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 Previous Article
The Journal of Neuroscience, July 1, 1999, 19(13):5683-5692
Direct Evidence for Biphasic cAMP Responsive Element-Binding
Protein Phosphorylation during Long-Term Potentiation in the Rat
Dentate Gyrus In Vivo
Stefan
Schulz,
Helge
Siemer,
Manfred
Krug, and
Volker
Höllt
Department of Pharmacology and Toxicology, Otto-von-Guericke
University, 39120 Magdeburg, Germany
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ABSTRACT |
Phosphorylation of the transcription factor cAMP responsive
element-binding protein (CREB) is thought to play a key role in synaptic plasticity and long-term memory. However, direct evidence for
CREB phosphorylation during hippocampal long-term potentiation (LTP)
in vivo is sparse. Here, we show that, in the intact
animal, CREB is rapidly phosphorylated in response to high-frequency
stimulation but not low-frequency stimulation of the perforant pathway.
CREB phosphorylation occurred in a biphasic manner, with a first peak at 30 min and a second long-lasting peak beginning 2 hr after tetanic
stimulation and lasting for at least 24 hr. Only stimuli that generated
nondecremental LTP promoted a sustained hyperphosphorylation of CREB
but not stimuli that produced decremental LTP. CREB phosphorylation was
specifically triggered in the dentate gyrus, as well as the CA1, but
not the CA3, hippocampal region. Pretreatment with the NMDA receptor
antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d]
cyclohepten-5,10-imine maleate completely prevented activation of CREB. Together, we have resolved the spatial and temporal dynamics of CREB phosphorylation during hippocampal LTP, showing that the transcription factor CREB is specifically recruited at two distinct time points in some forms of hippocampal synaptic plasticity in vivo.
Key words:
CREB; phosphorylation; LTP; hippocampus; antibodies; immunocytochemistry
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INTRODUCTION |
Hippocampal long-term potentiation
(LTP) is an activity-dependent strengthening of synaptic efficacy that
can last for days or weeks in intact animals and may be a useful model
for studying learning-induced changes in synaptic activity (Bliss and
Collingridge, 1993 ). In an interesting parallel to learning and memory,
LTP has at least two functionally and mechanistically distinct phases. Whereas the induction phase (1-3 hr) is generally believed to be
mediated via activation of existing proteins, i.e., by phosphorylation or isoprenylation (Bailey et al., 1996; Matthies et al., 1997a,b), maintenance of LTP requires de novo RNA transcription and
protein synthesis (Krug et al., 1984; Otani and Ben Ari,
1993).
Substantial evidence has evolved indicating that the cAMP-responsive
element-binding protein (CREB) plays a central and highly conserved
role in the formation of long-term memory (for review, see Frank and
Greenberg, 1994; Stevens, 1994; Bailey et al., 1996). Long-term
facilitation of sensory neurons in Aplysia is blocked by
injection of CRE oligonucleotides into the presynaptic nucleus (Dash et
al., 1990). Furthermore, relief of repression of the inhibitory CREB2
isoform converted transient facilitation in Aplysia into
long-term facilitation (Bartsch et al., 1995, 1998). In transgenic Drosophila, long-term memory disappears or is augmented
after induction of repressor or activator forms of CREB, whereas
short-term memory appears to be intact (Yin et al., 1994, 1995). Mutant
mice lacking CREB isoforms  and  have deficiencies in long-term
retention of several learning tasks and markedly attenuated LTP
(Bourtchuladze et al., 1994). In addition, creation of transgenic mice
carrying a CRE-regulated reporter construct revealed that CRE-mediated gene expression is indeed increased in response to stimuli that generate LTP (Impey et al., 1996, 1998a).
CREB is constitutively expressed and, after phosphorylation on
Ser-133, it becomes active in promoting transcription from the CRE. A
number of protein kinases, including cAMP-dependent protein kinase A
(PKA) and Ca2+/calmodulin-dependent kinase II
(CaMKII), has been implicated in CREB phosphorylation (Sheng et al.,
1991; Hagiwara et al., 1993; Ginty et al., 1994; Sun et al., 1994).
Thus, CREB integrates increases in cAMP and Ca2+ by
inducing synergistic increases in CRE-mediated transcription.
