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Previous Experiments

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 5352. pp.870-873. 1998. Previous Experiments. Synaptic Strength Related To Learning/Memory i.e. LTP

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Previous Experiments

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  1. Autophosphorylation at Thr286 of the αCalcium-Calmodulin Kinase II in LTP and LearningKarl Peter Giese, Nikolai B. Fedorov, Robert K. Filipkowski, Alcino J. Siilva*Science vol.279, no 5352. pp.870-873. 1998

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

  3. More Background • Autophosphorylation at Thr286 CaMKII from CaM-independent to CaM-dependent • LTP  Long-lasting Autophosphorylated form of CaMKII at Thr286

  4. Hypothesis The autophosphorylation at the Thr286 site of CaMKII is required for LTP and learning.

  5. CaMKII TT 305/306 Inhibitory B C

  6. Experiment 1 – T286A

  7. Results from Experiment 1 Result • The αCaMKIIT286A-129B6F2 mutation decreased the total CaM-independent CaMKII activity in the mutants • β-CaMKII?

  8. Verify Experiment 1 Result • The point mutations and the loxP site did not alter the expression of theαCaMKII gene

  9. Verify Experiment 1 Result Result • Total CaMKII (CaM independent + dependent) activity remained approximately equal.

  10. 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

  11. Testing for LTP in αCaMKIIT286A-129B6F2 mutants • Protocol • 100Hz tetanus (1s) • Check for potentiation60 minutes • Results • LTP deficient in αCaMKIIT286A-129B6F2 mutants

  12. Testing for LTP in αCaMKIIT286A-129B6F2 mutants  NO OVERLAP LTP impairments in the αCaMKIIT286A-129B6F2 mutants

  13. 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  LTP impairments NOT due to prepotentiation of Synaptic Transmission

  14. Experiment 3 - Pairing Protocol Background • To confirm the LTP deficiency of αCaMKIIT286A-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

  15. Pairing Protocol Result Pairing Protocol Mutant: 132 ± 8% WT: 277 ± 21% No overlap LTP deficits in the αCaMKIIT286A-129B6F2 mutants

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

  17. Experiment 4 – NMDAR-dependent LTP • Procedure • 100 Hz Tetanus for 1 s • NMDAR in the presence of AP5 • Results (after 30 min) Mutant Insensitive to AP5  Mutant NMDAR-dependent LTP already deficient

  18. 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

  19. 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

  20. 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 ± 0.053 mV • Wild-Type: 0.200 ± 0.020 mV  LTP impairments in the mutant αCaMKIIT286A-129B6F2 were not due to abnormal NMDAR function.

  21. Original Hypothesis: • The autophosphorylation at the Thr286 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?

  22. Autophosphorylation and Spatial Learning 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 Morris Water Maze

  23. Autophosphorylation and Spatial Learning

  24. Visible Platform Test • Visible Platform F. Transfer Test • Hidden Platform G. Transfer Test

  25. Result αCaMKIIT286A-129B6F2 mutants have deficits in spatial learning

  26. Conclusion • Autophosphorylation of aCaMKII is required LTP and Learning

  27. Criticism/Future Experimentation • What about bCaMKIIs? • Visible Platform Test Early Results QUESTIONS?

  28. Powerpoint created by: Wendy Lee Jill Marti Power point presented by: Jason Ly Parth Makker Powerpoint assisted by: Ruby Liu

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