1InstitutodeNeurocienciasdeAlicante(UniversidadMiguelHerna ´ndez–ConsejoSuperiordeInvestigacionesCientı ´ficas),CampusdeSantJoan,SantJoan
To investigate the role of CREB-mediated gene expression on the excitability of CA1 pyramidal neurons, we obtained intracellular
Studies in different model systems have established a critical role
for the cAMP signaling pathway and cAMP-responsive element
binding protein (CREB)-mediated gene expression in different
forms of synaptic plasticity related to learning (Lonze and Ginty,
2002; Barco et al., 2003; Josselyn and Nguyen, 2005). In particu-
phorylation of CREB in CA1 pyramidal neurons and the induc-
tion of CRE-driven gene expression (Bito et al., 1996; Deisseroth
et al., 1996; Impey et al., 1996; Lu et al., 1999). Furthermore, the
enhanced expression of CRE-driven genes favors the formation
and stability of LTP in this pathway (Barco et al., 2002, 2005;
sion by deleting specific CREB isoforms (Bourtchuladze et al.,
1994) or by overexpressing a dominant negative form of CREB
caused deficits in some forms of LTP (Pittenger et al., 2002;
ever, may cause the overexpression of other CRE-binding pro-
teins (Hummler et al., 1994; Blendy et al., 1996), which may
compensate the deficiency in CREB activity and reduce the im-
et al., 2003).
directly control neuronal excitability. The expression of the con-
stitutively active CREB variant, VP16–CREB, using viral vectors
increased the firing frequency and reduced the resting potential
of neurons in the locus ceruleus (Han et al., 2006) and the excit-
ability of spiny neurons in the nucleus accumbens (Dong et al.,
2006). The long-term effects of these modifications in neuronal
physiology and survival have not been investigated. Neither is it
known what the relationship is between the regulation of long-
term forms of synaptic plasticity by CREB and the changes in
To explore the role of CREB-mediated gene expression in
controlling the excitability of CA1 pyramidal neurons, we per-
in a regulated manner. Here we report that transgene expression
caused a rapid and reversible change of the firing properties of
CA1 pyramidal neurons by reducing the slow (sAHP) and me-
CT-2005-016343, Spanish Ministerio de Educacio ´n y Ciencia Grants BFU2005-00286 and SAF2005-24584-E, and
Herna ´ndez–Consejo Superior de Investigaciones Cientı ´ficas), Campus de Sant Joan, Apartado 18, Sant Joan
Disclosure of financial interest: E.R.K. is one of four founders of Memory Pharmaceuticals and Chairman of its
Scientific Advisory Board. Memory Pharmaceuticals is concerned with developing drugs for age-related memory
TheJournalofNeuroscience,December12,2007 • 27(50):13909–13918 • 13909
dium (mAHP) afterhyperpolarization. Furthermore, we found
that the sustained activation of CREB-mediated activity can trig-
the excitotoxic cell death of pyramidal and granular neurons in
and high levels of constitutive CREB activity allowed us to sepa-
rate these two processes.
mice expressing VP16–CREB under the control of tetO promoter were
generated by microinjection of the linear construct as previously de-
scribed (Barco et al., 2002). The founder mice were backcrossed to
C57BL6 F1/J mice more than 15 times to generate the transgenic lines
used in our study. We have previously described in detail the mice now
referred as VP16–CREBhigh(Barco et al., 2002, 2005). Those bitrans-
genic animals resulted of the crossing of pCaMKII-tTA mice (line B)
(Mayford et al., 1996) and tetO–VP16–CREB line VC27, the line that
also investigate the phenotype of bitransgenic animals resulted of the
crossing of pCaMKII-tTA mice (line B) and tetO–VP16–CREB line
this bitransgenic strain as VP16–CREBlowmice. In all our experiments,
we used as control littermate mice carrying either pCaMKII-tTA, tetO–
the indicated times before experimentation. VP16–CREBlowmice were
raised without dox. Experiments were performed in adult mice (2-4
month old), at least otherwise indicated in figure or table legends. All
mice were maintained and bred under standard conditions, consistent
with national guidelines and approved by the Institutional Animal Care
and Use Committees.
