Cell, Vol. 108, 689–703, March 8, 2002, Copyright 2002 by Cell Press
Expression of Constitutively Active CREB Protein
Facilitates the Late Phase of Long-Term
Potentiation by Enhancing Synaptic Capture
pathway experiments indicate that both short-term and
long-term facilitation can be synapse specific.
Frey and Morris (1997, 1998) first delineated synaptic
capture in the mammalian brain. They found that once
transcription-dependent LTP has been induced at one
pathway, the long-term process can be “captured” at
a second pathway receiving a single train, a stimula-
ion that would normally produce only E-LTP. Thus, the
stimulus for the short-term process serves not only to
produce transient facilitation at that synapse, but can
also mark and stabilize facilitation at any synapse of
the neuron by capturing, for that synapse, the newly
description, synaptic capture in mammalian hippocam-
pus has remained uncharacterized, and many aspects
remain unclear. Does capture share the same molecular
machinery as L-LTP? What gene products are distrib-
uted cell-wide when the long-term process is turned on
at one synapse? What is the molecular nature of the tag
that marks active synapses?
Studies of synaptic capture at the synapses between
the sensory and motor neurons of the gill-withdrawal
reflex in Aplysia have shed light on some of these ques-
tions. In Aplysia, synapse-specific facilitation requires
the activity of the transcriptional activator CREB-1 (the
cAMP responsive element binding protein) in the nu-
the stimulated synapses and local protein synthesis to
stabilize that mark (Casadio et al., 1999; Martin et al.,
1997). Furthermore, injection of CRE sequence oligonu-
cleotides in Aplysia neurons selectively inhibits long-
term facilitation with no effect in short-term synaptic
plasticity (Dash et al., 1990), suggesting that the cAMP
responsive element (CRE)-driven gene products are
good candidates for the priming molecules that need
to be captured at the marked synapse. Indeed, injection
into the cell body of phosphorylated CREB-1 gives rise
neuron by seeding these synapses with the protein
is not maintained unless the synapse is marked by the
short-term process (Casadio et al., 1999).
from mollusks to humans suggests that CREB acts as
one of the core components in the molecular switch
that converts short- to long-term synaptic plasticity and
short- to long-term memory (reviewed in Mayford and
Kandel, 1999; Silva et al., 1998). In Aplysia, opposing
forms of CREB (CREB1a activator and CREB1b and
CREB2 repressor) produce opposite effects on long-
term facilitation (Bartsch et al., 1998, 1995). Similarly,
opposing forms of CREB also produce opposite effects
on long-term memory in transgenic flies (Yin et al., 1995,
Mammals also seem to require activation of CRE-
pocampal infusion of CREB antisense oligonucleotides
disrupts long-term spatial memory in rats, but does not
affect short-term memory (Guzowski and McGaugh,
1997). In mice, there is increased expression of a CRE-
Angel Barco,2,4Juan M. Alarcon,2,4
and Eric R. Kandel1,2,3
1Howard Hughes Medical Institute and
2Center for Neurobiology and Behavior
College of Physicians and Surgeons
of Columbia University
1051 Riverside Drive
New York, New York 10032
Restricted and regulated expression in mice of VP16-
CREB, a constitutively active form of CREB, in hippo-
campal CA1 neurons lowers the threshold for elicit-
ing a persistent late phase of long-term potentiation
(L-LTP) in the Schaffer collateral pathway. This L-LTP
has unusual properties in that its induction is not de-
pendent on transcription. Pharmacological and two-
pathway experiments suggest a model in which VP16-
CREB activates the transcription of CRE-driven genes
and leads to a cell-wide distribution of proteins that
prime the synapses for subsequent synapse-specific
capture of L-LTP by a weak stimulus. Our analysis
indicates that synaptic capture of CRE-driven gene
products may be sufficient for consolidation of LTP
and provides insight into the molecular mechanisms
The encoding of new memories in the nervous system
tant synaptic model for encoding memories is long-term
potentiation (LTP) (Martin et al., 2000b). In LTP, as in
memory storage, it is possible to distinguish between
stages of storage. There is an early, short-term stage
(E-LTP), which lasts minutes, and a later, long-term
(LTM) the requirement for the synthesis of new mRNA
and protein (Frey et al., 1988; Montarolo et al., 1986;
Nguyen et al., 1994). The finding of a transcriptional
requirement for long-lasting forms of synaptic plasticity
has raised a fundamental question in the study of learn-
in the nucleus mean that the critical unit of long-term
synaptic plasticity is the cell nucleus, or can long-term
synaptic plasticity somehow be restricted to the single
synapse? If the unit of long-term storage is the synapse,
what mechanisms restrict the action of the newly ex-
pressed gene products to some synapses but not to
others? Studies of these questions in Aplysia (Martin et
al., 1997) and rats (Frey and Morris, 1997) using two-
4These authors contributed equally to this work.
driven lacZ reporter construct following stimuli that pro-
duce L-LTP (Impey et al., 1996) and after training on a
hippocampus-dependent task (Impey et al., 1998).
cells by electrical stimuli that induce LTP (Bito et al.,
1996; Lu et al., 1999) and after training in hippocampus-
dependent tasks (Taubenfeld et al., 1999). CREB is a
target of PKA, CaMKIV, and the MAPK cascade, all of
which have been implicated in L-LTP. Therefore, CREB
is a strong candidate for the activation of CRE-driven
gene expression observed during memory formation.
Indeed, LTP and long-term, but not short-term, memory
of ?? isoforms of CREB (Bourtchuladze et al., 1994).
However, in contrast to the evidence for the direct role
the situation in mammals is less clear. Both memory
and the deficits in LTP in mice with genetic deletion of
the ?? isoform of CREB have been found to be sensitive
to gene dosage and genetic background, indicating that
the activity of other genes can compensate for loss
of CREB (Gass et al., 1998). In fact, the CREB partial
knockout mice show strong upregulation of other CRE
ler et al., 1994). In addition, transgenic mice overex-
pressing a dominant-negative mutant of CREB in amyg-
et al., 2000), although overexpression of CREB in the
same region, using viral expression vectors, enhanced
memory (Josselyn et al., 2001).