Genetic evidence for the importance of CREB in long-term memory by far
exceeds knowledge of the physiology of its activation. Progress on this
front has been hampered by the difficulty of monitoring CREB activation
after electrically stimulated synaptic activity or plasticity. Although
we and others have detected nuclear CREB phosphorylation in response to
LTP-inducing synaptic stimuli in hippocampal slices (Matthies et al.,
1997b), as well as in dissociated hippocampal neurons in
vitro (Bito et al., 1996; Deisseroth et al., 1996), so far
activation of CREB during hippocampal LTP has not been shown in intact
animals in vivo. In the present study, we addressed this
question by using a phospho-specific CREB antibody for
immunocytochemistry on brain tissue from chronically implanted rats.
This approach allowed us to define the spatiotemporal pattern of
hippocampal CREB phosphorylation after tetanic stimulation of the
perforant pathway.
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MATERIALS AND METHODS |
Animals. Experiments were performed with male Wistar
rats [Shoe:Wist (Shoe)] from Tierzucht Schönwalde, aged 8 weeks
at the beginning of the experiment. Animals were kept in groups of five rats per cage under controlled laboratory conditions (12 hr light/dark cycle, temperature 20 ± 2°C, humidity 55-60%). After
surgery, animals were housed individually in transparent plastic cages throughout the experiment and had access to food and water ad libitum.
Surgery. For all procedures, ethical approval was sought
before the experiments according to the requirements of the
German National Act on the Use
of Experimental Animals. To record the monosynaptic evoked field potential and to induce LTP in the
dentate gyrus after stimulation of the entorhinal-dentate pathway,
animals were chronically implanted with a bipolar stimulation electrode in the ipsilateral perforant pathway (angular bundle) and a monopolar recording electrode in the dorsal blade of the ipsilateral dentate gyrus. The electrodes were made from polyurethane-coated stainless steel wires with a diameter of 125 µm. The animals were deeply anesthetized with pentobarbital (40 mg/kg, i.p.) and mounted on a David
Kopf (Tujunga, CA) stereotaxic instrument with  1 mm below bregma. Small holes were drilled into the scull at the following stereotaxic coordinates: anteroposterior (AP),  2.8 mm; lateral (L), 1.8 mm; and AP, 6.8 mm; L, 4.1 mm (according to Paxinos and Watson, 1982). Electrodes were placed under
electrophysiological guidance. Two wires connected to miniature screws
were placed into the nasal bone and served as ground and reference
electrodes. All electrodes were connected to a flexible miniature
socket and fixed with acrylic dental cement. Animals were allowed to
recover for at least 10 d after surgery.
Electrophysiological recording. For electrophysiological
recording, animals were connected to a model 2100 stimulator (A-M Systems, Inc., Everett, WA) and an AC-coupled amplifier (frequency range of 2 Hz to 10 kHz) through flexible, shielded cable and a swivel,
thus allowing free movement. A MUSYCS interface and personal computer
controlled by FAMOS software (Integrated measurement control
Me systeme, Berlin, Germany) were used to generate trigger signals
for driving the stimulator and calibration signals at the amplifier
input. To elicit evoked potentials, a series of eight biphasic square
wave impulses with a duration of 100 µsec/half-wave was generated
with a frequency of 0.2 Hz. The field potentials were digitized with a
resolution of 100 µsec, averaged, and stored on a hard disk. The
slope function of the field EPSP (fEPSP) and population spike amplitude
were automatically calculated as the steepest 400 µsec segment of the
initial potential upstroke and the peak to peak difference,
respectively. Nondecremental LTP was induced by stimulating the
ipsilateral perforant pathway with 20 trains of impulses. Each train
consisted of 15 pulses having the same duration as the test pulse. The
frequency within the train was 200 Hz for high-frequency stimulation or
0.2 Hz for low-frequency stimulation, and the distance between
trains was 5 sec. For induction of decremental LTP, animals received
only two stimulus trains. Rats were removed from the recording cages either 5 min, 8 min, 15 min, 30 min, 1 hr, 2 hr, 6 hr, or 24 hr after
tetanization and killed by vascular perfusion. A total of 59 animals
were used, and groups of at least three to six animals were analyzed
per time point and treatment condition. In addition, one group of three
animals received (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d]
cyclohepten-5,10-imine maleate (MK801) (0.3 mg/kg, i.p.) 15 min before tetanization.
