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• W2084824599 abstract "Long lasting changes in the strength of synaptic transmission in the hippocampus are thought to underlie certain forms of learning and memory. Accordingly, the molecular mechanisms that account for these changes are heavily studied. Postsynaptically, changes in synaptic strength can occur by altering the amount of neurotransmitter receptors at the synapse or by altering the functional properties of synaptic receptors. In this study, we examined the biochemical changes produced following chemically induced long term depression in acute hippocampal CA1 minislices. Using three independent methods, we found that this treatment did not lead to an internalization of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Furthermore, when the plasma membrane was separated into synaptic membrane-enriched and extrasynaptic membrane-enriched fractions, we actually observed a significant increase in the concentration of AMPA receptors at the synapse. However, phosphorylation of Ser-845 on the AMPA receptor subunit GluR1 was significantly decreased throughout the neuron, including in the synaptic membrane-enriched fraction. In addition, phosphorylation of Ser-831 on GluR1 was decreased specifically in the synaptic membrane-enriched fraction. Phosphorylation of these residues has been demonstrated to control AMPA receptor function. From these data, we conclude that the decrease in synaptic strength is likely the result of a change in the functional properties of AMPA receptors at the synapse and not a decrease in the amount of synaptic receptors. Long lasting changes in the strength of synaptic transmission in the hippocampus are thought to underlie certain forms of learning and memory. Accordingly, the molecular mechanisms that account for these changes are heavily studied. Postsynaptically, changes in synaptic strength can occur by altering the amount of neurotransmitter receptors at the synapse or by altering the functional properties of synaptic receptors. In this study, we examined the biochemical changes produced following chemically induced long term depression in acute hippocampal CA1 minislices. Using three independent methods, we found that this treatment did not lead to an internalization of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Furthermore, when the plasma membrane was separated into synaptic membrane-enriched and extrasynaptic membrane-enriched fractions, we actually observed a significant increase in the concentration of AMPA receptors at the synapse. However, phosphorylation of Ser-845 on the AMPA receptor subunit GluR1 was significantly decreased throughout the neuron, including in the synaptic membrane-enriched fraction. In addition, phosphorylation of Ser-831 on GluR1 was decreased specifically in the synaptic membrane-enriched fraction. Phosphorylation of these residues has been demonstrated to control AMPA receptor function. From these data, we conclude that the decrease in synaptic strength is likely the result of a change in the functional properties of AMPA receptors at the synapse and not a decrease in the amount of synaptic receptors. Rapid excitatory synaptic transmission in the hippocampus is mediated predominantly by glutamate-activated AMPA 2The abbreviations used are: AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazoleproponic acid; AMPAR, AMPA receptor; LFS, low frequency stimulation; LTD, long term depression; cLTD, chemically induced LTD; PSD, postsynaptic density; NMDA, N-methyl-d-aspartate; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; PP2B, protein phosphatase 2B; fEPSP, field excitatory postsynaptic potential; BS3, bis(sulfosuccinimidyl) suberate; αCaMKII, the α subunit of calcium/calmodulin-dependent protein kinase II; ACSF, artificial cerebrospinal fluid; BSA, bovine serum albumin; AIR, arbitrary immunoreactive; X-linked, cross-linked; un-X-linked, uncross-linked; P, postnatal. 