Autophosphorylation at Thr 286 of the α Calcium-Calmodulin Kinase II in LTP and Learning Karl Peter Giese, Nikolai B. Fedorov, Robert K. Filipkowski, Alcino J. Siilva* Science vol.279, no pp

Previous Experiments Synaptic Strength Related To Learning/Memory –i.e. LTP Inhibit CaMKII  Inhibit LTP and Learning Increasing tCaMKII affects Learning/Memory

More Background Autophosphorylation at Thr 286  CaMKII from CaM-independent to CaM- dependent LTP  Long-lasting Autophosphorylated form of CaMKII at Thr 286

Hypothesis The autophosphorylation at the Thr 286 site of CaMKII is required for LTP and learning.

CaMKII TT 305/306 Inhibitory BC

Experiment 1 – T286A

Results from Experiment 1 Result The αCaMKII T286A-129B6F2 mutation decreased the total CaM-independent CaMKII activity in the mutants CaM-Independent Activity Mutant 0.40 ± 0.06 pmol -1 min Wild Type 1.09 ± 0.12 pmol -1 min β-CaMKII?

Verify Experiment 1 Result  The point mutations and the loxP site did not alter the expression of the αCaMKII gene

Verify Experiment 1 Result Total CaMKII Activity Mutant 4.74 ± 1.12 pmol-1 min Wild Type 5.07 ± 0.91 pmol-1 min Result Total CaMKII (CaM independent + dependent) activity remained approximately equal.

Experiment 2 – Testing Mutants for LTP Method – Extracellular field recordings in the stratum radiatum of hippocampal slices. –Transverse hippocampal slices (400 μm) from 5-10 month old mice placed in a submerged recording chamber perfused continuously with artificial cerebrospinal fluid (ACSF). –Extracellular fEPSPs recorded with an electrode filled with ACSF in CA1 stratum radiatum. –Schaeffer collaterals were stimulated

Testing for LTP in αCaMKII T286A-129B6F2 mutants Protocol –100Hz tetanus (1s) –Check for potentiation 60 minutes Results –LTP deficient in αCaMKII T286A-129B6F2 mutants PotentiationSample Mutant ± 6.2% 7 Mice, 7 Slices Wild Type ± 7.5 % 10 Mice, 10 Slices

LTP impairments in the αCaMKII T286A-129B6F2 mutants  NO OVERLAP Testing for LTP in αCaMKII T286A-129B6F2 mutants

Verify LTP Deficiency in Mutant  Test that LTP impairments not due to defect in synaptic connectivity in the CA1 region.  Synaptic Transmission during tetanus  Biphasic Change at 10 Hz Stimulus Maximum IncreaseMaximum Decrease Mutant146.7 ± 4.4 %91.6 ± 6.9 % Wild Type140.3 ± 3.2 %79.3 ± 6.9 %  LTP impairments NOT due to prepotentiation of Synaptic Transmission

Experiment 3 - Pairing Protocol Background To confirm the LTP deficiency of αCaMKII T286A-129B6F2 mutants Method EPSP currents recorded from CA1 pyramidal neurons from 6-12 month old mice with a patch electrode in the whole-cell voltage clamp mode. Postsynaptic depolarization up to +10mV 2 Hz synaptic stimulation for 50s

Pairing Protocol Result Pairing Protocol Mutant: 132 ± 8% WT: 277 ± 21% No overlap LTP deficits in the αCaMKII T286A-129B6F2 mutants

Robust Protocol γ-aminobutyric acidA (GABA A ) receptors blocked with picrotoxin (PTX) during these recordings.  LTP impairments not due to abnormalities in inhibition.

Experiment 4 – NMDAR-dependent LTP Procedure –100 Hz Tetanus for 1 s –NMDAR in the presence of AP5 Results (after 30 min) (+) AP5 - Potentiation(-) AP5 - Potentiation Mutant ± 3.5 %113.9 ± 2.8 % Wild Type ± 3.9 %158.1 ± 8.7 % Mutant Insensitive to AP5  Mutant NMDAR-dependent LTP already deficient

Early Potentiation via NMDAR 2 Theta Burst Tetanus – 2 high-frequency bursts of four stimuli at 100 Hz, with 200 ms separating the onset of each burst After 2 seconds –WT + AP5:113.7 ± 2.0 % Potentiation –Mutant:108.8 ± 2.6 % Potentiation After 10 Seconds –WT + AP5:106.3 ± 2.0 % Potentiation –Mutant:112.4 ± 3.3 % Potentiation  Mutants without AP5 had very similar potentiation as Wild Type + AP5  Thus, early mutant potentiation was not NMDAR dependent

Possible Problem The autophosphorylation of αCaMKII at Thr286 leads to trapping of Calmodulin. Calmodulin can reduce the opening probability of NMDARs. Proposed Problematic Model: –T286A  no phosphorylation  more Calmodulin  reduced probability of NMDARs opening  reduced LTP

Check NMDAR opening probability Results –  Amplitude of NMDAR currents normal in mutants when compared to wild type –  Voltage dependence of NMDAR currents normal in mutants when compared to wild type. Extracellular Field Recordings – 50 μA Stimulus –Mutants:0.159 ± mV –Wild-Type:0.200 ± mV  LTP impairments in the mutant αCaMKIIT286A-129B6F2 were not due to abnormal NMDAR function.

Original Hypothesis: –The autophosphorylation at the Thr 286 site of CaMKII is required for LTP and learning. Conclusion Thus Far: –Autophosphorylation of αCaMKII is required for LTP. Next Question: –Is autophosphorylation of αCaMKII required for learning?

Autophosphorylation and Spatial Learning Morris Water Maze Hidden Platform Version Hippocampal Dependent 2-5 Month old mice 5 days 12 trials per day Blocks of 4 trials Transfer tests at the end of days 3, 5 Visible Platform Version Hippocampal Independent Tested for 2 days 12 trials per day Transfer test at the end Hidden Platform Version Performed after Visible Platform Version to verify data

Autophosphorylation and Spatial Learning

D.Visible PlatformF. Transfer Test E.Hidden PlatformG. Transfer Test Visible Platform Test

αCaMKII T286A-129B6F2 mutants have deficits in spatial learning Result

Conclusion Autophosphorylation of  CaMKII is required LTP and Learning

Criticism/Future Experimentation What about  CaMKIIs? Visible Platform Test Early Results QUESTIONS?

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