Immunocytochemistry. Animals were deeply anesthetized with
chloralhydrate (350 mg/kg, i.p.) and transcardially perfused with Tyrode's solution, followed by a fixative containing 4%
paraformaldehyde and 0.2% picric acid in 0.1 M phosphate
buffer, pH 6.9. Brains were rapidly dissected and post-fixed in the
same fixative for 2-4 hr at room temperature. Brains were then
cryoprotected in 30% sucrose, and 40 µm coronal sections were cut
from the hippocampal plane near the recording site using a freezing
microtome. Free-floating sections were washed in TPBS (10 mM Tris, 10 mM phosphate buffer, 137 mM NaCl, and 0.05% thimerosal, pH 7.4), placed in methanol containing 0.3% H2O2 for 30 min, and incubated
in 3% normal goat serum in TPBS with 0.3% Triton X-100 for 1 hr.
Subsequently, the sections were incubated with either phospho-specific
anti-CREB antibody, phosphorylation state-independent anti-CREB
antibody or phospho-specific anti-mitogen-activated protein kinase
(MAPK) antibody (all from New England Biolabs, Beverly, MA) at a
dilution of 1:1000 for 72 hr. Staining of primary antibody was detected using the biotin amplification procedure as described previously (Matthies et al., 1997b; Schulz et al., 1998). Briefly, tissue sections
were transferred to biotinylated goat anti-rabbit IgG (1:1000 in TPBS
containing 0.3% Triton X-100 and 1% NGS; Vector Laboratories,
Burlingame, CA) for 1.5 hr, incubated in an avidin-biotinylated horseradish peroxidase complex (ABC) solution (1:200 in TPBS plus 0.3%
Triton X-100; Vector Laboratories ABC Elite kit) for 45 min, transferred to biotinylated tyramine (BT) solution [1:1000 in TPBS
plus 0.01% H2O2; BT was prepared as
described by Adams (1992)] for 20 min, followed by a final
incubation step in streptavidine-cyanin 3.18 (1:200 in TPBS plus 0.3%
Triton X-100; Amersham, Braunschweig, Germany) overnight at 4°C.
Sections were then mounted onto chrome alum gelatin-subbed glass
slides, dehydrated through several concentrations of alcohol, cleared
in xylol, and coverslipped with DPX. Specimens were examined
using a Leica (Nussloch, Germany) TCS-NT confocal laser scanning
microscope. Cyanin 3.18 was imaged with 561 nm excitation and 590 nm
long-pass emission filters. Images were recorded with 2.5× or 5×
objectives. Staining for serine 133 phosphorylated CREB (pCREB) was
quantitated using the Leica TCS-NT software. The integrated pixel
intensity was determined in the ipsilateral dentate gyrus from three
adjacent sections per animal and averaged. Pixel intensity was
normalized to the corresponding integrated pixel intensity in the area
between stratum radiatum and stratum lacunosum.
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RESULTS |
Time course of CREB phosphorylation during hippocampal LTP
To monitor CREB activation, chronically implanted animals were
tetanized and removed from the recording cages at different time
intervals ranging from 5 min to 24 hr. When perfusion-fixed brain
sections from these animals were immunocytochemically stained using a
pCREB-specific antibody, phosphorylation of the transcription factor
CREB became apparent as quickly as 5 min after tetanization (Figs. 1,
2). At this early time point, pCREB-like
immunoreactivity (Li) was confined to the granule cell nuclei of the
ipsilateral dentate gyrus. After 8 min, the staining intensity in the
dentate gyrus strongly increased. In addition, pCREB-Li was also
observed in a few isolated nuclei of CA1 pyramidal cells. The intensity of this staining further increased within the next 30 min. At 30 min
after tetanization, we also observed nuclear CREB phosphorylation in
the contralateral dentate gyrus. One hour after tetanization, pCREB-Li
in both hippocampi almost completely disappeared. A second and more
sustained peak of CREB activation was observed beginning after 2 hr and
lasting for at least 24 hr. This peak followed a very similar spatial
and temporal pattern as the earlier one in that it first involved the
ipsilateral dentate gyrus, then CA1 region, and at 24 hr also the
contralateraldentate gyrus. At 30 min, 2 hr, 6 hr, and 24 hr, CREB
activation was also seen in the hilar region. In contrast to the
dentate gyrus and the CA1 region, nuclear CREB phosphorylation in
response to high-frequency stimulation was not observed in the CA3
region during the various time points studied. Figure
3C depicts the quantification
of pCREB-Li in the ipsilateral dentate gyrus of all animals examined,
clearly demonstrating the biphasic manner of pCREB. In contrast,
population spike (pop-spike) amplitude, as well as fEPSP, were
continuously enhanced during the same time period, clearly
showing that LTP actually occurred in these experiments (Fig.
