Autophosphorylation at Thr 286 of the α Calcium-Calmodulin Kinase II in LTP and Learning
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A. J. Silva C. F. Stevens S. Tonegawa Y. Wang ibid. p. 201.
Pettit D. L., Perlman S., Malinow R., ibid. 266, 1881 (1994);
Miller S. G., Kennedy M. B., ibid. 44, 861 (1986);
Waxham M. N., Aronowski J., Westgate S. A., Kelly P. T., Proc. Natl. Acad. Sci. U.S.A. 87, 1273 (1990);
Two point mutations (ACCGTGGAC was changed to GCCGTCGAC) were introduced by polymerase chain reaction (PCR) mutagenesis into the 866–base pair Hind III fragment containing the exon encoding Thr 286 (27). The wild-type Hind III fragment of the 6.1-kb Pvu II genomic clone (4) was substituted by the mutagenized Hind III fragment. A PGKneo cassette flanked by loxP sites was inserted into the Bgl II site. After transfection of R1 embryonic stem cells (28) and selection with G418 targeted clones were identified by Southern (DNA) blot analyses. Because the PGKneo cassette could interfere with the expression of neighboring genes (29) it was removed by transient transfection with pBS185 (30) a plasmid containing Cre recombinase DNA. Chimeras generated by injection of proper cells into blastocysts were mated with C57BL/6J mice and crosses of F1 heterozygotes yielded a Mendelian distribution of F2 offspring. The mutants were identified by PCR (primers: 5′-CTGTACCAGCAGATCAAAGC-3′ and 5′-ATCACTAGCACCATGTGGTC-3′). Polymerase chain reaction (primers: 5′-GATGCTGACCATCAACCCAT-3′ and 5′-CCCATTGTGACTCTACACCT-3′) followed by a Hinc II restriction indicated the presence of the point mutations in the homozygous mutants.
The immunoblot analysis was done with protein from adult brain with monoclonal antibodies to αCaMKII and synaptophysin (Boehringer). The secondary antibody was labeled with 125 I. Blots analyzed with an antibody to βCaMKII (Zymed Laboratories) showed that βCaMKII is expressed at normal amounts in adult brain of the αCaMKII T286A-129B6F2 mutants. For the immunocytochemical analysis coronal sections of the adult brain were incubated with the monoclonal antibody to αCaMKII. Antigen-antibody complexes were visualized with the glucose oxidase–diaminobenzidine-nickel method (31).
The kinase activity was determined from hippocampal slices with autocamtide-3 as substrate as previously described (11 32). For wild-type mice ( n = 3) the total activity was 5.07 ± 0.91 pmol − 1 μg − 1 min and the CaM-independent activity was 1.09 ± 0.12 pmol − 1 μg − 1 min (22.1 ± 1.8%). For the homozygous mutants ( n = 3) the total activity was 4.74 ± 1.12 pmol − 1 μg − 1 min and the CaM-independent activity was 0.40 ± 0.06 pmol − 1 μg − 1 min (9.2 ± 1.9%).
Transverse hippocampal slices (400 μm) from 5- to 10-month-old mice were placed in a submerged recording chamber perfused continuously with artificial cerebrospinal fluid (ACSF) equilibrated with 95% O 2 and 5% CO 2 at 31°C. Extracellular fEPSPs were recorded with an electrode filled with ACSF in CA1 stratum radiatum and the Schaffer collaterals were stimulated. A second pathway was used to control the stability of the recordings. The stimulus duration was 100 μs. The ACSF contained 120 mM NaCl 3.5 mM KCl 2.5 mM CaCl 2 1.3 mM MgSO 4 1.25 mM NaH 2 PO 4 26 mM NaHCO 3 and 10 mM d -glucose.
Synaptic transmission during the 10-Hz tetanus undergoes biphasic (increase minus decrease) changes. For wild-type mice ( n = 6) the maximal increase was 140.3 ± 3.2% and for mutants it was 146.7 ± 4.4% ( n = 5). The maximal decrease (at the end of the tetanus) was 79.2 ± 6.9% for wild-type mice and 91.6 ± 8.9% for mutants.
Excitatory postsynaptic currents were recorded from CA1 pyramidal neurons from 6- to 12-month-old mice with a patch electrode (7 to 10 Mohm) in the whole-cell voltage-clamp mode. The pipette solution contained 122.5 mM cesium methanosulphonate 17.5 mM CsCl 10 mM Hepes buffer 0.1 mM EGTA 8 mM NaCl 2 mM Mg–adenosine triphosphate and 0.3 mM Na 3 –guanosine triphosphate (pH 7.25 290 to 300 mosm). A second pathway was used to control for the stability of the recordings. Picrotoxin (100 μM) was present in all experiments.
Similar results were obtained 10 s after a 2 theta burst (two high-frequency bursts of four stimuli at 100 Hz with 200 ms separating the onset of each burst) tetanus (wild-type mice without d -AP5: 131.3 ± 5.6%; six mice 12 slices; wild-type mice with d -AP5: 106.3 ± 2.0%; four mice eight slices; mutants without d -AP5: 112.4 ± 3.3%; five mice five slices).
Extracellular field recordings (in the presence of CNQX and PTX with a stimulation strength of 50 μA) indicated that the NMDAR potentials did not differ between mutant (0.159 ± 0.053 mV; three mice four slices) and wild-type mice (0.200 ± 0.020 mV; three mice five slices).
Two- to five-month–old mice were studied in the spatial version of the water maze for 5 days (12 trials per day; blocks of four trials) as previously described (33). Transfer tests were given at the end of days 3 and 5. In another experiment the mice were tested for 2 days with a visible platform in a fixed location (12 trials per day); then the visible platform was replaced by a hidden platform and the mice were tested for another 2 days (12 trials per day). A transfer test was given at the end of training. Data were studied with one-way and two-way analysis of variance.
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K. P. Giese N. B. Fedorov R. K. Filipkowski A. J. Silva data not shown.
We thank P. Chen K. Fox P. W. Frankland J. H. Kogan J. E. Lisman R. Malinow K. Mizuno A. Nagy M. N. Waxham and D.-J. Zuo for helpful discussions and materials. K.P.G. was supported by a European Molecular Biology Organization and Deutsche Forschungsgemeinschaft fellowship. R.K.F. was supported by the Foundation for Experimental and Clinical Oncology Poland. A.J.S. was supported by grants from the Whitehall Beckman Klingenstein and McKnight Foundations and NIH (AG13622).