814) Google Scholar). In addition, overexpression of wild type CREB in Drosophila facilitates the formation of long-term memory (16Yin J.C.P. Del Vecchio M. Zhou H. Tully T. Cell. 1995; 81: 107-115Abstract Full Text PDF PubMed Scopus (552) Google Scholar). These studies indicate a role for CREB-mediated transcription in long-term memory rather than for transient or short-term memory. cAMP response element-binding protein phosphate-buffered saline 12-O-tetradecanoylphorbol-13-acetate response element phenylmethylsulfonyl fluoride calmodulin-dependent protein kinase pentylenetetrazol protein kinase A cAMP response element. The CREB/ATF (activating transcription factor) family of proteins binds to the CRE sequence located in the promoter regions of Ca2+/cAMP-inducible genes (17Montminy M.R. Sevarino K.A. Wagner J.A. Mandel G. Goodman R.H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6682-6686Crossref PubMed Scopus (1046) Google Scholar, 18Hyman S.E. Comb M. Lin Y.S. Pearlberg J. Green M.R. Goodman H.M. Mol. Cell. Biol. 1988; 8: 4225-4233Crossref PubMed Scopus (108) Google Scholar, 19Hoffler J.P. Meyer T.E. Yun Y. Jameson J.L. Habener J.F. Science. 1988; 242: 1430-1432Crossref PubMed Scopus (520) Google Scholar, 20Sheng M. Thomson M.A. Greenberg M.E. Science. 1991; 252: 1427-1430Crossref PubMed Scopus (1262) Google Scholar). Increases in intracellular cAMP result in the activation of protein kinase A (PKA) by the dissociation of the catalytic from the regulatory subunits. Prolonged increase in cAMP causes translocation of the catalytic subunits into the nucleus (21Adams S.R. Harootunian A.T. Buechler Y.J. Taylor S.S. Tsien R.Y. Nature. 1991; 349: 694-697Crossref PubMed Scopus (536) Google Scholar). It has also recently been shown that isozymes of Ca2+/calmodulin-dependent protein kinase (CaMK IV and certain isoforms of CaMK II) are localized to the nucleus (22Mathews R.P. Guthrie C.R. Wailes L.M. Zhao X. Means A.R. McNight G.S. Mol. Cell. Biol. 1994; 14: 6107-6116Crossref PubMed Scopus (489) Google Scholar, 23Tokumitsu H. Brickey D.A. Glod J. Hidaka H. Sikela J. Soderling T.R. J. Biol. Chem. 1994; 269: 28640-28647Abstract Full Text PDF PubMed Google Scholar). Once activated, PKA or CaMK can phosphorylate CREB on Ser133 (20Sheng M. Thomson M.A. Greenberg M.E. Science. 1991; 252: 1427-1430Crossref PubMed Scopus (1262) Google Scholar, 24Gonzalez G.A. Yamamoto K.A. Fisher W.H. Karr D. Menzel P. Bigg III, W. Vale W.W. Montminy M.R. Nature. 1989; 337: 749-752Crossref PubMed Scopus (643) Google Scholar, 25Dash P.K. Karl K.A. Colicos M.A. Prywes R. Kandel E.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5061-5065Crossref PubMed Scopus (467) Google Scholar, 26Enslen H. Sun P. Brickey D. Soderling S.H. Klamo E. Soderling T.R. J. Biol. Chem. 1994; 269: 15520-15527Abstract Full Text PDF PubMed Google Scholar). Phosphorylated CREB protein then binds to CREB-binding protein (27Chrivia J.C. Knok R.P. Lamb N. Hagiwara M. Montminy M.R. Goodman R.H. Nature. 1993; 365: 855-859Crossref PubMed Scopus (1740) Google Scholar, 28Lundblad J.R. Kwok R.P.S. Laurance M.E. Harter M.L. Goodman R.H. Nature. 1995; 374: 85-88Crossref PubMed Scopus (526) Google Scholar), and this complex is thought to induce transcription of genes containing CRE sequences such as c-fos (7Sheng M. Greenberg M.E. Neuron. 1990; 4: 477-485Abstract Full Text PDF PubMed Scopus (1938) Google Scholar), tyrosine hydroxylase (29Kim K.S. Lee M.K. Carroll J. Joh T.H. J. Biol. Chem. 1993; 268: 15689-15695Abstract Full Text PDF PubMed Google Scholar), enkephalin (18Hyman S.E. Comb M. Lin Y.S. Pearlberg J. Green M.R. Goodman H.M. Mol. Cell. Biol. 1988; 8: 4225-4233Crossref PubMed Scopus (108) Google Scholar), somatostatin (17Montminy M.R. Sevarino K.A. Wagner J.A. Mandel G. Goodman R.H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6682-6686Crossref PubMed Scopus (1046) Google Scholar), and vasoactive intestinal peptide (30Deutsch P.J. Hoeffler J.P. Jameson J.L. Lin J.C. Habener J.F. J. Biol. Chem. 1988; 263: 18466-18472Abstract Full Text PDF PubMed Google Scholar). Many models of neuronal activity, including seizure, produce long-lasting changes in both the structure and function of the brain (31Cotman C.W. Nieto-Sampedro M. Science. 