2The abbreviations used are: AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazoleproponic acid; AMPAR, AMPA receptor; LFS, low frequency stimulation; LTD, long term depression; cLTD, chemically induced LTD; PSD, postsynaptic density; NMDA, N-methyl-d-aspartate; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; PP2B, protein phosphatase 2B; fEPSP, field excitatory postsynaptic potential; BS3, bis(sulfosuccinimidyl) suberate; αCaMKII, the α subunit of calcium/calmodulin-dependent protein kinase II; ACSF, artificial cerebrospinal fluid; BSA, bovine serum albumin; AIR, arbitrary immunoreactive; X-linked, cross-linked; un-X-linked, uncross-linked; P, postnatal. receptors (AMPARs). Theoretically, long term changes in the strength of synaptic transmission, as occur during long term potentiation and long term depression (LTD), can arise from changes in the amount of glutamate that is released from the presynaptic terminal, changes in the number of synaptic AMPARs, or changes in the functional properties of synaptic AMPARs. Experimentally, LTD of synaptic responses has been shown to be induced by several different treatments, the most common of which include several low frequency stimulation (LFS) paradigms (1Dudek S.M. Bear M.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4363-4367Crossref PubMed Scopus (1300) Google Scholar), N-methyl-d-aspartate (NMDA) application (2Lee H.K. Kameyama K. Huganir R.L. Bear M.F. Neuron. 1998; 21: 1151-1162Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar), 3,5-dihydroxyphenylglycine application (3Palmer M.J. Irving A.J. Seabrook G.R. Jane D.E. Collingridge G.L. Neuropharmacology. 1997; 36: 1517-1532Crossref PubMed Scopus (278) Google Scholar, 4Huber K.M. Roder J.C. Bear M.F. J. Neurophysiol. 2001; 86: 321-325Crossref PubMed Scopus (317) Google Scholar), and insulin application (5Man H.Y. Lin J.W. Ju W.H. Ahmadian G. Liu L. Becker L.E. Sheng M. Wang Y.T. Neuron. 2000; 25: 649-662Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar). The biochemical mechanisms that underlie these different types of synaptic depression have been the subject of numerous studies in many different experimental systems.A number of studies have demonstrated that protein phosphatase activity is a requirement for long term depression. This was originally demonstrated through the use of protein phosphatase 1 (PP1), 2A (PP2A), and 2B (PP2B/calcineurin) inhibitors, which were shown to inhibit both the induction and the maintenance of LTD (6Mulkey R.M. Herron C.E. Malenka R.C. Science. 1993; 261: 1051-1055Crossref PubMed Scopus (538) Google Scholar, 7Mulkey R.M. Endo S. Shenolikar S. Malenka R.C. Nature. 1994; 369: 486-488Crossref PubMed Scopus (898) Google Scholar). Furthermore, it has been suggested that synaptically localized PP1 is critical for the induction of LTD and that enhancing the synaptic targeting of PP1 facilitates long term depression (8Hu X.D. Huang Q. Yang X. Xia H. J. Neurosci. 2007; 27: 4674-4686Crossref PubMed Scopus (52) Google Scholar). In addition, activation of protein kinase A inhibits the induction of LFS and NMDA-induced LTD, and protein kinase A inhibition has been shown to result in a rundown of synaptic responses that occludes subsequent LTD (9Kameyama K. Lee H.K. Bear M.F. Huganir R.L. Neuron. 1998; 21: 1163-1175Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). The translocation of protein kinase A away from synaptic substrates may be an important step in LTD because NMDA-induced depression has been correlated with a redistribution of the kinase away from the synapse (10Snyder E.M. Colledge M. Crozier R.A. Chen W.S. Scott J.D. Bear M.F. J. Biol. Chem. 2005; 280: 16962-16968Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 11Smith K.E. Gibson E.S. Dell'Acqua M.L. J. Neurosci. 2006; 26: 2391-2402Crossref PubMed Scopus (123) Google Scholar), and disrupting the binding of protein kinase A to one of its anchoring proteins causes synaptic response rundown (12Tavalin S.J. Colledge M. Hell J.W. Langeberg L.K. Huganir R.L. Scott J.D. J. Neurosci. 2002; 22: 3044-3051Crossref PubMed Google Scholar) that occludes induced synaptic depression (10Snyder E.M. Colledge M. Crozier R.A. Chen W.S. Scott J.D. Bear M.F. J. Biol. Chem. 2005; 280: 16962-16968Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Importantly, one of the substrates being dephosphorylated during synaptic depression appears to be the AMPAR. Several groups using different LTD induction protocols and experimental systems have reported decreases in the phosphorylation of GluR1 Ser-845, which is a protein kinase A substrate (2Lee H.K. Kameyama K. Huganir R.L. Bear M.F. Neuron. 