3A,B).
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Figure 1.
Rapid induction of CREB phosphorylation in
hippocampal LTP. Left, Immunofluorescent confocal images
of coronal hippocampal sections of animals that were subjected to
high-frequency stimulation 5 (n = 4), 8 (n = 3), 15 (n = 4), or 30 (n = 6) min before vascular perfusion. Sections
were immunocytochemically stained with anti-pCREB antibodies.
Representative results from one of three independent experiments
performed using the same method. Total number of animals used per time
point is given above. Right,
Corresponding electrophysiological recording before tetanization and at
indicated time points after tetanization. Note that CREB
phosphorylation is rapidly induced in response to stimuli that generate
LTP. HFS, High-frequency stimulation. Scale bar, 1 mm.
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Figure 2.
Second peak of CREB phosphorylation in hippocampal
LTP. Left, Immunofluorescent confocal images of coronal
hippocampal sections of animals that were subjected to high-frequency
stimulation 1 (n = 6), 2 (n = 3), 6 (n = 3), or 24 (n = 3) hr
before vascular perfusion. Sections were immunocytochemically stained
with anti-pCREB antibodies. Representative results from one of three
independent experiments performed using the same method. Total number
of animals used per time point is given above.
Right, Corresponding electrophysiological recording
before tetanization and at indicated time points after tetanization.
Note that CREB phosphorylation declines 1 hr after tetanization, and a
second long-lasting peak occurs from 2 to 24 hr. HFS,
High-frequency stimulation. Scale bar, 1 mm.
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Figure 3.
Biphasic increase in CREB phosphorylation during
hippocampal LTP. A, Quantitative analysis of pop-spike
potentiation in the ipsilateral dentate gyrus in response to
high-frequency stimulation. B, Quantitative analysis of
fEPSP potentiation in the ipsilateral dentate gyrus in response to
high-frequency stimulation. C, Quantification of pCREB
immunostaining in the ipsilateral dentate gyrus in response to tetanic
stimulation at 0 (n = 3), 5 (n = 4), 8 (n = 3), 15 (n = 4), 30 (n = 6), 60 (n = 6), 120 (n = 3), and 360 (n = 3) min
and 24 hr (n = 3).
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Stimulus-specificity of hippocampal CREB activation
If CREB is specifically involved in hippocampal plasticity, it
should be selectively activated in response to stimuli that induce a
long-lasting enhancement of synaptic strength. Therefore, in a separate
series of experiments, rats were subjected to either high- or
low-frequency stimulation conditions known to either generate or not
generate hippocampal LTP, respectively. Again, a robust induction of
pCREB-Li was observed in the dentate gyrus, as well as the CA1 region,
30 min after high-frequency stimulation. In contrast, virtually no
pCREB-Li was seen in animals that were either subjected to
low-frequency stimulation or not stimulated (Fig.
4). Moreover, treatment of animals with
the NMDA receptor antagonist MK801 not only prevented the induction of
hippocampal LTP but also blocked the enhancement of pCREB-Li in
response to high-frequency stimulation (Fig. 4).
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Figure 4.
Stimulus specificity of hippocampal CREB
activation. Left, Immunofluorescent confocal images of
coronal hippocampal sections of animals that were not stimulated
(n = 3) or subjected to either low-frequency
(n = 3) or high-frequency (n = 3) stimulation 30 min before vascular perfusion. In addition, one group
of animals was treated with MK801 (0.3 mg/kg, i.p.;
n = 3) 15 min before high-frequency stimulation.
Sections were immunocytochemically stained with anti-pCREB antibodies.
Representative results from one of three independent experiments
performed using the same method. Total number of animals used per time
point is given above. Right,
Corresponding electrophysiological recording before tetanization and at
indicated time points after tetanization. Note that CREB
phosphorylation occurs selectively in response to stimuli that generate
LTP in an NMDA-dependent manner. CON, Control;
LFS, low-frequency stimulation; HFS,
high-frequency stimulation; HFS+MK801, high-frequency
stimulation plus MK801 treatment. Scale bar, 1 mm.