1984; 225: 1287-1294Crossref PubMed Scopus (210) Google Scholar, 32Dichter M.A. Ayala G.F. Science. 1987; 237: 157-164Crossref PubMed Google Scholar, 33Greenough W.T. Bailey C.H. Trends Neurosci. 1988; 11: 142-147Abstract Full Text PDF Scopus (204) Google Scholar). Several of these studies have demonstrated that c-fos, fra-1, nur/77, c-jun, prodynorphin, enkephalin, and nerve growth factor are induced during stimulation of intact nervous systems, supporting the idea that these genes are important regulators of nerve cell responses in vivo (34Anokhin K. Mileusnic R. Shamakina I. Rose S.P.R. Brain Res. 1991; 544: 101-107Crossref PubMed Scopus (151) Google Scholar, 35Cole A.J. Abu-Shakra S. Saffen D.W. Baraban J.M. Worley P.F. J. Neurochem. 1990; 55: 1920-1927Crossref PubMed Scopus (182) Google Scholar, 36Herdegen T. Sandkuhler J. Gass P. Kiessling M. Bravo R. Zimmermann M. J. Comp. Neurol. 1993; 333: 271-288Crossref PubMed Scopus (134) Google Scholar, 37Wisden W. Errington M.L. Williams S. Dunnet S.B. Waters C. Hitchcock D. Evans G. Bliss T.V.P. Hunt S.P. Neuron. 1990; 4: 603-614Abstract Full Text PDF PubMed Scopus (606) Google Scholar). For example, long-term potentiation in the hippocampus, a proposed model for spatial learning and memory, is associated with increased expression of zif/268 (6Cole A.J. Saffen D.W. Baraban J.M. Worley P.F. Nature. 1989; 340: 474-476Crossref PubMed Scopus (885) Google Scholar). Direct electrical stimulation as well as kindling stimulation increase c-fos mRNA (38Dragunow M. Robertson H.A. Nature. 1987; 329: 441-442Crossref PubMed Scopus (502) Google Scholar, 39White Gall C. Brain Res. 1987; 427: 21-29Crossref PubMed Scopus (163) Google Scholar). c-Fos also is increased in the paraventricular and supraoptic nuclei involved in thirst control following 24-h water deprivation (40Sagar S.M. Sharp F.R. Curran T. Science. 1988; 240: 1328-1331Crossref PubMed Scopus (1742) Google Scholar) and in the dorsal horn neurons of the spinal cord following peripheral sensory stimulation (40Sagar S.M. Sharp F.R. Curran T. Science. 1988; 240: 1328-1331Crossref PubMed Scopus (1742) Google Scholar, 41Hunt S.P. Pini A. Evan G. Nature. 1987; 328: 632-634Crossref PubMed Scopus (1731) Google Scholar). Taken together, these in vivo studies are consistent with the idea that alteration in gene expression may underlie longlasting changes in brain function. Many of the genes induced by neuronal activity have CRE-like sequences in their promoter regions and therefore are likely to be induced by CREB phosphorylation (7Sheng M. Greenberg M.E. Neuron. 1990; 4: 477-485Abstract Full Text PDF PubMed Scopus (1938) Google Scholar). Using pentylenetetrazol (PTZ)-induced seizure to induce maximal neuronal activity (42Qian Z. Gilbert M.E. Colicos M.A. Kandel E.R. Kuhl D. Nature. 1993; 361: 453-457Crossref PubMed Scopus (634) Google Scholar), we have examined the phosphorylation of the CREB protein. We have utilized an antibody that specifically detects CREB when phosphorylated on Ser133 (10Ginty D.D. Kornhauser J.M. Thompson M.A. Bading H. Mayo K.E. Takahashi J.S. Greenberg M.E. Science. 1993; 260: 238-241Crossref PubMed Scopus (731) Google Scholar). In this study, we report that the phosphorylation of CREB is maximal between 3 and 8 min after the onset of seizure both in the hippocampus and cortex and declines slowly thereafter. This enhanced phosphorylation of CREB can be reduced by pretreatment with sodium pentobarbital, a GABA receptor agonist which blocks neuronal activity (43Sanna E. Garau F. Harris R.A. Mol. Pharmacol. 