1998; 21: 1151-1162Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar, 8Hu X.D. Huang Q. Yang X. Xia H. J. Neurosci. 2007; 27: 4674-4686Crossref PubMed Scopus (52) Google Scholar, 11Smith K.E. Gibson E.S. Dell'Acqua M.L. J. Neurosci. 2006; 26: 2391-2402Crossref PubMed Scopus (123) Google Scholar, 13Lee H.K. Barbarosie M. Kameyama K. Bear M.F. Huganir R.L. Nature. 2000; 405: 955-959Crossref PubMed Scopus (878) Google Scholar, 14Ehlers M.D. Neuron. 2000; 28: 511-525Abstract Full Text Full Text PDF PubMed Scopus (884) Google Scholar). Furthermore, mice in which Ser-845 has been mutated to alanine have deficits in long term depression (15Lee H.K. Takamiya K. Han J.S. Man H. Kim C.H. Rumbaugh G. Yu S. Ding L. He C. Petralia R.S. Wenthold R.J. Gallagher M. Huganir R.L. Cell. 2003; 112: 631-643Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar). The mechanisms by which Ser-845 dephosphorylation may contribute to LTD are unknown, but two principal hypotheses have been presented and are discussed below.Phosphorylation of Ser-845 is known to influence AMPAR functional properties (16Roche K.W. O'Brien R.J. Mammen A.L. Bernhardt J. Huganir R.L. Neuron. 1996; 16: 1179-1188Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar). Specifically, phosphorylation of this residue has been demonstrated to control peak open probability of GluR1-containing receptors (17Banke T.G. Bowie D. Lee H. Huganir R.L. Schousboe A. Traynelis S.F. J. Neurosci. 2000; 20: 89-102Crossref PubMed Google Scholar). Because of this, several researchers have postulated that a decrease in AMPAR function as the result of Ser-845 dephosphorylation may play a major role in the depression of synaptic responses (18Song I. Huganir R.L. Trends Neurosci. 2002; 25: 578-588Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar). However, a key issue for this model is whether the stoichiometry of Ser-845 phosphorylation is decreased on synaptic receptors following LTD induction.Another postsynaptic mechanism suggested by many to be involved in synaptic depression is the internalization of AMPARs. A role for endocytic processes in general for LTD is provided by studies that have shown that inhibitors of clathrin mediated endocytosis prevent LTD induction (5Man H.Y. Lin J.W. Ju W.H. Ahmadian G. Liu L. Becker L.E. Sheng M. Wang Y.T. Neuron. 2000; 25: 649-662Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar, 19Luscher C. Xia H. Beattie E.C. Carroll R.C. von Zastrow M. Malenka R.C. Nicoll R.A. Neuron. 1999; 24: 649-658Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, 20Wang Y.T. Linden D.J. Neuron. 2000; 25: 635-647Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 21Xiao M.Y. Zhou Q. Nicoll R.A. Neuropharmacology. 2001; 41: 664-671Crossref PubMed Scopus (143) Google Scholar). There have also been many studies using cultured neurons that have directly demonstrated AMPAR internalization following LTD-inducing treatments (8Hu X.D. Huang Q. Yang X. Xia H. J. Neurosci. 2007; 27: 4674-4686Crossref PubMed Scopus (52) Google Scholar, 10Snyder E.M. Colledge M. Crozier R.A. Chen W.S. Scott J.D. Bear M.F. J. Biol. Chem. 2005; 280: 16962-16968Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 14Ehlers M.D. Neuron. 2000; 28: 511-525Abstract Full Text Full Text PDF PubMed Scopus (884) Google Scholar, 15Lee H.K. Takamiya K. Han J.S. Man H. Kim C.H. Rumbaugh G. Yu S. Ding L. He C. Petralia R.S. Wenthold R.J. Gallagher M. Huganir R.L. Cell. 2003; 112: 631-643Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar, 19Luscher C. Xia H. Beattie E.C. Carroll R.C. von Zastrow M. Malenka R.C. Nicoll R.A. Neuron. 1999; 24: 649-658Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, 21Xiao M.Y. Zhou Q. Nicoll R.A. Neuropharmacology. 2001; 41: 664-671Crossref PubMed Scopus (143) Google Scholar, 22Carroll R.C. Lissin D.V. von Zastrow M. Nicoll R.A. Malenka R.C. Nat. Neurosci. 1999; 2: 454-460Crossref PubMed Scopus (379) Google Scholar, 23Snyder E.M. Philpot B.D. Huber K.M. Dong X. Fallon J.R. Bear M.F. Nat. Neurosci. 2001; 4: 1079-1085Crossref PubMed Scopus (451) Google Scholar, 24Man H.Y. Sekine-Aizawa Y. Huganir R.L. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 3579-3584Crossref PubMed Scopus (238) Google Scholar, 25Oh M.C. Derkach V.A. Guire E.S. Soderling T.R. J. Biol. Chem. 2006; 281: 752-758Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, 26Lin D.T. Huganir R.L. J. Neurosci. 2007; 27: 13903-13908Crossref PubMed Scopus (136) Google Scholar, 27Ashby M.C. De La Rue S.A. Ralph G.S. Uney J. Collingridge G.L. Henley J.M. J. Neurosci. 2004; 24: 5172-5176Crossref PubMed Scopus (186) Google Scholar). Furthermore, some LTD studies have demonstrated a reduction in surface AMPARs in the more physiologically relevant acute hippocampal slice system (11Smith K.E. Gibson E.S. Dell'Acqua M.L. J. Neurosci. 2006; 26: 2391-2402Crossref PubMed Scopus (123) Google Scholar, 28Huang C.C. You J.L. Wu M.Y. Hsu K.S. J. Biol. Chem. 2004; 279: 12286-12292Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 29Huang C.C. Lee C.C. Hsu K.S. J. Neurochem. 2004; 89: 217-231Crossref PubMed Scopus (93) Google Scholar, 30Holman D. Feligioni M. Henley J.M. Neuropharmacology. 2007; 52: 92-99Crossref PubMed Scopus (20) Google Scholar, 31Davidkova G. Carroll R.C. J. Neurosci. 2007; 27: 13273-13278Crossref PubMed Scopus (93) Google Scholar) and in vivo (32Heynen A.J. Quinlan E.M. Bae D.C. Bear M.F. Neuron. 2000; 28: 527-536Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Interestingly, several of these studies have found a correlation between the dephosphorylation of GluR1 Ser-845 and AMPAR endocytosis in cultured neurons (14Ehlers M.D. Neuron. 2000; 28: 511-525Abstract Full Text Full Text PDF PubMed Scopus (884) Google Scholar, 15Lee H.K. Takamiya K. Han J.S. Man H. Kim C.H. Rumbaugh G. Yu S. Ding L. He C. Petralia R.S. Wenthold R.J. Gallagher M. Huganir R.L. Cell. 2003; 112: 631-643Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar, 24Man H.Y. Sekine-Aizawa Y. Huganir R.L. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 3579-3584Crossref PubMed Scopus (238) Google Scholar, 25Oh M.C. Derkach V.A. Guire E.S. Soderling T.R. J. Biol. Chem. 2006; 281: 752-758Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar) and in hippocampal slices (11Smith K.E. Gibson E.S. Dell'Acqua M.L. J. Neurosci. 2006; 26: 2391-2402Crossref PubMed Scopus (123) Google Scholar), suggesting a role for this phosphorylation site in AMPAR trafficking. However, to date, there is no known mechanism by which Ser-845 might influence trafficking as this site is not part of any known endocytic motifs. In the present report, we have attempted to explore these two hypotheses by directly examining AMPAR phosphorylation and localization following chemical induction of LTD.EXPERIMENTAL PROCEDURESCA1 Minislice Preparation and Treatment—All of the experiments used in this study were approved by the Institutional Animal Care and Use Committee at the University of Colorado Health Sciences Center. Twenty-one-(P21) to twenty-nine-(P29) day-old male Sprague-Dawley rats were used for all experiments. After sacrifice, the brain was immediately removed and placed in ice-cold oxygenated artificial cerebrospinal fluid (ACSF: 124 mm NaCl, 4 mm KCl, 1 mm MgCl2, 2.5 mm CaCl2, 10 mm dextrose, 1 mm KH2PO4, 25.7 mm NaHCO3). Both hippocampi were carefully dissected out and unrolled along the hippocampal fissure. Subsequently, area CA1 was isolated by two cuts and then sliced into 400-μm minislices using a McIlwain tissue chopper. On average, 27 minislices could be harvested from each animal. For all of the electrophysiology experiments and some of the biochemistry experiments, the minislices were transferred to chambers perfused with 32 °C ACSF that was bubbled with a 95% O2, 5%CO2 mix. The perfusion rate in these chambers was 3 ml/min. For the remainder of the biochemistry experiments, the minislices were transferred to static chambers and incubated at interface in 32 °C ACSF (which had been bubbled with a 95% O2, 5%CO2 mix) and humidified with 95% O2, 5%CO2. For both setups, minislices were recovered in ACSF for at least 90 min before treatment or recording, with ACSF being changed every 15 min in the static chambers. To induce cLTD in the perfused chambers, NMDA was perfused over the slices at a final concentration of 20 μm for 3 min. To induce cLTD in the interface chambers, the ACSF was replaced with ACSF containing 20 μm NMDA for 3 min. After 3 min, the