Src activation in the induction of long-term potentiation in CA1 hippocampal neurons

Science

Volumn 279, Issue 5355, 1998, Pages 1363-1367

  Lu, You Ming ^a   Roder, John C ^a   Davidow, Jonathan ^a   Salter, Michael W ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

ALPHA AMINO 3 HYDROXY 5 METHYL 4 ISOXAZOLEPROPIONIC ACID; CALCIUM; N METHYL DEXTRO ASPARTIC ACID RECEPTOR; PROTEIN TYROSINE KINASE;

ARTICLE; HIPPOCAMPUS; LEARNING; LONG TERM POTENTIATION; MEMORY; PRIORITY JOURNAL; PYRAMIDAL NERVE CELL; RECEPTOR BINDING; SYNAPTIC MEMBRANE;

AMINO ACID SEQUENCE; ANIMALS; CALCIUM; ELECTRIC STIMULATION; ENZYME ACTIVATION; EXCITATORY POSTSYNAPTIC POTENTIALS; HIPPOCAMPUS; LONG-TERM POTENTIATION; MOLECULAR SEQUENCE DATA; OLIGOPEPTIDES; PATCH-CLAMP TECHNIQUES; PEPTIDE FRAGMENTS; PROTO-ONCOGENE PROTEINS PP60(C-SRC); PYRAMIDAL CELLS; RATS; RATS, SPRAGUE-DAWLEY; RECEPTORS, AMPA; RECEPTORS, N-METHYL-D-ASPARTATE; RECOMBINANT PROTEINS; SRC-FAMILY KINASES; UP-REGULATION;

HIPPOCAMPUS HIPPOCAMPUS;

EID: 0001048711     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.279.5355.1363     Document Type: Article

References (38)