1995; 47: 213-217PubMed Google Scholar). Preceding CREB phosphorylation, we observed an increased activity of autophosphorylated CaMK in the cortex and enhanced concentrations of intracellular cAMP in the hippocampus. Finally, we report that subsequent to CREB phosphorylation, the expression of a CRE-containing gene c-fos and AP-1 complexes (heterodimers of Fos and Jun family members) is increased. These data suggest that neuronal activity stimulates PKA and CaMK, resulting in the phosphorylation of the CREB protein and induction of CRE-containing genes. PhosphoCREB and CREB antibodies were purchased from UpState Biotechnology (Lake Placid, NY). c-Fos antibodies were bought from Santa Cruz Biotechnology (Santa Cruz, CA). [32P]ATP was obtained from Amersham Corp. Male rats (180–200 g) were purchased from Harlan Sprague-Dawley (Indianapolis, IN). All procedures involving the use of animals were performed under the guidelines of the National Institutes of Health's Guide for Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee. Animals were injected intraperitoneally with 55 mg/kg of PTZ prepared in saline (42Qian Z. Gilbert M.E. Colicos M.A. Kandel E.R. Kuhl D. Nature. 1993; 361: 453-457Crossref PubMed Scopus (634) Google Scholar). Control animals were injected with saline. As an additional control, some animals were intraperitoneally injected with 30 mg/kg of sodium pentobarbital 15 min prior to PTZ injection. At various times following injection, animals were sacrificed by a guillotine. The cortical (parietal and half of the frontal cortex) and the hippocampal tissues were quickly removed in oxygenated ice-cold artificial cerebrospinal fluid (10 mM HEPES, pH 7.2, 1.3 mM NaH2PO4, 3 mM KCl, 124 mM NaCl, 10 mM dextrose, 26 mM NaHCO3, 2 mM CaCl2, and 2 mM MgCl2). Sample preparation was carried out at 4°C. The tissues were separately homogenized (10 strokes) in 5 volumes of a buffer containing 15 mM HEPES, pH 7.2, 0.25 M sucrose, 60 mM KCl, 10 mM NaCl, plus protease inhibitors (1 mM EGTA, 5 mM EDTA, and 1 mM PMSF), and phosphatase inhibitors (2 mM NaF, 2 mM NaPPi, and 5 µM microcystin-LR) in a Dounce homogenizer using a loose pestle. The cells were then pelletized at 2,000 × g for 10 min. To lyse the cells, the pelletized material was incubated in 5 volumes of 10 mM HEPES, pH 7.2, 1.5 mM MgCl2, 10 mM KCl, 1 mM PMSF, 5 µM microcystin-LR, 2 mM NaF, and 2 mM NaPPi for 5 min. The cell suspension was homogenized (7 strokes) in a Dounce homogenizer using a tight pestle. The nuclei were pelletized at 4,000 × g for 10 min. The nuclei were lysed in 1 bed volume of 100 mM HEPES, pH 7.2, 1.5 mM MgCl2, 1 mM EDTA, 0.8 M NaCl, 25% glycerol, 2 mM NaF, 2 mM NaPPi, 1 mM PMSF, and 5 µM microcystin-LR by gentle rocking for 30 min. Cell debris and genomic DNA were removed by centrifugation at 14,000 × g for 30 min. The supernatant solution was removed and frozen. At different time points following the onset of seizure, rats were sacrificed by decapitation. The cortex and hippocampus were immediately dissected while submerged under oxygenated ice-cold artificial cerebrospinal fluid without Ca2+ or Mg2+. The tissues were homogenized in 10 volumes of 10 mM Tris/HCl (pH 7.4), 1 mM EGTA, 1 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM PMSF, 10 µg/ml leupeptine, 0.5 mM isobutylmethylxanthine, and 5 µM microcystin-LR. After 20 strokes in a motor-driven Teflon-glass homogenizer, 0.6 ml of each sample was added to an equal volume of 12% trichloroacetic acid for cAMP measurements. The rest of the homogenate was aliquoted and centrifuged at 14,000 × g for 30 min at 4°C. The total homogenates, supernatant solutions, and pelletized materials were stored at −80°C. For cAMP sample preparation, the 0.6 ml of homogenate mixed with an equal volume of 12% trichloroacetic acid was allowed to sit on ice for 10 min. This was followed by centrifugation at 14,000 × g for 10 min. The supernatant solutions were collected and divided into three aliquots. Aliquots were washed five times with 2.5 volumes of water-saturated ether to remove the trichloroacetic acid. Each wash was performed by vigorous vortexing followed by centrifugation at 14,000 × g for 2 min to separate the two phases. The organic phase was discarded prior to