NMDA ACSF was replaced with regular ACSF. The control treatment for these experiments was ACSF containing vehicle (H20).Electrophysiology—Each n for the electrophysiology experiments represents data obtained from one minislice. Field excitatory postsynaptic potentials (fEPSPs) were evoked by stimulating the Schafer collateral-commissural pathway using a bipolar tungsten electrode. A silver chloride recording electrode inside a finely drawn glass capillary containing ACSF was placed in the dendritic layer of the minislice. Both the amplitude and the initial slope of the fEPSP were recorded at the frequency of 0.033 Hz. The stimulus intensity was adjusted to produce a fEPSP with 50–70% of the maximum achievable response. Following the recording of a stable baseline (at least 20 min), cLTD was induced (as described above). The stimulator was turned off during the treatment and the washout of the NMDA to ensure that depression was not dependent on electrical stimulation. Depression (percent change) at 45 min following the termination of the cLTD treatment was calculated by comparing the average of the responses recorded from 40–50 min following the termination of the treatment to the average of the responses recorded 10 min immediately prior to the treatment.Subcellular Fractionation—For experiments in which the lysed crude synaptosomal membrane fraction (LP1) was collected, each n represents all of the minislices that could be harvested from one animal divided equally between control and cLTD conditions. For experiments in which the postsynaptic density (PSD)/synaptic membrane-enriched fraction (TxP) was collected, each n represents all of the minislices that could be harvested from two animals divided equally between control and cLTD conditions (the pooling of animals was required to ensure that each fraction contained a sufficient amount of protein for analysis). In both cases, minislices were harvested 45 min after the treatment into ice-cold homogenization buffer containing 320 mm sucrose, 10 mm Tris (pH 7.4), 100 μm Na3VO4, 40 mm NaF, 300 nm okadaic acid, and 1 mm EDTA. Following harvesting, the slices were immediately homogenized in a glass grinding vessel by a Teflon pestle rotating at 1000 rpm. Subsequently, a subcellular fractionation protocol was employed (11Smith K.E. Gibson E.S. Dell'Acqua M.L. J. Neurosci. 2006; 26: 2391-2402Crossref PubMed Scopus (123) Google Scholar, 33Carlin R.K. Grab D.J. Cohen R.S. Siekevitz P. J. Cell Biol. 1980; 86: 831-845Crossref PubMed Scopus (599) Google Scholar, 34Huttner W.B. Schiebler W. Greengard P. De Camilli P. J. Cell Biol. 1983; 96: 1374-1388Crossref PubMed Scopus (884) Google Scholar, 35Cho K.O. Hunt C.A. Kennedy M.B. Neuron. 1992; 9: 929-942Abstract Full Text PDF PubMed Scopus (1001) Google Scholar, 36Davies K.D. Alvestad R.M. Coultrap S.J. Browning M.D. Brain Res. 2007; 1158: 39-49Crossref PubMed Scopus (24) Google Scholar). The homogenate was spun at 1000 × g for 10 min, and the pellet (P1), which contains nuclei and incompletely homogenized material, was discarded. The supernatant (S1) was then spun at 10,000 × g for 15 min. For the LP1 experiments, the pellet from this spin (P2) was resuspended in homogenization buffer lacking sucrose and hypoosmotically lysed for 30 min on ice. The lysed P2 was then respun at 10,000 × g for 15 min, and the resulting pellet, LP1 (lysed crude synaptosomal membrane fraction), was resuspended in homogenization buffer. For TxP experiments, the P2 was resuspended in homogenization buffer (lacking sucrose) containing 0.5% Triton X-100, homogenized with a rotating plastic pestle, incubated on ice for 40 min, and then spun at 32,000 × g for 20 min. The pellet from this spin, TxP, was resuspended in homogenization buffer. The supernatant from this spin was subjected to acetone precipitation to concentrate the protein. Eight volumes of acetone were added to the supernatant, which was then incubated at –20 °C overnight. The precipitate was collected by centrifugation at 3000 × g for 15 min. The pellet from this spin, extrasynaptic membrane-enriched fraction (TxS), was dried and then resuspended in homogenization buffer. The supernatant from the second spin (S2) was spun at 100,000 × g for 1 h. The pellet from this spin, microsome/light