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CREB is hyperphosphorylated during nondecremental, but not
decremental, LTP
Genetic evidence suggests that CREB may play a crucial role
for generation of a late-phase of LTP, as well as consolidation of
long-term memory. Thus, experiments were conducted to determine how
tightly CREB phosphorylation correlates with nondecremental and
decremental LTP. When animals received a strong stimulus (20 trains)
that generated nondecremental LTP, a robust and sustained hyperphosphorylation of CREB was seen in the ipsilateral dentate gyrus
(Fig. 5, left panels). This
increase in CREB phosphorylation lasted for at least 6 hr. In contrast,
animals that received a weak stimulus (2 trains) resulting in
decremental LTP as evidenced by the decline of both pop-spike and fEPSP
potentiation showed, if at all, a small and transient rise in
hippocampal CREB phosphorylation within 2 hr after tetanus, and after 6 hr, none of the animals showed any significant CREB phosphorylation
(Fig. 5, right panels).
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Figure 5.
Differential CREB phosphorylation during
nondecremental and decremental LTP. Left, Nondecremental
LTP was induced by stimulating the ipsilateral perforant pathway with
20 stimulus trains as described in Materials and Methods. Animals were
removed from the recording cages after 0 (n = 3),
30 (n = 6), 120 (n = 3), or 360 (n = 3) min and subjected to vascular perfusion.
Right, Decremental LTP was induced by stimulating the
ipsilateral perforant pathway with two stimulus trains as described in
Materials and Methods. Animals were removed from the recording cages
after 0 (n = 3), 30 (n = 3),
120 (n = 3), or 360 (n = 3) min
and subjected to vascular perfusion. Sections were immunocytochemically
stained with anti-pCREB antibodies. A, B,
Quantitative analysis of pop-spike potentiation in the ipsilateral
dentate gyrus. C-J, Immunofluorescent confocal images
of coronal hippocampal sections. Representative results from one of
three independent experiments performed using the same method. Total
number of animals used per time point is given above.
Note that only stimuli that generate nondecremental LTP induced
sustained CREB phosphorylation but not stimuli that generate
decremental LTP. Scale bar, 1 mm.
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Comparison between CREB phosphorylation and MAPK activation
It has been shown recently that sequential activation of
the extracellular signal-regulated kinase (ERK)/MAPK pathway and the
CREB kinase ribosomal S6 kinase 2 (RSK2) may lead to phosphorylation and transactivation of CREB (Impey et al., 1998b). The
activation state of MAP kinase can easily be monitored using
immunocytochemistry with tyrosine 204 phosphorylation-specific MAPK
antibodies. If the ERK/MAPK cascade would contribute to CREB
phosphorylation, then activation of MAP kinase and CREB would be
expected to follow closely overlapping patterns. However, when animals
were perfused 30 min after tetanization and adjacent brain sections
were stained with either anti-pCREB or anti-pMAPK antibodies, we found
that pCREB-Li and pMAPK-Li showed strikingly different regional and subcellular distributions (Fig.
6A-D). Like pCREB-Li,
pMAPK-Li was prominent in the ipsilateral dentate gyrus. Unlike
pCREB-Li, pMAPK-Li was not seen in granule cell nuclei but rather was
confined to fibers and processes in the polymorph layer and hilus. In
the Ammon's horn of the hippocampus, pMAPK-Li was not restricted to the CA1 region but involved also the CA3 region. In contrast to pCREB-Li, pMAPK-Li was virtually absent from pyramidal cell nuclei but
was prominent in the stratum oriens, as well as stratum radiatum.
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Figure 6.
Comparison of pMAPK- and pCREB-like
immunoreactivities at 30 min after tetanus. Immunofluorescent confocal
images of coronal hippocampal sections of animals that were subjected
to high-frequency stimulation 30 min (n = 3) before
vascular perfusion. Sections were immunocytochemically stained with
either anti-pMAPK, anti-pCREB, or anti-CREB antibodies. Representative
results from one of three independent experiments performed using the
same method. Note that both MAPK and CREB are clearly activated in the
ipsilateral dentate gyrus but show strikingly different distributions
30 min after tetanization. Similar levels of total immunoreactive CREB
proteins were detected in all regions of both hippocampi and adjacent
brain regions, indicating that the pCREB-Li staining pattern reflects a
response to tetanic stimulation. Scale bars: (in E)
A, C, E, 1 mm; (in
F) B, D,
F, 1 mm.