decapitated, and coronal slices that included the dorsal hippocampus
(300 ?m) were cut in oxygenated (95% O2/5% CO2) ice-cold artificial
CSF (ACSF) containing (in mM): NaCl 118, KCl 2.5, NaHCO325,
NaH2PO41.2, MgCl2, 1.3, CaCl22.5 and glucose 10. After a recovery
ACSF. For recordings, slices were transferred to the recording chamber
from CA1 pyramidal neurons using infrared differential interference
were filled with intracellular solution containing (in mM): KMeSO4135,
NaCl 8, HEPES 10, Mg2ATP 2, Na3GTP 0.3 (pH 7.2 osmolarity 290
out the experiment. Experiments were discarded if the series resistance
changed by more than 10% during the course of the experiment. Signals
were recorded using a MultiClamp 700B amplifier (Molecular Devices,
1320A, Molecular Devices). All cells described in this study had a mem-
brane potential more negative than ?50 mV. Electrical activity was re-
(AxoGraph Scientific, Sydney, Australia). No series resistance compen-
sation was used and membrane potentials were corrected for junction
potentials (-4 mV). To investigate the firing properties of neurons, 15
mouse (left), in a VP16–CREBhighmouse (middle), and in a VP16–CREBhighmouse that ex-
old. Note the lack of spike frequency adaptation in CA1 VP16–CREB-expressing neurons. B,
VP16–CREBhigh(n ? 22) and from wild-type littermate (n ? 16) mice 10 d after inducing
Neuronal excitability is increased in VP16–CREB-expressing neurons. A, Repre-
13910 • J.Neurosci.,December12,2007 • 27(50):13909–13918LopezdeArmentiaetal.•CREBControlsNeuronalExcitability
50 pA increments from a holding potential of ?70 mV. Passive mem-
evoked by a 50 ms depolarizing voltage step to 0 mV from a holding
potential of ?50 mV. ImAHPand IsAHPamplitudes were measured at the
tively. Because unclamped APs during the depolarizing voltage step
ent groups. Only a small proportion of neurons in both VP16–CREB
(usually a second small spike) but these neurons did not exhibit bigger
command step and the contribution of the unclamped APs is not signif-
icant. For extracellular recordings transverse hippocampal slices (400
?m) were prepared, incubated in an interface chamber at 32° with oxy-
fEPSP in CA1 region both stimulating and recording electrodes were
placed in the stratum radiatum of CA1 area. Extracellular activity was
recorded simultaneously from stratum pyramidale of the CA1 and CA3
subregions with 1 M? pipettes filled with ACSF, filtered at 3 KHz and
and its area was measured between 0 and 1 KHz as an index of neuronal
et al., 2002). In all electrophysiological experiments, ‘n‘ indicates the
number of cells or slices tested. The number of mice is also indicated in
figure legends and Table 1. Two-way ANOVA and Student’s t test were
used for data analysis. Experimenters were blind to mice genotype.
Histological techniques. Nissl staining was realized as previously de-
scribed (Mayford et al., 1996). For Timm’s staining, mice were anesthe-
tized with ketamine/xylazine, transcardially perfused with buffered
night in 10% NBF. 50 ?m thick sections were cut with a Leica vibration
microtome, collected in 0.1M PB and mounted on the slides. Developer
viter, 1982). The slides were incubated in developing solutions in total
darkness for 60 min and then rinsed in dH2O and cleared with ethanol/
xylene. For immunohistochemistry, mice were anesthetized, perfused
with 4% paraformaldehyde, postfixed overnight, and 50 ?m sections
were obtained. The following primary antibodies were used: ?-VP16
(Santa Cruz), ?-Synaptophysin, ?-MAP-2, ?-Calbindin and ?-GAP-43
(Sigma). Secondary biotinylated antibodies, streptavidin-peroxidase
conjugate and DAB substrate were obtained from Sigma.
7300 real-time PCR unit using the SYBR mix (Invitrogen) and primers
specific for VP16 (forward: 5? cctacggcgctctggatatg 3?, reverse: 5?cggta-
aacatctgctcaaactcg 3?) and GAPDH (forward: 5? cttcaccaccatggagaaggc
3?, reverse: 5?catggactgtggtcatgagcc 3?) sequences. Each independent
sample was assayed in duplicate and VP16 levels were normalized using
Microarray analysis. U74Av2 genechips were used to analyze changes
in gene expression in the hippocampus of VP16–CREBhighat different
times postinduction, data were processed, normalized and statistically
analyzed using GCOS 1.2 software as previously described (Barco et al.,
2005). This dataset is accessible at the GEO database (accession no.