To explore the role played in hippocampal synaptic
plasticity by CRE-driven genes, we generated trans-
genic mice in which we can induce, in a regulated man-
ner, the expression of a constitutively active CREB pro-
tein. This chimeric protein, VP16-CREB, was obtained
by replacing the first transactivation domain of CREB
virus (HSV) VP16. Equivalent chimeric proteins bind to
CRE sequences in tissue-specific promoters and be-
have like CREB activated by phosphorylation in both
adipocytes (Reusch et al., 2000) and in cultured neurons
(Riccio et al., 1999).
We find that when expressed in the postsynaptic neu-
rons of the Schaffer collateral pathway, VP16-CREB
binds to CREs and regulates transcription of several
downstream genes thought to play an important role in
LTP and memory formation. Expression of VP16-CREB
is sufficient to facilitate establishment of hippocampal
L-LTP in an input-specific manner by enhancing synap-
tic capture, much as is the case with phospho CREB-1
in Aplysia. The findings provided us with an opportunity
notypic characteristics of capture in both transgenic
and wild-type synapses.
is 25-fold more active than wild-type CREB (p ? 0.005)
and slightly less active than wild-type CREB when co-
transfectedwith PKAcatalyticsubunit(Figure 1A,differ-
ence is not significant). Cotransfection of VP16-CREB
with PKA elicits an even greater activation of the CRE-
driven reporter (p ? 0.05), presumably mediated by
phosphorylation of the KID domain of CREB present in
VP16-CREB. This stimulation was specific to a CRE-
containing promoter, as it was not observed in promot-
ers bearing a number of different response elements,
such as SRE, HRE, or GRE (Figure 1A and results not
To investigate the consequences of constitutive ac-
tivation of CRE-dependent transcription for synaptic
plasticity, we generated transgenic mice expressing the
chimeric VP16-CREB protein. To regulate and limit the
expression of the transgene to neurons in the forebrain,
we used the double transgenic system developed in our
laboratory (Mayford et al., 1996). Transgene expression
pattern was examined by in situ hybridization. One of
the transgenic lines (VC27) was particularly advanta-
geous for studying hippocampal function and Schaffer
collateral LTP, because VP16-CREB was expressed se-
lectively in CA1 cells, but not in CA3 neurons (Figure
1B). This provided selective expression in the postsyn-
aptic, but not the presynaptic, neurons of the Schaffer
collateral pathway. The transgene was also detected in
dentate gyrus and, at lower levels, in striatum and in
of expression using anti-VP16 antibody. Immunohisto-
chemical analyses showed that most of the neurons in
CA1 express the transgeneand the intracellular location
of the protein is mostly nuclear (Figure 1C).
precise temporal regulation of transgene expression
through the ability of doxycycline (dox) to block the
binding of tTA to DNA (Mayford et al., 1996). To avoid
possible developmental problems due to early expres-
sion of CREB activity and possible compensation by
other CRE binding transcription factors, we bred the
animals in the presence of dox and removed the drug
to induce expression of the transgene at specific times
before our experiments. We did not observe any gross
difference between the brains of transgenic and wild-
Western blot analysis of hippocampal extracts from
transgenic mice revealed that removal of dox induces
the synthesis of a protein recognized by anti-CREB and
anti-VP16 antibodies at a level similar to that of endoge-
nous CREB (Figure 2A and result not shown). Transgene
induction was detected three to four days after removal
of dox and reached a plateau level after a week. In turn,
repression of VP16-CREB required two to four days of
feeding with dox. Therefore, in the double transgenic
CREB in the pyramidal neurons of the CA1 region and
to rapidly turn its activity on and off in a few days.
Regulated Expression in the Forebrain of VP16-
CREB, a Constitutively Active CREB
We created a chimeric protein by replacing the first
Gln-rich domain of CREB with the acidic transactivation
domain of HSV VP16. Cotransfection of HEK293 cells
with a CRE reporter plasmid showed that VP16-CREB
VP16-CREB Stimulates Transcription
of CRE-Driven Genes in Mouse Brain
To determine whether the VP16-CREB fusion protein
was functional, we assayed CRE binding activity with
Facilitated LTP by Constitutively Active CREB
Figure 1. Expression of VP16-CREB, a Constitutively Active Form of CREB, in the Brain of Transgenic Mice
(A) HEK293 cells were cotransfected with 1 ?g of pCRE.luc or pSRE.luc reporter plasmids and 0.5 ?g of pRcRSV-derived plasmids encoding
wild-type CREB, VP16-CREB, or PKA. In all transfections, 0.05 ?g of pRL-SV40 was added for normalization and the total amount of DNA
was adjusted to 2.05 ?g with pRcRSV vector DNA. Firefly luciferase expression was normalized to Renilla luciferase activity and compared
for each reporter to the normalized luciferase activity for CREB in the absence of PKA (arbitrarily set at 1.0). Each bar represents the mean
of three independent experiments ? SEM.
(B) In situ hybridization on brain coronal (upper panel) and sagittal (lower panel) sections from a VP16-CREB mouse (VC) and a wild-type
littermate (WT) with a probe specific for the VP16/CREB junction (Hp: hippocampus, Cx: cortex, St: striatum).
(C) From low to high magnification, detail of transgene expression in hippocampus (Hp). Brain vibratome sagittal sections of a VP16-CREB-
expressing mouse were immunostained with anti-VP16 antibody. VP16-CREB is mainly located in CA1 and dentate gyrus (DG) neurons. Boxes
indicate amplified regions. Bar ? 200 ?m.
anti-VP16 antibody in hippocampal extracts of trans-
genic and control mice. Only animals expressing VP16-
CREB showed VP16-immunoreactive protein that was
able to bind to CRE oligonucleotide probe (Figure 2B,
p ? 0.05), demonstrating that the chimeric protein ex-
pressed in hippocampal neurons is functionally active
and able to bind the CRE sequence.