the addition of the next wash. Ether-extracted samples were frozen at −80°C until ready for use. For CREB and c-Fos Western blots, nuclear extracts were diluted in water to reduce the salt concentration to approximately 100 mM prior to use. For Western blots examining the α isoform of CaMK II, supernatant solutions were thawed on ice and sonicated briefly to ensure homogeneity. The pellet samples were resuspended in 200 µl of homogenization buffer (10 mM Tris/HCl (pH 7.4), 1 mM EGTA, 1 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM PMSF, 10 µg/ml leupeptin, 0.5 mM isobutylmethylxanthine, and 5 µM microcystin-LR) and sonicated briefly. The amount of protein in each sample was measured using a Micro BCA assay (Pierce). Samples were prepared by boiling in sample buffer, and equal amounts of protein were separated on a 8.5% SDS-polyacrylamide gel electrophoresis. The proteins were transferred to an Immobilon-P membrane (Millipore) using a semi-dry transfer apparatus (Millipore). Western blots were carried out using antibodies at a final concentration of 0.2 µg/ml as described previously (44Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). The blots were visualized using an alkaline phosphatase chemiluminescence detection system (Life Technologies, Inc.), which allowed the production of multiple fluorographs with different lengths of exposure. The quantification of the immunoreactive bands was carried out utilizing a Bio-Rad model GS-670 imaging densitometer. The Western blots were repeated in at least four independent experiments. Equal amounts of the nuclear extracts were used for gel retardation assays using the 12-O-tetradecanoylphorbol-13-acetate response element (TRE) sequence as a probe for measuring the AP-1 binding. The double-stranded oligonucleotide probe was synthesized using the metallothionein gene TRE sequence 5′-GATCTGTGAGTCAGCGCGA-3′ and its complement sequence (45Lee W. Haslinger A. Karin M. Tjian R. Nature. 1987; 325: 368-372Crossref PubMed Scopus (482) Google Scholar). Probe preparation and assays were carried out essentially as described previously (46Dash P.K. Moore A.N. Cell. Mol. Biol. 1993; 39: 35-43PubMed Google Scholar). For competition with oligonucleotides, the cold competitors were added to the reaction mixture prior to the addition of protein extracts. The oligonucleotides used for competition were 5′-GATCATCTCAATTAGTCAGCAA-3′ and its complement for TRE, and 5′-GGAGCGCGCCTCGAATGTTCTAGAAAAGGCTGCA-3′ and its complement for the heat shock element. Supershift assays were performed by preincubating the nuclear extracts with 0.2 µg of c-Fos antibodies in the absence of any thiol reagent for 15 min at room temperature. The resulting antibody-antigen complex was then incubated with the probe. Hippocampi were removed quickly as described under “Preparation of Nuclear Extracts.” The tissues were fixed in cold 4% paraformaldehyde and 15% picric acid in phosphate-buffered saline (PBS) for 8–10 h. The tissue was then placed in 30% sucrose in PBS overnight at room temperature. Tissues were briefly dried to remove any surface liquid and mounted in optimal cutting temperature (O.C.T., Miles Inc.) cryostat imbedding medium. 50-micron slices were prepared using a cryostat. Slices were washed twice with PBS and incubated with 5 µg/ml primary antibody in 2% bovine serum albumin, 0.3% Triton X-100, and 1.5% normal goat serum in PBS at room temperature overnight. Slices were washed four times with PBS, and immunoreactivity was detected using an avidin-biotin detection system from Vector Laboratory essentially as suggested by the supplier. Supernatant and total homogenate samples were thawed on ice and sonicated briefly to ensure homogeneity. Pellet samples were resuspended and sonicated as described under “Western Blots.” The protein concentration in each sample was determined using Micro BCA assays with bovine serum albumin as the standard. CaMK activity was measured using a synthetic peptide substrate (autocamtide-3; KKALHRQETVDAL) (47Hanson P.I. Schulman H. J. Biol. Chem. 1992; 267: 17216-17224Abstract Full Text PDF PubMed Google Scholar, 48Kolb S.J. Hudmon A. Waxham M.N. J. Neurochem. 