membrane-enriched fraction (P3), was resuspended in homogenization buffer. All of the above spins were conducted at 4 °C to inhibit enzyme action. The final pellets were sonicated and boiled in 1% SDS, 1 mm EDTA, and 10 mm Tris (pH 8) for 5 min and kept at –80 °C. We have previously characterized these fractionation procedures (36Davies K.D. Alvestad R.M. Coultrap S.J. Browning M.D. Brain Res. 2007; 1158: 39-49Crossref PubMed Scopus (24) Google Scholar, 37Goebel S.M. Alvestad R.M. Coultrap S.J. Browning M.D. Brain Res. Mol. Brain Res. 2005; 142: 65-79Crossref PubMed Scopus (46) Google Scholar).Cross-linking—For cross-linking experiments, each n represents all of the minislices that could be harvested from one animal divided equally between control un-X-linked (un-cross-linked), control X-linked (cross-linked), cLTD un-X-linked, and cLTD X-linked conditions. Minislices were harvested 45 min after the treatment into ice-cold ACSF (for un-X-linked) or ACSF containing 1 mg/ml bis(sulfosuccinimidyl) suberate (BS3) (for X-linked). Minislices were then incubated on ice while rocking for 40 min. Following 40 min, the cross-linking reaction was quenched by three washes with ice-cold ACSF containing 20 mm Tris (pH 7.6). The minislices were then sonicated and boiled in 1% SDS, 1 mm EDTA, and 10 mm Tris (pH 8) for 5 min and kept at –80 °C. To determine changes in the amount of internal receptor, cross-linked samples were directly compared. To determine changes in the percentage of surface expression, the following equation was used: ((un-X-linked – X-linked)/un-X-linked) × 100%.Semiquantitative Western Blotting—Protein concentrations were determined using the BCA™ protein assay kit from Pierce with bovine serum albumin (BSA) as a standard. Samples were prepared for gel loading such that they contained a known amount of total protein and 1× SDS-PAGE sample buffer (62.5 mm Tris, 2% SDS, 10% glycerol, bromphenol blue, and 5% β-mercaptoethanol). A five-point standard curve of known total protein concentration was prepared as above from SDS-homogenized recovered whole hippocampal slices. The samples, along with the standard curve, were run through 7.5% polyacrylamide gels at 150–200 V for 45–60 min. Separated proteins were transferred to PolyScreen polyvinylidene difluoride membranes. Blots were then blocked in 5% milk or 3% BSA in Tris-buffered saline with Tween (TBST: 140 mm NaCl, 20 mm Tris (pH 7.6), 0.1% Tween 20) for 1 h and then incubated overnight at 4 °C with primary antibody in 1% milk or 1% BSA. Following three 10-min washes in TBST, blots were incubated in secondary antibody conjugated to horseradish peroxidase in 1% milk or 1% BSA at room temperature for 1 h. After three 10-min washes in TBST, bands were detected using Pierce SuperSignal® chemiluminescence kits and the Alpha Innotech ChemiImager 4400 imaging system. In some cases, blots were stripped with Restore™ Western blot stripping buffer from Pierce for 60 min at 60 °C, and then subjected to six 10-min washes in TBST followed by blocking and antibody incubation. Primary antibodies were as follows: GluR1 and GluR2/3 (Chemicon) used at 1:3000 in BSA, phospho-Ser-845 and phospho-Ser-831 (PhosphoSolutions) used at 1:1000 in BSA, PP2Aα (BD Pharmingen) used at 1:5000 in milk, synapsin, NR2B, and NR1 (PhosphoSolutions) used at 1:2000 in BSA, the α subunit of calcium/calmodulin-dependent protein kinase II (αCaMKII) (BD Pharmingen) used at 1:3000 in BSA, phospho-Thr-286 (PhosphoSolutions) used at 1:3000 in milk, PSD-95 (Affinity Bioreagents) used at 1:5000 in BSA, and transferrin receptor (Zymed Laboratories Inc.) used at 1:1000 in milk.Western Blot Analysis—Quantitation of Western blots was performed using AlphaEase software. An integrated density value for samples and standards was determined as the sum of the intensity of the pixels constituting each band. A line was generated from the standard curve by plotting the integrated density value versus the total amount of protein loaded for each curve point. Using this line, the integrated density value of each sample was used to calculate its immunoreactivity, which was measured in arbitrary immunoreactive (AIR) units. This number was then divided by the amount of total protein loaded in micrograms for the sample to