A number of genes regulated by CREB are thought to
be important for the late phases of LTP and for memory
storage. We have examined three examples of genes
that have been shown previously to be regulated by
CREB and involved in neuronal function. First, expres-
promoter contains a CRE sequence, increases after
learning-related events and can modulate physiological
plasticity in CA1 area (Korte et al., 1995; Patterson et
al., 1992; Tao et al., 1998). Similarly, expression of the
peptide dynorphin is regulated by CREB, both in vitro
and in vivo (Carlezon et al., 1998). Finally, we examined
expression of c-fos, a commonly used marker of gene
induction associated with neuronal activity, whose pro-
moter alsocontains two CREs thatmediate its induction
by cAMP and Ca2?(Sheng et al., 1990). The concentra-
tion of BDNF and prodynorphin was clearly increased
after VP16-CREB expression and the time course of this
induction mirrored that of the VP16-CREB transgene
(Figure 2C). Using double labeling with anti-c-fos and
anti-VP16 antibodies, we also found a correlation of
VP16-CREB expression and c-fos induction at the cellu-
lar level (Figure 2D). Together, these data indicate that
VP16-CREB activates the expression of CREB-regu-
lated genes in hippocampal neurons.
VP16-CREB Expression Does Not Affect
Basal Synaptic Activity
We investigated the effect of postsynaptic expression
of VP16-CREB on synaptic plasticity in the Schaffer col-
lateral pathway by recording extracellular field poten-
tials. If CREB by itself serves as a unique switch for
the generation of LTP, overexpression of VP16-CREB
in transgenic animals might increase basal synaptic
transmission and occlude further attempts to elicit LTP.
This happens in Aplysia neurons, where injection of
Figure 2. Regulation of Transgene and Downstream Gene Expression by Doxycycline
(A) Western blot of hippocampal protein extracts from transgenic (T) and control (C) mice at different times (expressed in days after characters
“T” or “C”) after withdrawal (Days Off) or addition (Days On) of dox. One single band (CREB) was recognized by anti-CREB antibody in wild-
type brain extracts, but an additional band (VP16-CREB) was detected in double transgenics.
(B) IP of CRE/VP16-CREB complex: nuclear extracts from transgenics and control littermates (3 weeks of dox) were incubated with32P-labeled
CRE oligonucleotides and immunoprecipitated using anti-VP16 or anti-myc antibodies (as control for nonspecific binding). Each bar represents
the mean of three independent experiments ? SEM.
(C) Western blot of hippocampal protein extract from transgenic (T) and control (C) mice immunoassayed with anti-BDNF (upper panel) or
anti-prodynorphin (lower panel) at different times after dox withdrawal or addition expressed in days.
(D) Upregulation of c-fos gene expression. Double labeling of CA1 neurons of wild-type (WT) and VP16-CREB (VC) mice (2 weeks off dox)
using anti-c-fos polyclonal antibody and anti-VP16 monoclonal antibody. TO-PRO 3, a DNA stain, was used for counterstaining.
facilitation (about 50% of regular long-term facilitation)
that does not persist and is not accompanied by mor-
phological changes (Casadio et al., 1999). In mice, the
temporal regulation of a transgene expression by dox
does not allow this sort of acute activation, but we did
not find any significant difference in basal transmission
after sustained (2–4 weeks) activation of CRE-depen-
dent transcription. Stimulus-response curves, analyses
of the synaptic fiber volley, and paired-pulse facilitation
(PPF) weresimilar in wild-type andtransgenic mice (Fig-
ures 3A and 3B, and results not shown). However, we
cannot exclude the possibility that the expression of
synaptic transmission that was not revealed in our
wild-type littermates, this single train induced LTP last-
ing 1.5 hr. In VP16-CREB mice, the same stimulation
evoked an LTP with a similar initial amplitude, but with
an enhanced and sustained long-lasting phase (Figure
3C: first 5 min: 198% ? 22% for the VP16-CREB mouse,
and 186% ? 37% for the wild-type mouse; at 3 hr,
179% ? 21% for transgenic compared to 105% ? 15%
for wild-type; p ? 0.05). Thus, the presence of VP16-
CREB in the postsynaptic cell allows a stimulus that
only elicits E-LTP in wild-type mice to elicit L-LTP in
The temporal regulation of the transgene expression
by dox allowed us to test whether the reported changes
in LTP observed in VP16-CREB mice are permanent or
can be reversed. We fed mice with doxycycline for 2–3
weeks after expressing VP16-CREB for one month and
reversed to normal the facilitated LTP observed after
tetanic stimulation, so that they were indistinguishable
from wild-type littermates treated with dox (Figure 3D).
This result demonstrates that constitutive expression of
CRE-driven genes, at least in the time window used in
our experiment, does not cause permanent changes in
the expressing neurons.
The finding that postsynaptical and constitutive ex-
pression of CRE-driven genes produces a shift in the
threshold for the induction of L-LTP suggested the pos-
sibility that other stimulation protocols might also reveal
Mice Expressing VP16-CREB Show a Frequency-
Dependent Shift in Synaptic Plasticity and
a Lowered Threshold for the Elicitation
of L-LTP that Can Be Reversed by Turning
Off Transgene Expression with Doxycycline
Although the continuous activation of CREB-regulated
tic transmission, it might enhance the ease with which
L-LTP is elicited. To test this idea, we first used a stan-
dard 100 Hz tetanus train of 1 s duration that normally
produces a nonsaturating, short-lasting LTP (E-LTP). In
Facilitated LTP by Constitutively Active CREB
Figure 3. Facilitated L-LTP in VP16-CREB Mice
(A) Input-output curve of fEPSP slope (mV/ms) versus stimulus (V) at the Schaffer collateral pathway of hippocampal slices from transgenics
and control littermates.