GSE3965). I (increase), D (decrease), MI (mild increase), MD (mild
decrease) and NC (no change) calls were obtained according to the sta-
tistical thresholds defined by the software. We filtered and sorted the list
of genes using the change p value, change call, and Log Ratio Signal. For
early time, we requested that the change call for wild-type samples or for
whereas the change call for samples in which VP16–CREB expression
was turned-on for a week (2 samples) indicated a statistically significant
change in expression. For late time, we used the same criteria applied to
arrays corresponding to 3 or 5 weeks after induction (3 arrays). The two
lists of genes so obtained were then analyzed for pathway building and
GO group classification using Pathway Studio 5.0 software (Ariadne
normalized, modeled and filtered using DNA-Chip Analyzer (dChip)
the composition of the gene lists generated with each one of these ap-
proaches, the results of Pathway Studio analysis were equivalent.
Our previous research demonstrated that enhanced CREB-
reduced the threshold for obtaining the late phase of LTP in the
Schaffer collateral pathway (Barco et al., 2002), whereas recent
studies indicate that CREB may regulate neuronal excitability in
striatal and brainstem neurons (Dong et al., 2006; Han et al.,
2006). To investigate whether similar changes in excitability may
also occur in the hippocampus, we examined the firing pattern
and membrane properties of CA1 pyramidal neurons in VP16–
as VP16–CREBhighto facilitate its comparison with VP16–CRE-
Blow, a new bitransgenic strain which we first describe in a later
phase of this study.
The tTA/tetO system of double transgenic mice enabled us to
obtain precise temporal regulation of transgene expression
through the ability of doxycycline (dox) to block tTA binding to
DNA. To avoid possible developmental problems attributable to
imals in the presence of dox and induced the expression of the
able from their wild-type littermates. Intracellular recordings in
CA1 pyramidal neurons 10 d after dox removal revealed no dif-
the IsAHPand ImAHPwere reversed by silencing again transgene expression for 10 d with dox
LopezdeArmentiaetal.•CREBControlsNeuronalExcitabilityJ.Neurosci.,December12,2007 • 27(50):13909–13918 • 13911
ferences in passive membrane properties
between transgenic mice and their litter-
mates (Table 1). However, the expression
of VP16–CREB profoundly affected the
number of action potentials (APs) elicited
by depolarizing current injections (Fig.
1A). Thus, CA1 pyramidal neurons from
VP16–CREBhighmice (VP16–CREB On)
tion during a depolarizing pulse and trig-
gered on average more APs than their
wild-type littermates ( p ? 0.001) (Fig.
1B). We also observed a significant in-
crease in the threshold to elicit an AP and
decreased AP amplitude and maximum
rate of depolarization in CA1 pyramidal
neurons from VP16–CREBhighmice (Ta-
Na?currents that could balance the in-
crease in excitability observed in these
neurons (Davis, 2006).
These changes in firing pattern and
membrane properties were reversed when
VP16–CREB expression was repressed for
10 d with dox (Table 1, VP16–CREBhigh
On/Off; Fig. 1A,B). We also recorded
from CA1 pyramidal neurons of VP16–
presence of dox and never expressed the
any difference between those transgenics
and their littermates (Table 1, VP16–
CREB Off). Therefore, the reduced spike
of VP16–CREBhighmice results from the
acute expression of this chimeric protein.
What alterations in membrane properties
underlie the reduction in spike frequency
adaptation? Because firing frequency is
importantly modulated by the amplitude
and duration of the AHP (Madison and
Nicoll, 1982; Peters et al., 2005), we inves-
tigated the AHP in CA1 pyramidal neu-
rons of VP16–CREBhighmice. Slow and
medium AHP has been proposed to re-
terdepolarization and multiple action po-
performed voltage-clamp recordings in CA1 pyramidal neurons
of VP16–CREBhighmice and found that the sAHP current were
CREBhighmice was 36% of that observed in neurons of control
littermates. This change was accompanied by a 52% reduction
also in the ImAHP(Table 1; Fig. 2A,C). Both components of the
AHP recovered when the expression of the transgene was turned
off with dox (Fig. 2B,C, On/Off; Table 1, On/Off), suggesting
stream of CREB activation.