1995; 64: 2147-2152Crossref PubMed Scopus (45) Google Scholar). Phosphorylation reactions were initiated by adding 3 µg of protein to a 20-µl mixture resulting in the following final concentrations: 10 mM HEPES (pH 7.4), 0.5 mM dithiothreitol, 20 µM substrate peptide, 50 µM ATP, 3 mM EGTA, 5 mM MgCl2, and 3 µCi of [γ-32P]ATP (3,000 Ci/mmol). After a 1-min incubation at 30°C, the reactions were terminated by spotting 15 µl of the reaction mixture on P-81 phosphocellulose filters (Whatman). The filters were washed three times, 10 min each, in 75 mM phosphoric acid, rinsed with ethanol, and dried under air flow. The radioactivity on the phosphorylated peptide substrate was quantitated in a scintillation counter by the Cerenkov method. cAMP was measured using a radioimmunoassay kit purchased from Amersham. Frozen samples were completely dried in a vacuum centrifuge at ambient temperature. Dried samples were resuspended in diluted assay buffer as recommended by the vendor. cAMP measurements were performed utilizing the acetylation method and compared to a standard curve, which was prepared simultaneously. The amount of cAMP in each sample was normalized to the total amount of protein in the homogenate prior to trichloroacetic acid precipitation. The results from the three aliquots were averaged for each hippocampal or cortical sample. cAMP measurements were repeated in at least three independent experiments. An analysis of variance was computed to detect a significant main effect for groups (p < 0.05). This was followed by a Tukey's b post-hoc test to identify specific group differences. Statistical analyses were performed using the integrated optical densities (Western blots), counts per minute (kinase assays), or by comparison to standard curves (cAMP measurements). The data are presented as percent control. PTZ-induced seizure has been used as a model for robust neuronal activity (42Qian Z. Gilbert M.E. Colicos M.A. Kandel E.R. Kuhl D. Nature. 1993; 361: 453-457Crossref PubMed Scopus (634) Google Scholar, 58Ben-Ari Y. Represa A. Trends Neurosci. 1990; 8: 312-318Abstract Full Text PDF Scopus (306) Google Scholar). As described under “Experimental Procedures,” seizure activity was initiated by an intraperitoneal injection of PTZ at a dose of 55 mg/kg body weight (42Qian Z. Gilbert M.E. Colicos M.A. Kandel E.R. Kuhl D. Nature. 1993; 361: 453-457Crossref PubMed Scopus (634) Google Scholar). We consistently observed the physical manifestations of seizure activity in all animals approximately 58 s after injection of the drug. As previously reported, the activity began with acute facial and forelimb contraction progressing to animals running and bouncing as well as tonic convulsion (49Browning R.A. Wang C. Lanker M.L. Jobe P.C. Epilepsy Res. 1990; 6: 1-11Crossref PubMed Scopus (65) Google Scholar). No seizure activity was observed in either the saline or the sodium pentobarbital controls. Phosphorylation of the CREB protein following PTZ injection was examined using an antibody that detects Ser133-phosphorylated CREB protein (10Ginty D.D. Kornhauser J.M. Thompson M.A. Bading H. Mayo K.E. Takahashi J.S. Greenberg M.E. Science. 1993; 260: 238-241Crossref PubMed Scopus (731) Google Scholar). As an initial study, nuclear extracts were prepared 8 min after the injection of PTZ or saline, and the levels of total CREB and phosphorylated CREB were determined by Western blots. Fig. 1A shows that total amount of CREB protein remained unchanged by seizure activity both in the cortex and in the hippocampus. In contrast, the phosphorylation of CREB in the cortex and hippocampus is enhanced at this time point in experimental animals (Fig. 1B). In addition to the 43-kDa phosphoCREB, weakly cross-reacting bands were detected at 40 kDa and around 100 kDa. The identity of these cross-reactive bands is unknown at present. When animals were