(B) Comparison of PPF in VP16-CREB and wild-type. Data are presented as the mean ? SEM of the facilitation of the second response relative
to the first response (wild-type: n ? 7, VP16-CREB: n ? 6).
(C) A single 100 Hz train (1 s) evoked E-LTP that lasts up to 2 hr in wild-type animals, but L-LTP lasting more than 6 hr in VP16-CREB mice
(p ? 0.05) (wild-type: n ? 7, VP16-CREB: n ? 7).
(D) Facilitated L-LTP induced by one 100 Hz train in transgenics was reversed by dox (wild-type: n ? 5, VP16-CREB: n ? 6).
(E) Summary data at different times after stimulation for synaptic plasticity at different frequencies.
(F) Four 100 Hz trains stimulation. Inset: detail of the normalized fEPSP slope values during four trains stimulation (wild-type: n ? 7, VP16-
CREB ? 8).
(G) Columns represent the average amplitude response (110–130 min after stimulation) for progressively stronger stimulation protocols.
changes in synaptic plasticity in the Schaffer collateral
pathway. We therefore examined a range of lower fre-
quencies and consistently found changes favoring syn-
aptic facilitation. Thus, stimulation with one 10 Hz train
of 1.5 min duration evoked LTP with an amplitude 1.5-fold
higher in transgenic mice than in wild-type littermates
(Figure 3E, p ? 0.05). Doxycycline administration again
reversed this phenotype (result not shown). Stimulation
at 5 Hz (3 min) evoked a small potentiation in VP16-
CREB mice that was not statistically different from that
observed in wild-type animals (Figure 3E). Stimulation
at 1 Hz (15 min) induced a noticeable LTD in wild-type
animals, but not in VP16-CREB-expressing mice (Figure
3E). Finally, 0.5 Hz stimulation for 30 min induced LTD
in both genotypes, but LTD was larger in wild-type than
in VP16-CREB mice (Figure 3E, p ? 0.05). Therefore,
the expression of VP16-CREB produces a shift in the
above 10 Hz stimulation and obliterates LTD expression
at 1 Hz stimulation.
If a single tetanic train yields L-LTP in VP16-CREB
mice, what is the effect of repeated tetanic trains? To
examine this question, we used stimulation protocols
with several 100 Hz trains spaced 5 min apart. We found
that LTP elicited by four trains had the same duration
but an initial lower initial amplitude in VP16-CREB mice
than in wild-type mice (Figure 3F: 30 min: 213% ? 26%
218% ? 22%, not significant). LTP elicited in transgenic
slices with two trains showed a duration and amplitude
that was not statistically different from that observed in
wild-type or transgenic slices after 4 trains stimulation
(Figure 3G). Indeed, this ceiling is almost reached with
a single tetanus.
the expression or normal E-LTP are present and func-
tional in transgenic mice, but there are further mecha-
nisms of potentiation activated by PKA. Inhibition of
PKA activity during the sustained phase of LTP (30–60
min after LTP induction) had no effect in the transgenic
phenotype (Figure 4B2), indicating that PKA activity is
necessary only to establish the facilitated state, but not
for its maintenance.
Resistance to depotentiation: low-frequency stimula-
tion has little effect on basal synaptic transmission in
the adult hippocampus, but can depress synapses that
have recently undergone LTP (Staubli and Chun, 1996).
differs in some of its properties from LTD (O’Dell and
Kandel, 1994) and can only be elicited during early stages
of LTP (i.e., during the first 20 min after LTP induction)
the potentiated synapses become resistant to depoten-
We initially used one 100 Hz train stimulation to elicit
LTP and 5 Hz stimulation to produce depotentiation.
This low-frequency stimulation by itself had little effect
in fEPSP slope amplitude (Figure 3E), but when given 5
min after LTP induction, it depotentiates the previously
potentiated synapses in wild-type mice, but failed to
depotentiate the synapses in transgenic mice (Figure
4C1: 106% ? 25% versus 172% ? 20%). To extend our
analysis to stronger protocols, we used brief bursts of
stimulation (100 Hz, twice for 1 s with a 20 s interval
[Zhuo et al., 1999]) to produce a long-lasting enhance-
ment of synaptic response in both wild-type and trans-
found that5 Hzstimulation depotentiatedthe previously
potentiated synapses in wild-type mice, but failed to
depotentiate the synapses in transgenic mice (Figure
4C2: 106% ? 12% versus 188% ? 20%). Therefore, the
synaptic plasticity obtained immediately after induction
of LTP in mutants appears to be similar to that observed
during late phase LTP in wild-type animals.
Occlusion by forskolin-induced LTP: forskolin stimu-
lates adenylyl cyclase and the cAMP signaling pathway,
and induces long-lasting synaptic potentiation. Such
potentiation occludes L-LTP induced by repeated teta-
nization (Huang and Kandel, 1994). We found that bath
application of forskolin elicited LTP with similar kinetic
and amplitude in both wild-type and transgenic mice
(125% ? 20% and 130% ? 15%, 30 min after applica-
tion), indicating that the cAMP pathway remains intact
and functional in mutant animals. We next found that
forskolin occluded the facilitated L-LTP produced with
a single tetanus in VP16-CREB mice (Figure 4D1), just
as it occluded the L-LTP induced by four trains in wild-
type littermates (Figure 4D2).
One Train LTP in VP16-CREB Mice Is Dependent
on NMDA and PKA, Cannot Be Depotentiated
with Low-Frequency Stimulation, and Is Occluded
How do the properties of this facilitated L-LTP compare
to those of L-LTP in wild-type mice? To address this
question, we compared four features:
Dependence on the NMDA receptor: induction of LTP
in the Schaffer collateral pathway requires the activity
of N-methyl-D-aspartate (NMDA) receptors. Using APV,
a specific antagonist of the NMDA receptor, we found
that the induction of LTP was similarly blocked in trans-
genic and wild-type littermates (Figure 4A), indicating
that facilitated L-LTP observed in VP16-CREB is an
NMDA-dependent process that shares the same induc-
tive machinery for LTP in the mutant as the wild-type
Dependence on PKA: the consolidation of L-LTP re-
quires PKA activity and is blocked by KT5720, an in-
hibitor of this enzyme (Abel et al., 1997; Matthies and
Reymann, 1993). Incubation with KT5720 during LTP
induction did not affect one train-evoked LTP (E-LTP)
in wild-type mice, but eliminated the LTP facilitation
observed in mice expressing VP16-CREB (Figure 4B1).
