generation of silent synapses by acute in vivo expression of camkiv and creb hélène marie, wade...
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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
CREBCaMKIV Silent Synapses
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:
CREBCaMKIV Silent Synapses
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)
CREBCaMKIV Silent Synapses
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?
CREBCaMKIV Silent Synapses
Synaptic Effects of CaMKIVCA and CaMKIVDN
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)?
CREBCaMKIV Silent Synapses
Synaptic Effects of CaMKIVCA and CaMKIVDN
*Mini-EPSCs
CREBCaMKIV Silent Synapses
Time
Time
Time
Time
Baseline Frequency and Amplitude
Increased Amplitude
Increased Frequency
Increased Frequency and Amplitude*Assuming a postsynaptic locus of plasticity
Synaptic Effects of CaMKIVCA and CaMKIVDN
*Mini-EPSCs
CREBCaMKIV Silent Synapses
Time
Time
Time
Time
Baseline Frequency and Amplitude
Increased Amplitude
Increased Frequency
Increased Frequency and Amplitude*Assuming a postsynaptic locus of plasticity
Synaptic Effects of CaMKIVCA and CaMKIVDN
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.
CREBCaMKIV Silent Synapses
Synaptic Effects of CaMKIVCA and CaMKIVDN
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
CREBCaMKIV Silent Synapses
Questions Left Unresolved: Where are NMDA receptors being inserted? Which of these effects are the result of
CaMKIV phosphorylation of CREB?
CREBCaMKIV Silent Synapses
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
CREBCaMKIV Silent Synapses
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
CREBCaMKIV Silent Synapses
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:
CREBCaMKIV Silent Synapses
Synaptic Effects of CREBCA LTP:
Increased magnitude and maintenance
LTD: Uneffected
CREBCaMKIV Silent Synapses
Questions Left Unresolved How does CaMKIV activity lead to AMPAR
insertion into synapses? How does CREB phosphorylation lead to
changes in NMDAR synaptic expression?
CREBCaMKIV Silent Synapses
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.
CREBCaMKIV Silent Synapses
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
CREBCaMKIV Silent Synapses
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.
CREBCaMKIV Silent Synapses
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
CREBCaMKIV Silent Synapses
Generation of Silent Synapses by CREBCA Minimal Stimulation technique:
Stimulate Schaffer Collaterals with very weak current
Activates a small number of axons
CREBCaMKIV Silent Synapses
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)
CREBCaMKIV Silent Synapses
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
CREBCaMKIV Silent Synapses
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
CREBCaMKIV Silent Synapses
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
CREBCaMKIV Silent Synapses
Morphological effects of CamKIV and CREBCA
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
CREBCaMKIV Silent Synapses
Morphological effects of CamKIV and CREBCA
Synaptic NMDAR density increases following CREBCA expression
CREBCaMKIV Silent Synapses
Morphological effects of CamKIV and CREBCA
Synaptic AMPAR density remains unchanged following CREBCA expression
CREBCaMKIV Silent Synapses
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
CREBCaMKIV Silent Synapses
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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