injected with 30 mg/kg of sodium pentobarbital prior to PTZ injection to block neuronal activity, the increased phosphoCREB signal was reduced both in the hippocampus and the cortex (Fig. 1, B and C). In the cortical samples, the signal for phosphoCREB was lower in comparison to saline controls. However, this difference was not statistically significant. In addition, no significant difference was detected between naive or saline-injected animals (data not shown). Fig. 1C shows the summary of results from several such experiments. The signal for phosphoCREB is increased by approximately 62% (saline = 99.5 ± 16.6%, experimental = 161.8 ± 34.1%, p < 0.05) in the hippocampus and by approximately 76% (saline = 99.7 ± 13.3%, experimental = 175.8 ± 13.3%, p < 0.05) in the cortex of experimental animals compared to saline-injected animals. Injection of sodium pentobarbital 15 min prior to PTZ injection blocked the enhanced CREB phosphorylation in both the hippocampus and cortex (Fig. 1C). We next examined the time course of CREB phosphorylation. Fig. 2A shows that phosphorylation of CREB protein is increased in the hippocampus as early as 3 min after the onset of seizure activity. The phosphorylation was maximal between 3 and 8 min and declined slowly. At 60 min following the onset of seizure, the phosphorylation was higher compared to the control. A similar time course for CREB phosphorylation was observed in the cortical tissue. The total amount of CREB protein remained unchanged both in the cortex and hippocampus at these time points (Fig. 2B). The spatial pattern of CREB phosphorylation in the hippocampus was investigated by immunohistochemistry. Animals for this study were sacrificed 8 min after the injection of PTZ or saline. Fig. 3A shows the cellular staining for total CREB protein. Both pyramidal and granular neurons show CREB immunoreactivity. When examined under high magnification, the signal for CREB immunoreactivity was found to be localized to the nucleus (data not shown). Consistent with the Western blot data, immunohistochemistry did not show any difference in total CREB protein between the control and experimental hippocampi. No staining was detected when the primary antibody was eliminated from the incubation solution (data not shown). Fig. 3B shows the low level of phosphorylated CREB-like proteins present in a control hippocampus. Seizure activity increases the phosphorylation of CREB-like proteins in the CA1, CA3 subfields and to a lesser extent in the dentate gyrus. CREB protein when phosphorylated on Ser133 either by PKA or CaMK can induce transcription of CRE-containing genes (20Sheng M. Thomson M.A. Greenberg M.E. Science. 1991; 252: 1427-1430Crossref PubMed Scopus (1262) Google Scholar, 24Gonzalez G.A. Yamamoto K.A. Fisher W.H. Karr D. Menzel P. Bigg III, W. Vale W.W. Montminy M.R. Nature. 1989; 337: 749-752Crossref PubMed Scopus (643) Google Scholar, 26Enslen H. Sun P. Brickey D. Soderling S.H. Klamo E. Soderling T.R. J. Biol. Chem. 1994; 269: 15520-15527Abstract Full Text PDF PubMed Google Scholar). To examine PKA activation, the amount of cAMP in both the cortical and hippocampal tissues at various time points following the onset of seizure activity were measured using a radioimmunoassay kit as described under “Experimental Procedures.” It has been reported that increased levels of cAMP correlate well with enhanced PKA activity (50Keely S.L. Corbin J.D. Park C.R. J. Biol. Chem. 1975; 250: 4832-4840Abstract Full Text PDF PubMed Google Schol" @default.
• W2130270377 created "2016-06-24" @default.
• W2130270377 creator A5063633345 @default.
• W2130270377 creator A5077004323 @default.
• W2130270377 creator A5087765123 @default.
• W2130270377 date "1996-06-01" @default.
• W2130270377 modified "2023-10-11" @default.
• W2130270377 title "Neuronal Activity Increases the Phosphorylation of the Transcription Factor cAMP Response Element-binding Protein (CREB) in Rat Hippocampus and Cortex" @default.
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