To investigate whether the alterations in neuronal excitability
and synaptic plasticity were dependent of the level of enhance-
ment of CREB-activity, we compared our results in VP16–CRE-
Bhighmice with those obtained in a lower-expressing strain, re-
Methods). As with VP16–CREBhighmice, VP16–CREBlowmice
expressed the transgene in the hippocampus selectively in gran-
ular cells in the dentate gyrus (DG) and CA1 pyramidal neurons,
excluding CA3 neurons (Fig. 3A). The quantification of VP16–
CREB mRNA levels by real-time RT-PCR revealed that the hip-
pocampal expression of this construct was fivefold higher in
domain. DAB reaction was longer in the case of VP16–CREBlowto facilitate the identification of positive neurons; therefore,
et al. (2002) and 3E in this article (L-LTP, corresponding to the average amplitude response 90-120 min after LTP induction).
The increase in neuronal excitability and inhibition of IAHPis dose dependent. A, Comparison of the pattern of
13912 • J.Neurosci.,December12,2007 • 27(50):13909–13918 LopezdeArmentiaetal.•CREBControlsNeuronalExcitability
VP16–CREBhighthan in VP16–CREBlowmice (Fig. 3B) ( p ?
0.04). We found that, despite the reduced level of expression,
these mice also exhibited a significant increase in neuronal excit-
in VP16–CREBhighmice. Moreover, VP16–CREBlowmice also
presented reduced IsAHP(Fig. 3D) (IsAHPin VP16–CREBlow?
5 mice; p ? 0.001). Interestingly, mAHP was, however, not af-
fected in these mutants (Fig. 3D) (ImAHPin VP16–CREBlow?
265 ? 18.9 pA, n ? 38, 5 mice; ImAHPin WT ? 260 ? 11.7 pA,
n ? 42, 5 mice; p ? 0.81).
the induction of L-LTP in both VP16–CREBhighand VP16–
CREBlowmice. Like VP16–CREBhighmice (Barco et al., 2002),
VP16–CREBlowanimals showed an enhanced response to one
standard 100 Hz tetanus train of 1 s duration. This stimulation
in mutant mice, but only produced a nonsaturating shorter-
lasting LTP (E-LTP) in wild-type littermates (Fig. 3E) (90-120
min: VP16–CREBlow: 126 ? 11%, n ? 4; WT: 109 ? 6%, n ? 4;
p ? 0.034).
The comparison of the results obtained in both bitransgenic
strains (Fig. 3F) shows that the magnitude of the alterations in
neuronal excitability and synaptic plasticity correlates well with
the level of expression of VP16–CREB.
We next turned to examine the long-term consequences of en-
tinuous expression of this chimeric transcription factor for sev-
ally showed spontaneous seizures during handling. These sei-
4), although ?50% of the animals survived for more than 8
weeks. In contrast, the low-expressing strain VP16–CREBlow
(results not shown).
The premature death of VP16–CREBhighmice could be com-
pletely prevented by turning off transgene expression with dox
during the first month of its induction (data not shown). Even
when transgene expression was repeatedly induced several times
during the life of the animal by switching between regular food
and food supplemented with dox, the deleterious effects were
3-4 weeks (Fig. 4). Conversely, when VP16–CREBhighmice were
kept off dox during their whole life, including embryonic devel-
described for mice in which we induced VP16–CREB expression
during adulthood, we occasionally observed severe seizures that
preceded the premature death of the animal.
acterization of these mice (one-two weeks after transgene induc-
transgenic and control littermates (see time course in Fig. 5). In
contrast, VP16–CREBhightransgenic mice expressing this chi-
meric transcription factor for several weeks showed progressive
cell loss in the CA1 and DG regions. This neurodegenerative
process could be easily visualized by Nissl staining (Fig. 5A) and
by immunostaining with a number of neuronal markers (Fig.
weeks of transgene expression as revealed by Timm’s staining
Although we observed significant variability in the onset of
investigated exhibited severe cell loss 6 weeks after dox removal.
