Generation of Silent Synapses by Acute In Vivo Expression of CaMKIV and CREB

1. Generation of Silent Synapses by Acute In Vivo Expression of CaMKIV and CREB • Hélène Marie, Wade Morishita1, Xiang Yu1, Nicole Calakos and Robert C. Malenka • Nancy Pritzker Laboratory, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Palo Alto, California 94304 Received 16 August 2004;  revised 23 November 2004;  accepted 26 January 2005.  Published: March 2, 2005.  Available online 2 March 2005. • Group 6: • Britta Mason, Mayank Mehrotra, Cynthia Meyer, Frances Miles, Ashely Mo, Coel Momita, Ryan Natan, Linh Nguyen, Nam Nguyen, Trang Nguyen, Albert Noniyeu, Alan Okada

2. Background • L-LTP requires protein synthesis (Frey et al. 1997, 1988) • Requires synapse to nucleus signal • How does a synapse communicate with a nucleus? • 3 ideas:

3. Background • Synaptic depolarization could spread to the soma and activate Voltage-Gated Ca++ channels (VGCC) (Otis et al. 2006, Thompson et al. 2004) • Somal Ca++ current could induce rapid signaling to the nucleus (Otis et al. 2006, Thompson et al. 2004) mV VGCC Ca++ Ca++ Ca++ Ca++

4. Background • Endoplasmic Reticular signaling using regenerative Ca++ waves mediated by Ryanodine Receptors or IP3 Receptors (Otis et al. 2006, Thompson et al. 2004) • Somal Ca++ current could induce rapid signaling to the nucleus (Otis et al. 2006, Thompson et al. 2004) Ca++ Ca++ Ca++ Ca++ Ca++ Ca++ Ca++ Ca++ Ca++ Ca++ Ca++

5. Background • Soluble molecules could diffuse or transport from distal sites to somal/nuclear sites • Kinases, CaM, etc… • RasRafMEKERK • Importin-mediated nuclear transport could function as a signal carrier (Otis et al. 2006, Thompson et al. 2004) CaM CaMKIV Ca++ • Calmodulin transports into the nucleus where CaMKIV is localized(Deisseroth et al. 1998) • Local Ca++ flux driven • L-Type Ca++ Channels • NMDA Receptors (Deisseroth et al. 1998) • Does NOT involve the spread of free Ca++from synapse to soma. (Deisseroth et al. 1998)

6. Background • Nuclear expressed CaMKIV Phosphorylates CREB at Ser133 • Phospho-CREB-S133 initiates transcription at CRE sequences

7. Putative Pathway CaMKIV CREB Silent Synapses

8. CaMKIV Tools CaMKIV-Consitutively Active (CaMKIVCA) CREB Silent Synapses • CaMKIVCA: • Deletion of autoinhibitory domain (aa 1 – 317) Construct Phosphorylated CREB (Phospho-S133) signal from CaMKIVCA is 2-fold stronger than GFP infected cells

9. CaMKIV Tools -CaMKIV-Dominant Negative (CaMKIVDN) CREB Silent Synapses • CaMKIVDN • Loss of function mutation at ATP Binding site (K75E) • KCl – depolarization-induced phosphorylation of CREB-S133(Ginty et al. 1993) (Deisseroth et al 1996) Construct:

10. CaMKIV Tools –CaMKIVCA and CaMKIVDN CREB Silent Synapses Paired-Pulse Ratio Test: Inversly correlates with changes in presynaptic release probability 50 msec Inter Stimulus Interval Data argues of a non-presynaptic effect by the constructs (postsynaptic)

11. CaMKIV Synaptic Effects of CaMKIVCA and CaMKIVDN CREB Silent Synapses • Decrease in AMPAR/NMDAR ratio Implications • NMDAR population increase? • Removal of AMPARs from synpases? • NMDAR increase masks changes in AMPAR populations (or opposite)? GFP • Next logical question: How are the receptor contributions changing?

12. CaMKIV Synaptic Effects of CaMKIVCA and CaMKIVDN CREB Silent Synapses • Where are NMDA and AMPA receptors being inserted? Implications • NMDAR population increase? • Removal of AMPARs from synpases? • NMDAR increase masks changes in AMPAR populations (or opposite)?