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13. 4, 10 mM Hepes, 0.2 mM EGTA, 2 mM Mg-ATP, 0.3 mM guanosine triphosphate, and 5 mM QX-314 (pH 7.25, 290 mosM). The intracellular solution was supplemented as required with peptides or antibodies that were stored as 100X single-use stock solutions prepared just before use. All peptides used in the study were from J. Bell, Ottawa Regional Cancer Centre, Ottawa, Canada. The peptides and antibodies were numbered and the experimenter (Y.M.L.) was unaware of which was applied in all experiments except those shown in Figs 3 and 4. Patch recordings were done using the "blind" patch method [M. G. Blanton, J. J. LoTurco, A. R. Kriegstein, J. Neurosci. Methods 30, 203 (1989)]. Raw data were amplified with an Axopatch 1-D, sampled at 3 to 6 kHz, and analyzed with Pclamp6, software (Axon Instruments, Foster City, CA). Synaptic responses were evoked by a bipolar tungsten electrode located about 50 μm from the cell bodies. Test stimuli were delivered at a frequency of 0.1 Hz, and the stimulus intensity was set to produce 25% of the maximum response. The tetanic stimulation consisted of two trains of 100-Hz stimulation lasting 500 ms at the intertrain interval of 10 s. In controls, this produced LTP that was at a stable level by 30 min after tetanus and persisted for more than 1.5 hours. For clarity, we show records of only the first 30 min after tetanus. EPSP slope was calculated as the slope of the rising phase 10 to 65% of the peak response. Average EPSP slope values were determined for each 5-min period of recording. The baseline value of EPSP slope was that from the first 5-min period and was defined as 100%. Series resistance ranged from 15 to 19 megohms. Input resistance was monitored every 5 min during the course of all experiments by measurement of responses to 0.2-nA injection for 400 ms. The average value of input resistance was 169 ± 13 megohms. The resting membrane potential was -60 ± 4 mV. Extracellular field potentials were simultaneously recorded with a micropipette filled with ACSF (1 to 3 megohms) placed in the stratum radiatum within 100 μm of the single cell studied. The averaged field EPSP slope was calculated every 1 min.
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15. The amino acid sequence of scrambled Src(40-58) was AGSHAPFPSPARAGVAPDA (13); it was created by random ordering of the sequence of Src(40-58). With scrambled Src(40-58), EPSP slope increased to 205 ± 13% of baseline [n = 5 cells, P < 0.01 versus Src(40-58)] 30 min after tetanus. As an additional control, Src(40-58) was tested in mice lacking src and had no effect on LTP induction (EPSP slope 30 min after tetanus was 195 ± 17% of baseline, n = 5 cells). Src(40-58) prevented LTP in wild-type mice from the same genetic background (EPSP slope was 110 ± 8% of baseline, n = 4 cells), and thus the effect of this peptide required Src.
16. S. Roche, S. Fumagalli, S. A. Courtneidge, Science 269, 1567 (1995). Anti-Src1 was obtained from S. Courtneidge, SUGEN, Redwood City, CA.
17. For voltage-clamp experiments, the patch pipette solution contained 132.5 mM Cs-gluconate, 17.5 mM CsCl, 10 mM Hepes, 0.2 mM EGTA, 2 mM Mg-ATP, 0.3 mM guanosine triphosphate, and 5 mM QX-314 (pH 7.25, 290 mosM). With this solution, the input resistance was 243 ± 19 megohms and series resistance was 20 ± 2 megohms. The holding potential was -60 mV except where otherwise indicated. The amplitude of the AMPAR EPSCs was taken as the peak of the inward current, and the 10 to 90% rise times were 0.6 to 2 ms. This rapid inward current was blocked by bath-applied 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 5 μM) and was unaffected by D,L,-2-amino-5-phosphonovaleric acid (APV, 50 μM). NMDAR EPSCs were pharmacologically isolated by bath application of 5 μM CNQX; these EPSCs were abolished by 50 μM APV.
18. c-Src (UBI) in kinase buffer were run in parallel. All of the samples were incubated for 5 min at 37°C, and the reaction was stopped by addition of 4X Laemmli sample buffer. Samples were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE; 10% gel). Proteins were transferred to a nitrocellulose membrane and exposed overnight on a Phosphor Screen (Molecular Dynamics, Sunnyvale, CA) for quantitation and analysis with ImageQuant software. Membranes were then immunoblotted with antibody to Src (1:500 dilution); the secondary antibody was coupled to horseradish peroxidase and was visualized by enhanced chemiluminescence.
19. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
20. X Liu et. al., Oncogene 8, 1119 (1993). The minimum concentration required to increase Src activity was 1 mM.
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25. 2.
26. Y. M. Lu, J. C. Roder, J. Davidow, M. W. Salter, data not shown.
27. - mice correlates with decreasing levels of Src expression. We suggest that in wild-type individuals, Src is a required mediator for LTP induction, whereas in mutants that develop without src it is likely that another member of the src family, possibly fyn, may substitute for src [S. M. Thomas, P. Soriano, A. Imamoto, Nature 376, 267 (1995); P. L. Stein, H. Vogel, P. Soriano, Genes Dev. 8, 1999 (1994)].
28. - mice correlates with decreasing levels of Src expression. We suggest that in wild-type individuals, Src is a required mediator for LTP induction, whereas in mutants that develop without src it is likely that another member of the src family, possibly fyn, may substitute for src [S. M. Thomas, P. Soriano, A. Imamoto, Nature 376, 267 (1995); P. L. Stein, H. Vogel, P. Soriano, Genes Dev. 8, 1999 (1994)].
29. - mice correlates with decreasing levels of Src expression. We suggest that in wild-type individuals, Src is a required mediator for LTP induction, whereas in mutants that develop without src it is likely that another member of the src family, possibly fyn, may substitute for src [S. M. Thomas, P. Soriano, A. Imamoto, Nature 376, 267 (1995); P. L. Stein, H. Vogel, P. Soriano, Genes Dev. 8, 1999 (1994)].
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32. 2+ entry through NMDARs.
33. 2+ entry through NMDARs.
34. 2+ entry through NMDARs.
35. 2+ entry through NMDARs.
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38. We thank S. Courtneidge and J. Bolen for providing antibodies and J. F. MacDonald, T. Pawson, and Y. T. Wang for critical comments on the manuscript. This work was supported by grants from the Medical Research Council of Canada to J.C.R. and to M.W.S. and by the Nicole Fealdman Memorial Fund (M.W.S.). J.D. was supported by a summer research bursary from Novartis Canada Ltd.