The onset of cell loss in the CA1 pyramidal layer seemed to coin-
cide with the occasional observation of epileptic seizures, sug-
gesting that these two events might be related. Mice in which we
alternated 2 weeks of regular mouse diet with 2 weeks of food
supplemented with dox never showed epileptic attacks (Fig. 4)
and exhibited normal hippocampal anatomy (data not shown)
despite accumulating several months of transgene expression.
turning transgene expression off, although this did not cause a
gene (Fig. 5D). Interestingly, no cell loss was observed in VP16–
CREBlowmice even after one year of transgene expression, indi-
cating that a milder increase in neuronal excitability was
compatible with neuronal survival (Fig. 5A, VP16–CREBlow).
What are the consequences of the early increase in excitability
and the late cellular loss in the activity of hippocampal circuits?
To investigate this issue we performed field recordings in acute
hippocampal slices from VP16–CREBhighmice 1 week after dox
increase in spontaneous activity at the CA1 subfield (Fig. 6B)
( p ? 0.01), as estimated by the power spectrum analysis, that
in the presence of dox (data not shown), or in mice with periodic induction of VP16–CREB
LopezdeArmentiaetal.•CREBControlsNeuronalExcitabilityJ.Neurosci.,December12,2007 • 27(50):13909–13918 • 13913
correlated with the increased neuronal ex-
citability observed in CA1 pyramidal neu-
rons. In contrast, activity in the CA3 sub-
field, in which the transgene is not
expressed, was unaltered (Fig. 6B).
When the expression of VP16–CREB
was sustained for more than 3 weeks, we
observed a significant reduction in the re-
ulation of afferent CA3 axons (Fig. 6C),
probably reflecting the incipient loss of
synapses in the CA1 subfield. This reduc-
tion may compensate the previously ob-
neurons and, consequently, no increase in
spontaneous activity in the CA1 subfield
was observed after 3 weeks (Fig. 6D).
Strikingly, we observed, however, an in-
crease in spontaneous activity in the CA3
subfield (Fig. 6D) ( p ? 0.002), suggesting
the existence of readjustments in the hip-
ing neuronal loss.
Because sAHP can be inhibited by glu-
tamate through kainate receptors activa-
tion (Melyan et al., 2002), we investigated
whether an increase in extracellular gluta-
mate concentration resulting from the
neurodegenerative process initiated in
VP16–CREBhighmice could underlie the
reduction of the sAHP. We measured
IsAHPof neurons from slices of VP16–
CREBhighmice that were either incubated
for 2 h in the presence of 5 ?M NBQX, an
AMPA/kainate antagonist, or maintained
in normal ACSF. No differences were
found between the two groups of neurons
(NBQX: IsAHP? 26 ? 6.2pA, n ? 15; veh:
gether, these results indicate that the in-
crease in excitability precedes the onset
and perhaps contributes to the neurode-
generative process observed in the hip-
pocampus of VP16–CREBhighmice.
The gene profiling analysis performed
in VP16–CREBhighmice (Barco et al.,
2005) also supports a dynamic scenario in
which a physiological CREB-mediated re-
sponse out of context eventually triggers a
analysis of our microarray data using Pathway Studio software
comparing early and late times (1 week vs ?3 weeks) after trans-
gene induction revealed clear differences between these two
stages. At early times, the groups more represented in the list of
significantly altered genes were signal transduction, transport,
at late times, there was a overrepresentation of gene groups re-
lated to pathological responses (immune and defense response,
proteolysis, apoptosis, inflammation) (Fig. 7A). Moreover, we
also used Pathway Studio software for pathway building and vi-
sualization and found that the overall connectivity of altered
genes to CREB decreased significantly at later times (Fig. 7B),
could be mediated by the altered expression of some early target
Our results indicate that the enhanced expression of CRE-driven
genes increases the neuronal excitability of CA1 pyramidal neu-
rons and reduces the threshold for LTP in the Schaffer collateral
a reduction of AHP currents. However, when a high level of
Floating vibratome sections (50 ?m) were stained with antibodies against calbindin, GAP-43, synaptophysin, and MAP2. No
CREBhighmice at different times after transgene induction and repression. All the experiments were realized in adult mice.