13. CaMKIV Synaptic Effects of CaMKIVCA and CaMKIVDN CREB Silent Synapses • *Mini-EPSCs Baseline Frequency and Amplitude Time Increased Amplitude Time Increased Frequency Time Increased Frequency and Amplitude *Assuming a postsynaptic locus of plasticity Time

14. CaMKIV Synaptic Effects of CaMKIVCA and CaMKIVDN CREB Silent Synapses • *Mini-EPSCs Baseline Frequency and Amplitude Time Increased Amplitude Time Increased Frequency Time Increased Frequency and Amplitude *Assuming a postsynaptic locus of plasticity Time

15. CaMKIV Synaptic Effects of CaMKIVCA and CaMKIVDN CREB Silent Synapses • mEPSC-test • Frequency increased Amplitude did not • Indicative of an increase in number of functional synapses • Implies insertion of AMPARs into naïve synapses • What’s happening to NMDA receptors? The increase is not resolved by this test.

16. CaMKIV Synaptic Effects of CaMKIVCA and CaMKIVDN CREB Silent Synapses Plasticity Test • Increased Magnitude and maintenance for LTP • No effect on LTD • Is CaMKIV doing something to facilitate early LTP? • What about Late-LTP? • What about minimal LTP-induction protocols to “titrate” the amount LTP is facilitated? • What about tests against the learning paradigm?

17. Questions Left Unresolved: CaMKIV CREB Silent Synapses • Where are NMDA receptors being inserted? • Which of these effects are the result of CaMKIV phosphorylation of CREB? • How does CaMKIV contribute to AMPAR insertion? • How does CaMKIV come to be activated?

18. CREB Tools – CREB-Consitutively Active (CREBCA) CaMKIV Silent Synapses • Note* • PPR test showed no presynaptic change • CREBCA - Control • C-fos-GFP construct: • C-fos is a known CREB target (West et al. 2002)(Lonze et al. 2002) • C-fos promotor controlled GFP gene in a mutant mouse (Barth et al. 2004) • Dissociated hippocampal cultures • Transfected with CREBWT/CA • Showed CREB activity by quanitfication of GFP signal in WT vs. CA transfected neurons Construct: Gain of Function mutation (Y134F)

19. CREB Synaptic Effects of CREBCA CaMKIV Silent Synapses • Similar decrease in AMPA/NMDA ratio to CaMKIVCA neurons • NMDAR contribution is 2-fold over control • AMPAR contribution is not significantly different • Implies that the AMPAR synaptic insertion observed following CaMKIV infection was not mediated by CREB activity

20. CREB Synaptic Effects of CREBCA CaMKIV Silent Synapses • CREBCA shows no significant change in amplitude or frequency of mEPSCs. • Consistent with the hypothesis that CREB is mediating changes in NMDA receptor synaptic insertion mEPSC-Test:

21. CREB Synaptic Effects of CREBCA CaMKIV Silent Synapses • LTP: • Increased magnitude and maintenance • LTD: • Uneffected

22. Questions Left Unresolved CaMKIV CREB Silent Synapses • How does CaMKIV activity lead to AMPAR insertion into synapses? • How does CREB phosphorylation lead to changes in NMDAR synaptic expression? • Where are NMDA receptors being inserted?

23. Mean Silent Synapses Generation of Silent Synapses by CREBCA CaMKIV CREB • Critical experiment 1: • Coefficient of variation √Variance • General Rule: • The lower the Coefficient of Variation (CV), the greater the number of synapses contributed to the synaptic response. • Coefficient of Variation • SqRt of Variance/Mean • SqRt of Variance = Standard Deviation • CV = StdDev/Mean • What would cause greater deviation from the mean? • Stochastic release.