In the presence of KT5720, the LTP evoked in VP16-
CREB mice had a similar amplitude and time course to
the one train-evoked LTP observed in the wild-type
mice, suggesting that all the components necessary for
Facilitated L-LTP in Mice Expressing VP16-CREB
Does Not Depend on the Synthesis of New RNA
We have shown that facilitated LTP in VP16-CREB mice
resembles the late phase of LTP in wild-type mice in a
synthesis, the most characteristic property of L-LTP?
As previously described (Frey et al., 1988; Nguyen et
al., 1994), inhibitors of transcription or translation had
Figure 4. Properties Shared by Facilitated LTP and L-LTP
(A) Dependence on NMDA-Receptor. LTP induced by 100 Hz stimulation in wild-type and VP16-CREB mice in the presence of APV.
(B) Dependence on PKA. (B1) E-LTP observed in vehicle-treated slices from wild-type mice after 100 Hz stimulation (result not shown) was
indistinguishable from that observed in slices treated with the PKA inhibitor KT5720. Facilitated L-LTP observed in vehicle-treated slices from
VP16-CREB mice was reversed to E-LTP by KT5720. (B2) Application of PKA inhibitor 30 min after 100 Hz stimulation did not affect facilitated
L-LTP in VP16-CREB mice.
(C) Resistance to depotentiation. (C1) Five min after evoking LTP with a single 100 Hz train, 5 Hz stimulation depotentiated the LTP elicited
in wild-type but not VP16-CREB mice (p ? 0.05). (C2) Equivalent results were obtained for depotentiation of LTP evoked by two 100 Hz trains
spaced by 20 s (p ? 0.05).
(D) Occlusion by forskolin. (D1) Forskolin-evoked LTP occludes facilitated L-LTP induced by one 100 Hz train in VP16-CREB mice. (D2)
Forskolin-evoked LTP occludes L-LTP evoked by four 100 Hz trains in wild-type mice.
Figure 5. Effect of Transcription and Translation Inhibition on LTP in VP16-CREB Mice
(A) Effect of transcription and translation inhibition on E-LTP induced by one 100 Hz train in wild-type mice.
(B) Effect of transcription and translation inhibition on facilitated L-LTP induced by one 100 Hz train in VP16-CREB mice.
(C) Anisomycin application 30 min after 100 Hz stimulation did not affect facilitated L-LTP in VP16-CREB mice.
(D) Effect of transcription and translation inhibition on L-LTP induced by four 100 Hz trains in wild-type mice.
(E) Effect of transcription and translation inhibition on L-LTP induced by four 100 Hz trains in VP16-CREB mice.
no effect on one 100 Hz train-elicited E-LTP in wild-type
animals (Figure 5A), but inhibited L-LTP obtained using
the four 100 Hz trains protocol (Figure 5D). In VP16-
CREB mice, we found that LTP induced either by 1 or
by 4 trains was not affected either by actinomycin D nor
two chemically distinct inhibitors of transcription (Fig-
ures 5B and 5E, and results not shown), and only was
partially inhibited by anisomycin, an inhibitor of transla-
tion (Figure 5B at 2 hr: vehicle 176% ? 13%, anisomycin
149% ? 22%, p ? 0.05 and Figure 5E at 2 hr: vehicle:
207% ? 15%; anisomycin: 150% ? 19%, p ? 0.05).
Inhibition of protein synthesis during the sustained
phase of facilitated L-LTP (30 to 60 min after LTP induc-
tion) evoked in slices from transgenic animals had no
effect on amplitude or duration of L-LTP (Figure 5C),
indicating that new protein synthesis is required for the
establishment of the facilitated LTP, but not for its main-
tenance. Therefore, the facilitated L-LTP elicited in
VP16-CREB expressing slices clearly differs from
L?LTP in wild-type in its requirement for transcription
andtranslation. Itseemstobe independentoftranscrip-
the other insensitive to inhibitors of protein synthesis.
A Model for Facilitated L-LTP in VP16-CREB Mice:
Cell-Wide Priming and Input-Specific Capture
of CRE-Driven Gene Products
by Active Synapses
Studies in Aplysia and mammals have shown that two
different phases are necessary for long-lasting changes
in synaptic plasticity (Frey and Morris, 1997; Martin et
al., 1997). In the first phase, synaptic activity produces
a signal that reaches the nucleus, activates gene tran-
scription, and leads, in the late phase, to the transport
Facilitated LTP by Constitutively Active CREB
Figure 6. Schematic Representation of E-LTP, L-LTP, Synaptic Capture and Cell-Wide Priming
One 100 Hz train stimulation elicits E-LTP in wild-type animal, a process that does not require gene activation but tags the stimulated synapse.
Four 100 Hz trains elicit L-LTP, a process that requires new gene expression. Once transcription-dependent LTP has been induced in one
pathway, the long-term process can be “captured” at a second pathway receiving a single train, a stimulation that would normally produce
only E-LTP. The expression of VP16-CREB may initiate the transcription and transport of gene products to the synapses, before the marking
has taken place. For these gene products to become functional, they must first be captured by marking the synapse.
tively at those synapses that have been marked (Fig-
ure 6). The expression of a constitutively active form of
CREB in the hippocampus of transgenic mice may initi-
ate the transcription and transport of gene products to
the synapses, before the marking has taken place. For
these gene products to become functional, they must
first be captured by marking the synapse (Figure 6).