13914 • J.Neurosci.,December12,2007 • 27(50):13909–13918LopezdeArmentiaetal.•CREBControlsNeuronalExcitability
pal circuit and causes neuronal loss. These deleterious effects are
express VP16–CREB at lower levels, although these mice also
showed increased excitability.
Two recent studies have found that a similar CREB variant,
when expressed using viral vectors, increased the excitability of
neurons in the locus ceruleus (Han et al., 2006) and the nucleus
intrinsic neuronal properties is a well conserved CREB function
through different neuronal types in the CNS. However, the spe-
cific changes in gene expression underlying these increments in
excitability remain unknown. We describe here that depression
of AHP might underlie the changes in neuronal excitability trig-
particular, sAHP was affected both in the low and in the high
a powerful mechanism for regulating NMDA-R-mediated plas-
ticity related to learning (Wu et al., 2004; Faber et al., 2005).
Blockade of the sAHP or mAHP converts short-lasting forms of
potentiation into L-LTP (Sah and Bekkers, 1996; Cohen et al.,
1999; Haug and Storm, 2000; Faber et al., 2005), whereas the
activation of the AHP increases the threshold for induction of
hippocampus-dependent tasks, such as eye blinking condition-
sAHP in CA1 pyramidal neurons (Moyer et al., 2000; Oh et al.,
2003), a phenomenon that may favor learning by reducing the
threshold for late forms of LTP.
Although our knowledge of the molecular underpinnings of
the plasticity of membrane properties is still limited, the prime
candidates to regulate these changes pri-
marily overlap with those thought to reg-
ulate LTP. These include adenylyl cyclase
and a number of kinases known to phos-
phorylate CREB (Zhang and Linden,
2003). In particular, variations in cAMP
directly linked to CREB activation in neu-
rons, play a major role in the regulation of
the sAHP current. Thus, forskolin inhibits
the current underlying sAHP by 90%,
whereas the PKA inhibitor Rp-cAMPS in-
creased the current, suggesting that PKA
maintains sAHP channels in the closed
state (Vogalis et al., 2003). Indeed, sup-
pression of sAHP seems to be mainly me-
diated by PKA in CA1 pyramidal neurons
(Pedarzani and Storm, 1993, 1995; Haug
and Storm, 2000). In the case of mAHP,
the inhibition observed in VP16–CREB-
highmice might result from modulation of
current (Gu et al., 2005), whose suppres-
sion in transgenic animals also increased
the excitability of CA1 pyramidal neurons
(Peters et al., 2005). Although neither
CREB nor de novo gene expression has
been so far directly involved in the modu-
lation of AHP, the persistence of the
changes in neuronal excitability and sAHP observed during be-
havioral training suggests the participation of molecular mecha-
expression and protein synthesis. We therefore propose that
types may play a relevant role regulating physiological neuronal
responses during addiction and learning and memory. Indeed, a
reemerging view in the learning and memory field proposes that
although synaptic changes, such as LTP or LTD, are likely to
represent the most important cellular mechanism for memory
storage given its computational properties, other forms of plas-
ticity, such as changes in neuronal intrinsic excitability, may also
and Linden, 2003). Although the encoding capacity of those
mechanisms is significantly lower than synaptic alterations, in-
trinsic changes might function as a trigger for consolidation or
adjustable gain control (Sah and Bekkers, 1996). Ascending
arousal and attention by suppressing this current (Deng et al.,
by CREB activation, which is known to be downstream of such
inputs (Berke and Hyman, 2000).
We have proposed that the sustained activation of CREB-
mediated gene expression causes a cell-wide facilitation that
tetanus (Barco et al., 2002; Alarcon et al., 2006). Now, we found
that in the same mice shortly after transgene induction there is a
significant reduction in the value of the IsAHP, a current that has
been related to learning and memory as much as has L-LTP. Are
these two phenomena two manifestation of the same molecular
process? The inhibition of sAHP may well represent the cellular
milliseconds) versus stimulus at the Schaffer collateral pathway of hippocampal slices from VP16–CREBhigh(n ? 31) and
Increased spontaneous activity in the hippocampal circuit. A, Input/output curve of fEPSP slope (millivolts per
LopezdeArmentiaetal.•CREBControlsNeuronalExcitability J.Neurosci.,December12,2007 • 27(50):13909–13918 • 13915
mechanism underlying cell-wide facilita-
not explain the resistance to protein and
mutants, suggesting that other processes
cific CREB target genes controlling these
changes are still unknown, but it has been
suggested that BDNF, a well known CREB
downstream gene and the main effector
filing analysis of VP16–CREBhighmice
(Barco et al., 2005), contributes to control
ronal networks to adapt to long-lasting
changes in activity (Desai et al., 1999).