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In addition, we compared the distribution of activated CREB proteins
detected using a Ser-133 phosphorylation-specific antibody and total
CREB proteins detected using a phosphorylation state-independent anti-CREB antibody. As shown in Figure 6, E and
F, similar levels of total immunoreactive CREB proteins were
detected in all regions of both hippocampi and adjacent brain regions,
indicating that the pCREB-Li staining pattern clearly reflects a
response to tetanic stimulation.
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DISCUSSION |
In the present work, we have resolved the spatiotemporal dynamics
of CREB serine 133 phosphorylation during hippocampal LTP in freely
moving rats. CREB phosphorylation occurs in a biphasic manner, with a
first short-lasting peak at 30 min and a second more sustained peak
beginning after 2 hr and lasting for at least 24 hr (Fig. 4). CREB was
selectively recruited in the dentate gyrus and the CA1, but not the
CA3, region of the ipsilateral hippocampus. CREB phosphorylation was
triggered in a stimulus-specific manner. Only stimuli that induce a
persistent enhancement of synaptic strength (high-frequency
stimulation) also produced robust increases in hippocampal pCREB-Li.
Stimuli that did not induce pop-spike or fEPSP potentiation
(low-frequency stimulation) also failed to trigger phosphorylation of
CREB. In fact, sustained CREB activation appears to be required for
maintenance of a late-phase of LTP. Only stimuli that generated
nondecremental LTP also promoted strong and long-lasting CREB
phosphorylation but not stimuli that induced decremental LTP. The
initial recruitment of CREB appears to depend on calcium influx via the
NMDA receptor because pretreatment of animals with MK801 completely
blocked the induction of hippocampal LTP and associated increases in
CREB phosphorylation at the 30 min time point.
Although our in vivo data strongly support the hypothesis
that CREB is an important component of a general switch that converts short-term into long-term synaptic plasticity, it is not known by which
signaling cascades CREB phosphorylation is mediated. The surrounding
sequences of serine 133 in mammalian CREB contain recognition sites for
PKA, CaMKII, and RSK2 (Sheng et al., 1991; Hagiwara et al.,
1993; Impey et al., 1998b). LTP is not a unitary phenomenon but
consists of several phases (Matthies et al., 1990). An early phase of
LTP that is blocked by inhibitors of CaMKII can be dissociated from
late LTP, which is sensitive to PKA inhibitors (Ito et al., 1991; Frey
et al., 1993; Matthies and Reymann, 1993; Huang et al., 1994).
It is thus very tempting to speculate that CaMKII may be
involved in the initial peak of CREB phosphorylation, whereas PKA may
primarily contribute to the second more sustained peak of CREB
phosphorylation. Recent work by Impey et al. (1998b) suggests that
sequential activation of the ERK/MAPK pathway and the CREB kinase RSK2
may lead to phosphorylation and transactivation of CREB. In the present
study, we monitored the activation state of the MAPK cascade using
immunocytochemistry with tyrosine 204 phosphorylation-specific MAPK
antibodies and observed strikingly different patterns of CREB
phosphorylation and MAPK activation. Whereas 30 min post-tetanus
pCREB-Li was clearly confined to the nuclei of granule and CA1
pyramidal cells, pMAPK-Li was prominent in fibers and processes of
these cells. These findings would suggest a model in that synaptic
activation of MAPK would trigger nuclear translocation of RSK2, which
in turn phosphorylates CREB. Alternatively, MAPK may
serve other functions during hippocampal LTP, i.e., phosphorylation of
neural cell adhesion molecules (Bailey et al., 1997).