The finding that a single train at 100 Hz produces a
facilitation, which resembles normal L-LTP induced by
repeated tetanic stimulation and the pharmacological
characterization of this L-LTP, are consistent with this
Thus, if VP16-CREB activates expression of CRE-
driven genes and these gene products are transported
to all the dendrites of a neuron, we should be able to
capture independently these gene products in different
synapses. To test this prediction, we stimulated two
independent synaptic inputs (S1 and S2) to the same
neuronal population in the CA1 region of hippocampal
slices of wild-type and transgenic mice using a protocol
similar to that originally described by Frey and Morris
(1997) for rats (Figure 7A). First, we found that in trans-
genic mice overexpressing VP16-CREB, as in wild-type
littermates, it is possibleto establish a pathway-specific
LTP without affecting the second pathway. Moreover,
we confirmed our previous finding, 100 Hz stimulation
elicited E-LTP in wild-type (Figure 7B: 30 min, S1:
175% ? 28%; 2 hr, S1: 106% ? 13%) and L-LTP in
mutants (Figure 7C: 30 min, S1: 222% ? 16%; 2 hr, S1:
and found that these plasticity changes were input spe-
cific. Weak tetanic stimulation leads to E-LTP in wild-
type mice (Figure 7D: 30 min, S1: 182% ? 18%, S2:
217% ? 20%; 2 hr, S1: 114% ? 12%, S2: 117% ? 14%)
and facilitated L-LTP in VP16-CREB mice (Figure 7E: 30
min, S1: 208% ? 20%, S2: 206% ? 26%; 2 hr, S1:
201% ? 32%, S2: 197% ? 24%) independently in every
pathway. In contrast, when the second tetanus is ap-
plied to the same pathway after return to baseline by
reducing the stimulus intensity, we did not observe any
difference between wild-type (Figure 7F, 30 min: first
LTP in S1: 189% ? 22%, S2: 112% ? 5%; second LTP
in S1: 172% ? 18%, S2: 115% ? 12%); and transgenic
littermates (Figure 7G: 30 min: first LTP in S1: 212% ?
30%, S2: 113% ? 6%; second LTP in S1: 156% ? 29%,
S2: 117% ? 6%). In agreement with previous observa-
tions (Frey et al., 1995), hippocampal neurons main-
tained their capacity for E-LTP immediately after long-
lasting potentiation, but the capacity for the induction
of longer lasting plastic changes was temporarily lost.
Characterization of New Features
of Synaptic Capture
If the model of cell-wide priming and input-specific cap-
ture is correct, then we can use our findings in VP16-
CREB mice to predict four previously uncharacterized
features of synaptic capture in wild-type animals: (1)
dependence on NMDA-R, (2) resistance to depotentia-
tivity to protein synthesis inhibition.
As previously described (Frey and Morris, 1997), we
found that weak tetanic stimulation, which normally
leads only to E-LTP, resulted in L-LTP when it followed
repeated tetanization at the other input to the same
population of neurons (Figure 8A: 30 min, S1: 223% ?
29%, S2: 225% ? 29%; 3.5 h, S1: 204% ? 25%, S2:
213% ? 32%), but not when the first pathway (S1) was
Figure 7. Synaptic Capture of CRE-Driven Genes
Recordings in S1 are represented in black (upper panels) and recordings in S2 are represented in red (lower panels).
(A) Hippocampal slice showing the positioning of the electrodes. The two independent inputs to the same neuronal population (electrodes
S1 and S2) and the recording site for fEPSP are shown.
(B) Input specific E-LTP in wild-type mice evoked by a single 100 Hz train.
(C) Input specific L-LTP in VP16-CREB mice evoked by a single 100 Hz train.
(D) Input specific E-LTP in wild-type mice can be independently evoked in the two pathways by a single 100 Hz train. E-LTP elicitation in S1
does not predispose the formation of L-LTP in S2.
(E) Input specific L-LTP in VP16-CREB mice can be independently evoked in the two pathways by a single 100 Hz train.
(F) Sequential tetanic stimulation of the same pathway in wild-type mice. Forty min after LTP elicitation with a single 100 Hz train in S1, the
stimulus intensity was reduced to baseline level and one additional 100 Hz train was given.
(G) Sequential tetanic stimulation of the same pathway in transgenic mice, as described for Figure 7F.
Facilitated LTP by Constitutively Active CREB
Figure 8. Features of Synaptic Capture in Wild-Type Mice
Recordings in S1 are represented in black (upper panels) and recordings in S2 are represented in red (lower panels). The 100 Hz-captured
LTP in the absence of inhibitors (taken from Figure 8A) is depicted in light gray for comparison in Figures 8B, 8D, and 8E.
(A) L-LTP elicitation in S1 facilitates the formation of L-LTP in S2 by 100 Hz stimulation in wild-type mice.
(B) Synaptic capture-mediated LTP in S2 is blocked in the presence of APV.
(C) Synaptic capture-mediated LTP in S2 cannot be depotentiated by low-frequency stimulation.
(D) PKA inhibition reduces the amplitude of synaptic capture-mediated LTP in S2, but does not noticeably affect the expression of L-LTP in
S1 (p ? 0.05).
(E) Anisomycin reduces moderately the amplitude of synaptic capture-mediated LTP in S2. Decrease is significant (p ? 0.05) since 3 hr after
stimulated only with one train (as shown in Figure 7D).
Incubation with APV during synaptic capture in S2 elimi-
nated the potentiation observed in S2, but did not affect
LTP in S1, indicating that elicitation of synaptic capture-
mediated LTP is an NMDA-dependent process (Figure
8B). In addition, we found that synaptic capture-medi-
ated L-LTP was resistant to depotentiation elicited by
5 Hz stimulation 5 min after capture (Figure 8C: 30 min,
S1: 222% ? 35%, S2: 212% ? 28%; 3.5 h, S1: 195% ?
21%, S2: 203% ? 26%) and clearly sensitive to PKA
inhibitors (Figure 8D: 30 min, S1: 221% ? 22%, S2:
206% ? 34%; 3.5 hr, S1: 197% ? 25%, S2: 138% ?
