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Vol. 61, Issue 6, 1453-1464, June 2002
Division of Molecular Psychiatry, Departments of Psychiatry and Pharmacology, Yale University School of Medicine and Connecticut Mental Health Center, New Haven, Connecticut (N.S., J.T., S.S.N., J.C., M.B.K., R.S.D.); and Department of Psychiatry, the University of Texas Southwestern Medical Center, Dallas, Texas (C.S., E.J.N.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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To investigate the role of cAMP response element-binding protein (CREB) in the adaptive responses to psychotropic drugs, we have developed inducible, brain region-specific CREB transgenic mice using the tetracycline-regulated gene expression system. The tetracycline transactivator (tTA) was placed under the control of 1.8-kilobase neuron-specific enolase (NSE) promoter for this purpose. Different patterns of CREB overexpression were found in striatum, nucleus accumbens, and cingulate cortex in different lines of bitransgenic mice, and CREB expression was blocked by addition of doxycycline, an analog of tetracycline. Overexpression of CREB influenced the expression of other members of the CREB/ATF family of transcription factors, consistent with previous reports. In addition, psychostimulant induction of dynorphin, a neuropeptide regulated by drugs of abuse, was up-regulated in striatum. Finally, there was a significant reduction in cocaine-induced locomotor activity in the CREB bitransgenic mice. These results are consistent with a role for CREB in mediating adaptive changes that occur in response to drugs of abuse.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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A
variety of neurotransmitter and endocrine signals which elevate
intracellular cAMP levels activate gene transcription via a cAMP
response element (CRE) found in the promoter region of target genes
(Ziff, 1990
). The cAMP response element binding protein (CREB), a
substrate of cAMP-dependent protein kinase A, is able to bind to the
CRE and trans-activate gene expression (Hoeffler et al.,
1988; Yamamoto et al., 1988; Gonzalez and Montminy, 1989). In addition
to CREB, several other CRE-binding proteins make up the CREB/activation
transcription factor (ATF) family (Hai et al., 1989; Maekawa et al.,
1989; Hai and Curran, 1991). Binding to a CRE is dependent on
dimerization at the basic/leucine zipper domain in the carboxyl
terminus of CREB/ATF proteins (Hai et al., 1989; Hai and Curran, 1991).
CREB can form functional homodimers, or heterodimers with other
CREB/ATF family proteins. After phosphorylation of CREB at the
Ser133 residue and binding to a CRE site, the
CREB binding protein, a coactivator of CREB, binds to the
phosphorylated CREB. This leads to activation of a transcription
factor/RNA polymerase II complex that directly
trans-activates target gene expression. Ser133 can also be phosphorylated by a number of
protein kinases other than protein kinase A, including calcium
calmodulin-dependent protein kinase, protein kinase C, and ribosomal
S-6 kinase. This suggests that CREB plays an important role in
integrating intracellular cAMP and calcium signaling as well as
responses to neurotrophic factors (Dash et al., 1991; Matthews et al.,
1994).
CREB has been shown to play a critical role in different types of
neuronal function, including memory formation and circadian rhythm
(Ginty et al., 1993; Milner et al., 1998; Obrietan et al., 1998).
Furthermore, region-specific regulation of CREB has been implicated in
the long-term neuronal plasticity that underlies the actions of
psychotropic drugs (Duman et al., 1997, 2000; Nestler and Aghajanian,
1997). For example, chronic opiate or psychostimulant administration
up-regulates the phosphorylation or expression of CREB in specific
brain regions, such as the locus ceruleus or nucleus accumbens
(Guitart et al., 1992; Widnell et al., 1994; Cole et al., 1995; Widnell
et al., 1996; Lane-Ladd et al., 1997; Nestler, 1997; Nestler and
Aghajanian, 1997). A role for CREB in the behavioral actions of drugs
of abuse is also supported by studies of CREB
mutant mice or
viral mediated gene transfer of CREB (Maldonado et al., 1996; Carlezon
et al., 1998). Another series of studies demonstrates that
administration of antidepressants, but not drugs of abuse, increases
the phosphorylation and expression of CREB in the hippocampus and
cerebral cortex but not in nucleus accumbens or locus ceruleus (Nibuya
et al., 1996; Thome et al., 2000). These findings demonstrate
that CREB is differentially regulated by psychotropic drugs and may
play a role in the cellular and behavioral responses to these agents.
To further elucidate the function of CREB, we have developed inducible,
brain region-specific CREB transgenic mice. Conventional transgenic
mice, which constitutively overexpress a transgene from early
development, can often exhibit compensatory or developmental adaptations that make it difficult to interpret the actions of transgene overexpression. In addition, expression of a transgene throughout the brain, as well as in peripheral tissues, can lead to
effects that indirectly influence the function of specific populations
of neurons. The use of inducible and region-specific transgenic mice
circumvents these problems. We have used the tetracycline-regulated system, in which the expression of CREB is placed under the control of
the tetracycline-operated promoter (TetOp). TetOp-CREB is regulated by
the tetracycline transactivator (tTA), which is under the control of
the neuron-specific enolase (NSE) promoter (Chen et al., 1998; Kelz et
al., 1999). Addition of tetracycline induces a conformational change in
tTA and thereby blocks its ability to bind TetOp and trans-activate CREB expression. This is referred to as the
tetracycline-off system (Fig. 1).