In freely moving animals, tetanic stimulation of the perforant path
projection is known to elicit long-term potentiation in the ipsilateral
dentate gyrus. Although CREB phosphorylation was first noted in the
ipsilateral dentate gyrus, it was not limited there. pCREB-Li
was also seen in the ipsilateral CA1 region (but not CA3), as well as
the contralateral dentate gyrus. The simplest explanation for this
finding would be that information about enhanced synaptic activity is
relayed via intrahippocampal circuits from the ipsilateral dentate
gyrus to the ipsilateral CA1 region, as well as to the contralateral
dentate gyrus. Alternatively, the entorhinal cortex may directly
project to the contralateral dentate gyrus (Amaral and Witter, 1989;
Berger et al., 1997). Indeed, we have found previously that, after
tetanic stimulation of the perforant path projection, a delayed form of
long-term potentiation can be recorded from the contralateral dentate
gyrus (M. Krug, unpublished observation). Excitation of the ipsilateral
CA1 region would be expected to occur via the "classical"
trisynaptic loop involving mossy fibers terminating at CA3 pyramidal
cells and the Schaffer collateral-commissural pathway. However,
significant CREB phosphorylation was not seen in CA3 pyramidal cell
nuclei. This is perhaps not surprising because the form of long-term
potentiation found at mossy fiber synapses is independent of NMDA
receptors and requires a rise in presynaptic Ca2+
(Collingridge and Bliss, 1995; Nicoll and Malenka, 1995). Here, we show that CREB phosphorylation is critically dependent on
Ca2+-influx via postsynaptic NMDA receptors.
Alternatively, a direct activation of CA1 pyramidal cells from the
entorhinal cortex seems possible (Amaral and Witter, 1989; Berger et
al., 1997).
Although substantial genetic evidence for a role of CREB in memory
formation exists and the present study clearly demonstrates that CREB
is specifically recruited during hippocampal LTP in vivo, it
is not known which of the many CREB/activating transcription factor
(ATF) or cAMP responsive element-binding modulator (CREM) protein
isoforms are involved in long-term neuronal plasticity. Here, we
selectively monitored CREB serine 133 phosphorylation showing that CREB
is recruited at two distinct time points. Whereas the first peak of
CREB phosphorylation appears to be rapidly terminated by
dephosphorylation, the second peak of CREB phosphorylation appears to
be more sustained and lasts for at least 24 hr. Interestingly, CRE-mediated gene expression can be detected as early as 2 hr after
tetanus and reaches a maximum at 4-6 hr, suggesting that sustained
CREB phosphorylation is required for induction of persistent CRE-mediated gene expression. However, it remains open to question what
role inhibitory CREM isoforms may play in the termination of the second
peak of CREB phosphorylation.
It is important to note that interesting correlations exist between the
time course of CREB phosphorylation and the expression of
immediate-early genes after induction of LTP. In awake animals, tetanic
stimulation of the perforant path resulted in a rapid induction of
Fos-related antigen but not of Fos or Fos-B proteins, which may
provide a link between CREB activation and nuclear gene expression (for
review, see Hughes and Dragunow, 1995). Subsequently, 1-6 hr after LTP
induction, a number of genes appear to be upregulated (Hevroni et al.,
1998). Moreover, induction of long-term facilitation in
Aplysia has been shown to be associated with multiple waves of protein synthesis. Here, a group of 10 proteins is rapidly induced
within the first hour after repeated exposure to 5-HT. These early
changes are followed by a second wave of changes in other proteins at 3 hr, and later by a third more persistent wave of protein synthesis
(Barzilai et al., 1989; Abel and Kandel, 1998). Similarly, a biphasic
time course of protein synthesis has also been observed in simple
learning paradigms (for review, see Matthies, 1989). It is thus
conceivable that the biphasic phosphorylation of CREB may participate
in triggering multiple waves of protein synthesis required for a
plastic change in synaptic efficacy.
In summary, the spatial and temporal pattern of CREB phosphorylation
during hippocampal LTP in vivo is more complex than
previously expected. The transcription factor CREB is specifically
recruited at two distinct time points in response to stimuli that
generate a persistent enhancement of synaptic efficacy. These findings strongly reinforce the notion that the CREB/ATF family of transcription factors is an important component of a molecular switch that converts short-term into long-term synaptic plasticity.
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FOOTNOTES |
Received Dec. 22, 1998; revised April 20, 1999; accepted April 21, 1999.
This work was supported by Deutsche Forschungsgemeinschaft Grant SCHU
924/4-1 to S.S., Kultusministerium des Landes Sachsen/Anhalt Grant
1908A/0025 to S.S., and Sonderforschungsbereich 426 Grant 125572-2 to
M.K. We thank Maria Wagner and Dana Wiborny for excellent technical assistance.
Correspondence should be addressed to Volker Höllt, Department of
Pharmacology and Toxicology, Otto-von-Guericke University, Leipziger
Strasse 44, 39120 Magdeburg, Germany.
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TOP
ABSTRACT
INTRODUCTION