During normal activity of the brain, the
ate transcriptional responses that are im-
portant for learning and memory. How-
ever, more intense
kindling model) may initiate inappropri-
ate gene expression response and lead to
the formation of epileptic neuronal cir-
(McEachern and Shaw, 1999). A physio-
logical. The deleterious effects observed at
late times in VP16–CREBhighmice could
be the undesired consequence of a physio-
logical function of CREB missing regula-
lular and molecular alterations associated
lobe, as well as to assay possible therapeu-
tic approaches. The reversibility and pos-
sibilities for regulation provided by the
tTA/tetO system of double transgenics
would be very useful in such studies.
The genechip analysis performed in
VP16–CREBhighmice provides some ad-
ditional clues for understanding this neu-
rodegenerative process. The list of genes
upregulated by VP16–CREBhighincluded
BDNF, a neurotrophin that, as CREB it-
self, is known to play an important role in
neuroprotection. Genetic and pharmaco-
logical studies have shown that increased
expression of BDNF leads to hyperexcit-
development of seizures (Kokaia et al., 1995; Elmer et al., 1997;
Binder et al., 1999; Croll et al., 1999; Tandon et al., 1999;
Lahteinen et al., 2002). These findings suggest that although
BDNF may have a neuroprotective role, too much BDNF may
also have adverse effects. VP16–CREBhighmice also overexpress
synaptic plasticity (Huh et al., 2000; Boulanger and Shatz, 2004;
significantly altered genes either early or late after expression. The main cellular process groups among the 84 probe sets
yellow, whereas genes related to pathological response are highlighted in red (red intensities reflect the overlapping of
Altered gene expression in VP16–CREBhighmice. A, Main cellular processes, as defined in gene ontology (GO),
13916 • J.Neurosci.,December12,2007 • 27(50):13909–13918 LopezdeArmentiaetal.•CREBControlsNeuronalExcitability
blood brain barrier prevents entry of leukocytes, antibodies,
complement factors and cytokines into the brain parenchyma, a
significant increase in MHCI expression, such as in chronic epi-
lepsy or after prolonged VP16–CREB expression, might make
both BDNF and MHC I molecules, individually or in combina-
tion with other molecules, could trigger the neurodegenerative
process and complex molecular changes observed in mice that
expressed VP16–CREB for several weeks or months.
CREB-dependent gene expression is necessary to maintain the
survival of different neuronal subtypes both in vitro and in vivo
(Bonni et al., 1999; Riccio et al., 1999; Lonze et al., 2002; Man-
tamadiotis et al., 2002; Papadia et al., 2005; Parlato et al., 2006).
Moreover, the transient expression of the constitutively active
CREB variant VP16–CREB has been shown to promote axon
regeneration (Gao et al., 2004), neurogenesis (Zhu et al., 2004)
and neuronal survival in vitro (Andreatta et al., 2004; Lee et al.,
in the pathogenesis of various neurodegenerative disorders, in-
cluding Alzheimer’s and Huntington’s diseases. Based in these
therapeutic approaches for neurodegenerative disorders (Barco
et al., 2003; Tully et al., 2003).
Our results in CREB mutant mice support the critical role of
CREB promoting neuronal survival and controlling learning-
related plasticity, but also highlight the dangers that may be as-
sociated to the manipulation of a high level of activity in this
pathway. This knowledge, however, does not reduce the promise
of drugs targeted to this pathway for treating memory or neuro-
degenerative disorders. A better understanding of the molecular
mechanisms underlying CREB activation and function should
make possible the design and development of pharmaceuticals
enhancement should have a beneficial impact in situations in
which this signaling cascade is impaired.
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