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In this study, we have characterized the expression of CREB in several lines of inducible and region-specific transgenic mice, and we have examined the effects of doxycycline, an analog of tetracycline, on the expression of CREB in vivo. In addition, we report the regulation of CREB/ATF1 family members, as well as possible target genes, in the CREB transgenic mice. Moreover, we investigate the behavioral consequences of CREB overexpression.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Development of Transgenic Mice and Drug Treatments.
The
methods for generating inducible and brain region-specific CREB
transgenic mice are described in our previous reports (Chen et al.,
1998; Kelz et al., 1999). The principle of the Tet-regulated system is
summarized in Fig. 1. In brief, plasmids that can drive tTA under the
control of 1.8-kilobase NSE promoter (designated pNSE-tTA) and an
another that can drive expression of rat CREB under the control of
the TetOp (designated pTetOp-CREB) were constructed. Linearized DNA
fragments from pNSE-tTA or pTetOp-CREB, which contain the promoter,
open reading frame, simian virus 40 intron, and poly(A)+ signal, were microinjected into
pronuclei of oocytes from SJL × C57BL6 mice. Transgenic mice with
a single transgene (either NSE-tTA or TetOP-CREB) were generated
independently. These two lines of mice were then crossed to develop
NSE-tTA × TetOp-CREB bitransgenic mice. Genotyping of generated
transgenic mice was performed by PCR with specific primer sets for the
NSE promoter or rat CREB cDNA using genomic DNA isolated from mouse
tail as template.
Immunohistochemistry.
Immunohistochemistry was conducted as
described previously (Hiroi and Graybiel, 1996; Hiroi et al., 1997). In
brief, mice were perfused with 0.9% NaCl followed by 4%
paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) after
anesthesia with sodium pentobarbital. Brains were removed and further
fixed with 4% paraformaldehyde in 0.1 M PBS for 16 h at 4°C.
The brains were stored in 20% (v/v) glycerol in 0.1 M PBS for 2 days
at 4°C. Coronal brain slices of 20 to 25 µm thickness were cut with
the use of a microtome. For CREB immunohistochemistry, slices were
blocked with 5% BSA in 0.01 M PBS for 30 min at room temperature and
then incubated with a polyclonal CREB antibody (1:500 dilution; Upstate
Biotechnology Incorporated, Lake Placid, NY) for 2 to 3 days at 4°C.
CREB immunoreactivity was detected using standard avidin-biotin
complex-diaminobenzidine method as described previously (Hiroi and
Graybiel, 1996; Hiroi et al., 1997).
Immunoblotting of CREB and Phospho-CREB.
Dissected striatum
and cerebellum from NSE-tTA × TetOp-CREB bitransgenic
(NSE-tTA+/TetOp-CREB+, +/+) mice or NSE-tTA monotransgenic (NSE-tTA+/TetOp-CREB
, +/) mice were homogenized and sonicated in
immunoprecipitation assay buffer (10 mM Tris-HCl, 1% Nonidet P-40,
0.1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 1 mM EDTA, 20 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride, pH 7.4) and
centrifuged at 19,000g for 15 min at 4°C. Supernatants were subjected to SDS-polyacrylamide gel electrophoresis and
immunoblotting using a polyclonal CREB antibody (1:500 dilution;
Upstate Biotechnology Incorporated) as described previously (Takahashi
et al., 1999).
). To compare the precise size of the
phospho-CREB band, a lysate of forskolin-treated CATH.a cells was used
as positive control. CREB immunoblotting was carried out simultaneously
to examine the level of total CREB immunoreactivity.
Phospho-CREB Immunohistochemistry. Immunohistochemistry for pCREB was performed using a slide-based protocol. Cryocut sections (14 µm thick) were briefly fixed in 4% paraformaldehyde followed by rinses in PBS. This was followed by a 5-min treatment with 1% hydrogen peroxide and two 5-min rinses in PBS. Sections were blocked to prevent nonspecific binding of antibody by a 30-min treatment in a blocking solution containing 2.5% BSA.
