generation of silent synapses by acute in vivo expression of camkiv and creb hélène marie, wade...

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

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:

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

Ca++

Ca++ Ca++

Ca++

VGCC

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++

Background Soluble molecules could diffuse or transport

from distal sites to somal/nuclear sites Kinases, CaM, etc…

RasRafMEKERK Importin-mediated nuclear transport could

function as a signal carrier (Otis et al. 2006, Thompson et al. 2004)

CaM

Ca++

CaMKIV

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)

Background

Nuclear expressed CaMKIV Phosphorylates CREB at Ser133

Phospho-CREB-S133 initiates transcription at CRE sequences

Putative Pathway

CREBCaMKIV Silent Synapses

Tools CaMKIV-Consitutively Active (CaMKIVCA)

CaMKIVCA: Deletion of

autoinhibitory domain (aa 1 – 317)

Construct

Phosphorylated CREB (Phospho-S133) signal from CaMKIVCA is 2-fold stronger than GFP infected cells


Tools - CaMKIV-Dominant Negative (CaMKIVDN)

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:


Tools – CaMKIVCA and CaMKIVDN

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)


Synaptic Effects of CaMKIVCA and CaMKIVDN

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?



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)?



*Mini-EPSCs


Time

Time

Time

Time

Baseline Frequency and Amplitude

Increased Amplitude

Increased Frequency

Increased Frequency and Amplitude*Assuming a postsynaptic locus of plasticity


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.



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?

Plasticity Test


Questions Left Unresolved: 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?

Tools – CREB-Consitutively Active (CREBCA)

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: Note*

PPR test showed no presynaptic change


Gain of Function mutation (Y134F)

Synaptic Effects of CREBCA 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


Synaptic Effects of CREBCA

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:


Synaptic Effects of CREBCA LTP:

Increased magnitude and maintenance

LTD: Uneffected


Questions Left Unresolved 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?

Generation of Silent Synapses by CREBCA

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.


Mean

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.

Generation of Silent Synapses by CREBCA 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


Generation of Silent Synapses by CREBCA 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

• With large variation, the CV becomes large

μ√V • With small

variation, the CV becomes small

μ√V

σ = √V = Std Dev

μ = MeanCV = μ

σ

Generation of Silent Synapses by CREBCA

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


Generation of Silent Synapses by CREBCA 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

Generation of Silent Synapses by CREBCA Suppose: Presynaptic release

probability of 50% (P=0.5)


NMDARP = 0.5

AMPAR/NMDAR

AMPAR/NMDAR

P = 0.5

P = 0.5

-65mV – 2 synapsesProbability of Failure = (0.5) 2 = 25%

Probability of Success = 75%

-65mV+40mV

+40mV – 3 synapsesProbability of Failure = (0.5) 3 = 12.5%

Probability of Success = 87.5%

Generation of Silent Synapses by CREBCA Imagine: CREBCA Silent Synapse

formation


AMPAR/NMDAR

AMPAR/NMDAR

P = 0.5

P = 0.5

-65mV – 2 synapsesProbability of Failure = (0.5) 2 = 25%

Probability of Success = 75%

-65mV+40mV

NMDARP = 0.5

NMDARP = 0.5

+40mV – 4 synapsesProbability of Failure = (0.5) 4 = 6.25%

Probability of Success = 93.75%Uninfected Success < Infected Success

Generation of Silent Synapses by CREBCA 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


Morphological effects of CamKIV and CREBCA

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



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



Synaptic NMDAR density increases following CREBCA expression



Synaptic AMPAR density remains unchanged following CREBCA expression


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


Other Targets AMPA receptor insertion

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

Citations Ginty DD, Kornhauser JM, Thompson MA, bading H, Mayo KE, Takahashi JS, Greenberg ME. Regulation

of CREB phosphorylation in the suprachiasmatic nucleus by light and a circadian clock. Science. 1993 Apr 9;260(5105):238-41.

K. Deisseroth, H. Bito and R.W. Tsien. Signaling from synapse to nucleus: postsynaptic CREB phosphorylation during multiple forms of hippocampal synaptic plasticity. Neuron 16 (1996), pp. 89–101.

Barth AL, Gerkin RC, Dean KL. Alteration of neuronal firing properties after in vivo experience in a FosGFP transgenic mouse. J Neurosci. 2004 Jul 21;24(29):6466-75.

A.E. West, E.C. Griffith and M.E. Greenberg. Regulation of transcription factors by neuronal activity. Nat. Rev. Neurosci. 3 (2002), pp. 921–931.

B.E. Lonze and D.D. Ginty. Function and regulation of CREB family transcription factors in the nervous system. Neuron 35 (2002), pp. 605–623.

Frey U, Morris RG. Synaptic tagging and long-term potentiation. Nature. 1997 Feb 6;385(6616):533-6. Frey U, Krug M, Reymann KG, Matthies H. Anisomycin, an inhibitor of protein synthesis, blocks late

phases of LTP phenomena in the hippocampal CA1 region in vitro. Brain Res. 1988 Jun 14;452(1-2):57-65.

Otis KO, Thompson KR, Martin KC. Importin-mediated nuclear transport in neurons. Curr Opin Neurobiol. 2006 Jun;16(3):329-35. Epub 2006 May 11. Review.

Thompson KR, Otis KO, Chen DY, Zhao Y, O’Dell TJ, Martin KC. Synapse to nucleus signaling during long-term synaptic plasticity; a role for the classical active nuclear import pathway. Neuron. 2004 Dec 16;44(6):997-1009.

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generation of silent synapses by acute in vivo expression of camkiv and creb hélène marie, wade...

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