obtain the AIR units/μg. The AIR units/μg is a measure of relative concentration that can be compared between samples. Relative phospho-stoichiometry was calculated by dividing the AIR units/μg determined with the phospho-specific antibody by the AIR units/μg determined with the pan antibody. To calculate total AIR units in the fractions, the AIR units/μg value was multiplied by the total protein concentration of the fraction and by the total volume of the fraction. The percentage of total GluR1 and GluR2/3 in the fractions (reported in Fig. 5, B and D) was calculated by dividing the total AIR units value for a particular fraction by the summation of the total AIR units for the three fractions (making this summation 100%). Only samples with an integrated density value within the standard curve were used for analysis.RESULTSLong term depression was first described following an LFS paradigm (1Dudek S.M. Bear M.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4363-4367Crossref PubMed Scopus (1300) Google Scholar), and LFS remains the most common method to induce LTD in hippocampal slices. However, stimulation by a single electrode only affects a limited region of the slice (38Dunwiddie T. Lynch G. J. Physiol. (Lond.). 1978; 276: 353-367Crossref Scopus (361) Google Scholar), and therefore, it is problematic to measure biochemical changes in the entire slice following LFS-induced depression. Because of this, many researchers have turned to inducing LTD by chemical means, which, in theory, should activate virtually all of the synapses in the slice. It has been well established that brief application of NMDA results in synaptic depression in hippocampal slices (2Lee H.K. Kameyama K. Huganir R.L. Bear M.F. Neuron. 1998; 21: 1151-1162Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar, 9Kameyama K. Lee H.K. Bear M.F. Huganir R.L. Neuron. 1998; 21: 1163-1175Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 30Holman D. Feligioni M. Henley J.M. Neuropharmacology. 2007; 52: 92-99Crossref PubMed Scopus (20) Google Scholar, 39Moult P.R. Gladding C.M. Sanderson T.M. Fitzjohn S.M. Bashir Z.I. Molnar E. Collingridge G.L. J. Neurosci. 2006; 26: 2544-2554Crossref PubMed Scopus (150) Google Scholar, 40Delgado J.Y. Coba M. Anderson C.N. Thompson K.R. Gray E.E. Heusner C.L. Martin K.C. Grant S.G. O'Dell T.J. J. Neurosci. 2007; 27: 13210-13221Crossref PubMed Scopus (44) Google Scholar). Here we confirm that a 3-min, 20 μm NMDA treatment (cLTD) induces a stable, long-lasting depression of the fEPSP recorded in the dendritic layer of CA1 minislices from P21–P29 rats (Fig. 1). As has been described previously, the synaptic response initially completely disappeared and then returned to a depressed level. Both the amplitude (Fig. 1B) and the initial slope (Fig. 1C) of the fEPSP were significantly depressed 45 min following the termination of the treatment (78.7 and 78.8% of baseline, respectively). All of the following biochemical experiments were performed at this time point, because here the synaptic response had stabilized.FIGURE 1Long-lasting depression of fEPSPs is induced in CA1 minislices by a 3-min, 20 μm NMDA treatment. A, sample traces of synaptically evoked fEPSPs before (average of 20 fEPSPs recorded in the 10 min prior to start of treatment) (baseline) and after (average of 20 fEPSPs recorded from 40 to 50 min following termination of treatment) (cLTD) NMDA treatment for one experiment. B and C, cumulative time course of all experiments (n = 10 (10 slices from 6 animals)) measuring both amplitude (B) and initial slope (C) of the fEPSP. Both the amplitude and the initial slope were significantly depressed during the time average stated in A as determined by Student's paired t test (#, p < 0.001, **, p < 0.01). Values represent the mean ± S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We wanted to examine molecula" @default.
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• W2084824599 date "2008-11-01" @default.
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• W2084824599 title "Long Term Synaptic Depression That Is Associated with GluR1 Dephosphorylation but Not α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Internalization" @default.
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