24. Silent Synapses Generation of Silent Synapses by CREBCA CaMKIV CREB • Coefficient of Variation Test: a statistical measure of silent synapse formation • Sample size is “inversely proportional” to variability of output data • Meaured EPSC StdDev  Normalized to mean • Relative variance  Coefficient of Variation (CV) • Vesicular release is stochastic • Variation about mean is due to the number of SYNAPSES, not the number of NMDA receptors

25. CV = • With small variation, the CV becomes small μ = Mean √V • With large variation, the CV becomes large σ √V μ μ σ = √V = Std Dev μ Silent Synapses Generation of Silent Synapses by CREBCA CaMKIV CREB • What does the CV value mean? • General Rule: • The lower the CV, the greater the number of synapses contributing to the synaptic response. • How does the CV change with changes in variability? • Mean remains relatively constant

26. Silent Synapses Generation of Silent Synapses by CREBCA CaMKIV CREB • Critical experiment 1: • CV-CREBCA at +40 mV dropped • CV is uneffected at -65 mV • Normalized CV ratio CV-NMDAR/CV-AMPAR • CREBCA CV-ratio is lower than control • CREBCA drives silent synapse formation

27. Silent Synapses Generation of Silent Synapses by CREBCA CaMKIV CREB • Minimal Stimulation technique: • Stimulate Schaffer Collaterals with very weak current • Activates a small number of axons • Presynaptic release is stochastic • Small sample occasional failure to release

28. P = 0.5 NMDAR P = 0.5 AMPAR/NMDAR P = 0.5 AMPAR/NMDAR Silent Synapses Generation of Silent Synapses by CREBCA CaMKIV CREB • Suppose: Presynaptic release probability of 50% (P=0.5) +40mV -65mV -65mV – 2 synapses Probability of Failure = (0.5) 2 = 25% Probability of Success = 75% +40mV – 3 synapses Probability of Failure = (0.5) 3 = 12.5% Probability of Success = 87.5%

29. NMDAR P = 0.5 P = 0.5 NMDAR P = 0.5 AMPAR/NMDAR P = 0.5 AMPAR/NMDAR Uninfected Success < Infected Success Silent Synapses Generation of Silent Synapses by CREBCA CaMKIV CREB • Imagine: CREBCA  Silent Synapse formation +40mV -65mV -65mV – 2 synapses Probability of Failure = (0.5) 2 = 25% Probability of Success = 75% +40mV – 4 synapses Probability of Failure = (0.5) 4 = 6.25% Probability of Success = 93.75%

30. Silent Synapses Generation of Silent Synapses by CREBCA CaMKIV CREB • Percent Silent Synapses: • CREBCA – 41% ± 4.8% • Uninfected – 19% ± 7.2% • *Assuming equal probability of release at the presynaptic terminals, the percent of silent synapses can be estimated

31. Silent Synapses Morphological effects of CamKIV and CREBCA CaMKIV CREB • What if the constructs are causing retrograde signaling that causes an overspill of quanta, ultimately activating extra NMDA receptors? • Possible contaminant of CV and failure/success rate tests • Solution: • Immunocytochemical spine analysis • Spine density • Receptor density

32. Silent Synapses Morphological effects of CamKIV and CREBCA CaMKIV CREB • Perfused Alexa Fluor 568 into GFP expressing CA1 pyramidal cells • An increase in spine density is consistent with data indicating an increase in silent synapses

33. Silent Synapses Morphological effects of CamKIV and CREBCA CaMKIV CREB • Synaptic NMDAR density increases following CREBCA expression

34. Silent Synapses Morphological effects of CamKIV and CREBCA CaMKIV CREB • Synaptic AMPAR density remains unchanged following CREBCA expression

35. Summary • Unanswered questions: • How does CaMKIV activity lead to AMPAR insertion into synapses? • How does CREB phosphorylation lead to changes in NMDAR synaptic expression? • What is the compliment of proteins produced by CREB that leads to silent synapse formation CaMKIV CREB Silent Synapses Other Targets AMPA receptor insertion

36. Concerns • Over-expression experiments don’t necessarily represent endogenous activity • Broader range of interpulse (interstimulus) intervals (ISI) to detect changes in release probability from presynaptic cell. • Test to rule out an increase in quantal release due to post-synaptic contruct expression for all constructs

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