26%). After PKA inhibition, some residual potentiation
was still detectable even 8 hr after stimulation, but it
should be taken into account that repeated tetanization
in S1 itself has a small potentiation effect in S2 baseline.
facilitated L-LTP in VP16-CREB mice (Figure 8E: 30 min,
S1: 219% ? 29%, S2: 202% ? 31%; 3.5 hr, S1: 180% ?
35%, S2: 169% ? 24%, plateauobserved for 6 hr, differ-
ence was significant since 3 hr after S2 stimulation).
Therefore, the results of these experiments are consis-
tent with our observations in VP16-CREB mice, validate
our model, and allow the enunciation of some new key
features of the capture process in mammalian hippo-
a second prerequisite for LTP consolidation. This result
is similar to that in Aplysia where the injection of phos-
pho-CREB into sensory neurons paired to a single pulse
tation that persists for several days (Casadio et al.,
In turn, the finding in VP16-CREB mice that a single
tetanus to any branch can capture the long-term pro-
cess has allowed us to investigate properties of “synap-
tic capture” in transgenic animals and then to confirm
its features in wild-type animals using two-pathway ex-
periments. This analysis has allowed us to delineate
(1) L-LTP in one pathway appears not to affect basal
transmission in the second pathway, but reduces in that
pathway the threshold for establishing L-LTP.
(2) L-LTP mediated by synaptic capture is dependent
on the activity of NMDA receptors, as is L-LTP mediated
by four trains stimulation.
pathway is sustained for several hours and resistant to
depotentiation, again resembling L-LTP obtained by
(4) Once CRE-driven gene products have been effi-
tional sustained reinforcement of synaptic connections.
This last feature is suggested by the ceiling effect ob-
served after repeated tetanization (Figures 3F and 3G)
and the occlusion of facilitated L-LTP by forskolin (Fig-
ure 4D1) or after previous potentiation of the same path-
way (Figure 7G).
(5) The tag appears to involve PKA. Our result with
the PKA inhibitor KT5720 suggest that PKA is necessary
fortaggingthe synapse,butisnot necessaryformainte-
nance of the captured potentiation process, a view con-
sistent with the resistance of synaptic capture to depo-
tentiation. Once the gene products have been captured,
PKA activity is no longer needed and can be obliterated
by activation of phosphatases during depotentiation
with no effect on potentiated synapses. PKA seems to
play a double role in the consolidation of LTP. First, it
acts locally to mark a synapse that occurs following one
tetanus. Two, it leads to nuclear activation with stronger
activation following four tetani (Abel et al., 1997; Frey
et al., 1993).
(6) New protein synthesis is necessary for maximal
that translation is required whereas transcription is not
for the facilitated LTP observed in VP16-CREB mice,
suggesting that stabilization of the maximal synaptic
change might require new protein synthesis. Among the
mRNAs induced by VP16-CREB, some might be trans-
et al., 2000a; Steward and Schuman, 2001). These re-
sults are consistent with studies in Aplysia suggesting
that tagging has two components: a mark for capture
zation mediated by local protein synthesis (Casadio et
(7) The proteins necessary for priming the captured
L-LTP represent CRE-driven gene products. We find
that as a consequence of VP16-CREB expression, cer-
Role of CRE Binding Proteins and CRE-Driven
Genes in LTP Consolidation
Studies of CREB hypomorphic (Gass et al., 1998) and
dominant-negative transgenic mice (Rammes et al.,
2000) have cast doubt on the role for CREB in L-LTP in
mammals and have suggested that other CRE binding
proteins, such as CREM or ATF1, might compensate for
the loss of CREB (Blendy et al., 1996; Hummler et al.,
1994). Our experiments with VP16-CREB mice were de-
signed to cast a broader net and focus not only on
CREB-1, but on the whole family of CREB transcription
factors by exploring the general role in LTP of all the
CRE binding proteins and their downstream genes. We
found that expression of this chimeric transcription fac-
tor in postsynaptic neurons of the Schaffer collateral
pathway facilitates the establishment of long-lasting
LTP in hippocampal slices by allowing a single teta-
nic train, which normally produces E-LTP, to produce
L-LTP. Although these data do not specify what role
CREB-1 by itself plays in hippocampal LTP, it is clear
by the CREB family of transcription factors plays an
essential role in the consolidation of LTP. Our results
also indicate that the input specificity of persistent
changes in synaptic strength is determined not only by
nuclear events but also by synaptic events, such as the
interactions between plasticity proteins and synaptic
tags. Thus, the activation of the CREB family of tran-
scription factors that takes place under physiological
conditions in hippocampal neurons might only reflect
the potential to induce a lasting change, rather that the
commitment to do so (Martin et al., 2000b).
Cell-Wide Priming, Synaptic Tagging, and Capture
of CRE-Driven Gene Products
Our data indicate that CRE-mediated transcription is
one of the prerequisites for the consolidation of long-
term synaptic changes. Specifically, VP16-CREB activ-
synaptic terminals with proteins and mRNAs required
for the stabilization and capture of L-LTP. These gene
products can then be used productively for L-LTP when
a given synapse is tagged by brief synaptic stimulation
of the sort normally needed for E-LTP. Thus, in addition
to CRE-mediated gene expression, synaptic tagging is
Facilitated LTP by Constitutively Active CREB
Assay System (Promega) for measuring luciferase activity in our
tain CRE-driven genes, such as BDNF, dynorphin, and
c-fos, are upregulated. Some of these genes, such as
BDNF, play a role in synaptic plasticity. Other genes
remain to be identified. To this end, we are currently
proceeding with a genome-wide expression analysis of
VP16-CREB mice using microarrays.
Generation and Maintenance of Transgenic Mice
Ten lines of transgenic mice were generated by microinjection of
the linear construct as previously described (Mayford et al., 1996).
blotting using a VP16 probe. The founder mice were backcrossed
to C57BL6 F1/J mice four to six times to generate the transgenic
line used in our study. We designated as VP16-CREB mice those
bitransgenic animals that resulted from the crossing of pCaMKII-
tTA mice (line B; Mayford et al., 1996) and tetO-VP16-CREB (line
VC27) transgenics and as wild-type mice those littermates carrying
either pCaMKII-tTA, tetO-VP16-CREB, or none transgene. For all
experiments, dox was administrated at 40 mg/kg of food and re-
moved at specific times before experimentation. Mice were main-
tained and bred understandard conditions, consistent with National
Institutes of Health guidelines and approved by the Institutional
Animal Care and Use Committees.
Molecular Gating and the Threshold
for Memory Storage
The gene expression necessary for the consolidation of
LTP, for synaptic growth and for memory storage is
regulated by two types of balancing mechanisms (Abel
et al., 1998). In a given synaptic terminal, a balance
between both phosphatase and kinase activities gates
the synaptic signals that reaches the nucleus (Winder
et al., 1998). In the nucleus, a second balance exists
between transcriptional activators and repressors. This
balance gatestranscription activation.The useof acon-
stitutively activeform ofCREB hasallowed usto bypass
both gates and to act directly on the nuclear output by
activating expression of a specific set of genes required
fore determined locally by the threshold for synaptic
tagging instead of that for nuclear activation.
What is the consequence of this threshold shift in
memory and learning capability? In VP16-CREB mice,
of persistent changes in synaptic efficacy shows an
upward shift for a range of frequencies. For example,
1 Hz stimulation produced depression in wild-type mice
but not in the mutants, and 10 Hz induced a much larger
LTP in mutants than in wild-type mice. In addition, po-
tentiated synapses in these animals cannot depotenti-
ate, making these synaptic changes irreversible. Train-
ing these mice in a spatial learning task might cause
too many synapses within the hippocampal network to
become strongly and irreversibly potentiated, pre-
venting the storage of new information. However, regu-
lated expression of this protein shortly before or during
the task might allow one trial learning and flashbulb
memory. To clarify this question, we are currently char-
acterizing VP16-CREB mice in a number of behavioral
In conclusion, the line of transgenic mice described
here represents a useful tool for deciphering the genetic
program required for LTP consolidation. We have used
this tool to provide some initial molecular insights into
hippocampus, and suggest that synaptic capture of
CRE-driven gene products may be sufficient for the es-
tablishment of the late phase of LTP.
CRE Binding Activity
Ten ?g of hippocampal nuclear extracts, obtained from wild-type
and transgenic mice using the NE-PER kit (Pierce), was incubated
at RT for 30 min in the presence of the
and immunoprecipitated with anti-VP16 or c-myc antibodies (Santa
Cruz) and protein A Sepharose. Radioactivity in the immunoprecipi-
tate was measured using a scintillation counter.
32P-labeled CRE dsDNA
In Situ Hybridization, Immunohistochemistry,
and Western Blot
In situ hybridization was performed as described in Wisden and
Morris (1994) using a35S ATP-labeled oligonucleotide (5?-GTCCTTA
CAGGAGGATCCACCGTACTCGTCAATTCC-3?) specific for the trans-
mine/xylamine, perfused with 4% paraformaldehyde, postfixed in
sections).Staining withDABor fluorescencewas realizedaccording
to the M.O.M.or Elite Immunodetection kits(Vector) using anti-c-fos
(Oncogene) or anti-VP16 (Sta. Cruz, Inc.) antibodies. Hippocampal
protein extracts and Western blot analysis were realized as de-
anti-BDNF (Sta. Cruz, Inc.), and anti-CREB (Cell signaling) anti-
unless otherwise indicated in the figure legend. Hippocampi were
collected following cervical dislocation of 2.5 to 3.5 months old
mice of either sex. Transverse hippocampal slices (400 ?m) were
prepared using conventional techniques. Slices were incubated in
cerebrospinal fluid (ACSF, containing 119 mM NaCl, 2.3 mM KCl,
1.3 mM MgSO4, 2.5 mM CaCl2, 26.2 mM NaHCO3, 1 mM NaH2PO4,
and 11 mM glucose), and allowed to equilibrate for at least 90 min.
phonovaleric acid (Sigma), anisomycin (Sigma), forskolin (Calbio-
chem), DRB (Calbiochem), actinomycin-D (ICN Biomedicals, Inc.),
or KT5720 (Biomol). For recording of fEPSP in the CA1 region of
the hippocampus, both the stimulating and recording electrodes
were placed in the stratum radiatum of CA1 area. The stimulation
intensity (0.05 ms duration) was adjusted to give fEPSP slopes ap-
proximately 40% of the maximum, and baseline responses were
elicited once per minute at this intensity. In two-pathway experi-
ments, the two stimulating and recording electrodes were placed
in the stratum radiatum of CA1 area as showed in Figure 7A, and we
analyzed interpathway PPF to assure minimal cross-contamination.
Two-way ANOVA and Student’s t test were used for electrophysio-
logical data analysis. In all electrophysiological experiments, “n”
indicates the number of slices. In the text and column graphs, the
electrophysiological data were presented as mean ? SD, whereas
in other figures the values were expressed as mean ? SEM. Experi-
menter was blind to mice genotype in physiological studies.
Cloning, Transient Transfections, and Reporter Assays
Standard manipulations of E. coli, cell culture, proteins, and nucleic
VP16-CREB, fusion protein between HSV VP16 (aas 363 to 490),
and CREB (aas 88 to 341), was cloned in pRcRSV (Invitrogen) for
transfection assays and in pMM400 (Mayford et al., 1996) for the
generation of transgenic mice. pCRE.luc, pSRE.luc, and other re-
porter constructswere obtainedfrom the Mercuryluciferase system
(Clontech). HEK293 were lipofected with Pfx-2 (Invitrogen) following
manufacturer instructions. We used the Dual-Luciferase Reporter
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