Sections were incubated overnight at 4°C in antibody solution (0.25% Triton X-100, 1% BSA in PBS) containing anti-phospho-CREB antibody (Upstate Biotechnology) at 1:500 dilution. Antibody-treated sections were given three 5-min washes with PBS to remove unbound primary antibody and were incubated in peroxidase-conjugated goat anti-rabbit IgG at 1:400 dilution for 1 h at room temperature. Unbound secondary antibody was removed by three 5-min rinses in PBS. Sections were then incubated in a preformed avidin and biotinylated horseradish peroxidase macromolecular complex (ABC reagent; Vector Labs, Burlingame, CA) for 1 h. After this incubation, sections were rinsed three times in PBS and then stained with the 3,3'-diaminobenzidine staining kit (Vector Labs) according to manufacturer's directions. Slides were air dried, dehydrated in an alcohol series, and coverslipped with distyrene/plasticizer/xylene mountant.RNase Protection and in Situ Hybridization Analysis. Total RNA was extracted from various regions of mouse brain using the RNAqueous kit (Ambion, Austin, TX) according to the manufacturer's protocol. RPA was performed with the RPAII kit (Ambion) according to the manufacturer's protocol and using total RNA extracts from mouse brain as templates.
To generate the CREB-specific riboprobes that distinguish the three major CREB isoforms (CREB, CREB, and CREB
; Ruppert et al.,
1992; Blendy et al., 1996), reverse transcriptase (RT)-polymerase chain
reaction was first carried out to obtain partial mouse CREB cDNA. A
sense primer that recognizes exon 2 (designated primer 6) of the mouse
CREB gene and an antisense primer that recognizes exon 7 (designated
primer 7) were designed for RT-PCR. The sequences of primers 6 and 7 were 5'-TAAATGACCATGGAATCTGGAGCA-3' and 5'-AGTTACACTATCCACAGACTCCTG-3', respectively. RT-PCR was performed using the Access RT-PCR system (Promega, Madison, WI), according to the manufacturer's recommended method. Total striatal RNA was used as template for RT-PCR, followed by
agarose gel separation and ethidium bromide staining to visualize the
products. A DNA band of 318 bp, which corresponds to the CREB isoform, was isolated using a gel extraction kit (QIAGEN, Valencia, CA), subcloned into pGEM-T Easy vector (Promega), and verified by
sequencing. The resulting plasmid was designated pCREB 6-7. A
riboprobe was generated by linearizing pCREB 6-7 with
SpeI and 32P-labeled using SP6 RNA
polymerase (Roche Applied Science, Indianapolis, IN). Protected
fragments were loaded onto an 8% acrylamide-Tris/borate/EDTA gel and the separated bands were detected by autoradiography. Measurement and quantification of protected band density was carried out using the Macintosh version of the NIH image analysis program (version 1.52; http://rsb.info.nih.gov/nih-image/).
For CREM (CRE modulator)-specific RPA analysis, RT-PCR was also
performed, as described above, to obtain partial cDNA encoding the
CREM
isoform (Foulkes et al., 1992). The sequences of the sense and
antisense primers were 5'-GAAACAGTTGAATCACAGCAGGAT-3' and
5'-TGATTGAATAACCGATGGATGTGG-3', respectively. RT-PCR products were
subcloned (designated as pCREM). This riboprobe can distinguish between CREM and -, repressor isoforms of CREM, as well as
CREM, an activator isoform of CREM, by the size of protected
fragments. CREM and - are recognized as a single band of 105 bp
and CREM is a protected fragment of 231 bp.
To generate a probe for RPA of ATF1, RT-PCR was performed using mouse
ATF1 specific primers (Lee et al., 1992). The sense and antisense
primers were 5'-ATAGGCTCCTCACAGAAAGCTCAC-3' and 5'-TAATGTCTGCAGTGCCTGCACTCC-3', respectively. The resulting subcloned plasmid was designated pATF1. RPA using the pATF1 riboprobe recognizes an ATF1 mRNA band of 258 bp. Probes used for RPA of inducible cAMP
early repressor (ICER) was carried out as described previously (Fitzgerald et al., 1996a).
For RPA and in situ hybridization of dynorphin, a riboprobe was
generated from a plasmid encoding the major exon of rat prodynorphin gene, which was provided by Dr. C. R. Gerfen (see Carlezon et al.,
1998). Experimental procedures for in situ hybridization of dynorphin
were as described previously (Nibuya et al., 1996; Takahashi et al.,
1999). After ISH, sections were dipped in nuclear track emulsion (NTB2;
Eastman Kodak, Rochester, NY) after diluting emulsion (1:1) in 600 mM
ammonium acetate at 42°C. Slides were developed after 1 week exposure
in emulsion, counter-stained in cresyl violet (10-15 min), dehydrated
in an alcohol series, cleared in Histochoice (15 s; Amresco, Solon,
OH), and coverslipped with distyrene/plasticizer/xylene mountant.
Analysis of Cocaine-Induced Locomotor Activity in Bitransgenic
Mice.
Locomotor sensitization was conducted in mice as described
previously (Hiroi et al., 1997). Subjects were habituated to the test
apparatus over 5 consecutive days and then received cocaine (10 mg/kg,
i.p.) or saline injections for 5 consecutive days. Horizontal locomotor
activity was quantified by automated beam crossing for 60 min each day.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Expression Pattern of CREB in NSE-tTA × TetOp-CREB
Bitransgenic Mice.
We have developed six lines of TetOp-CREB mice,
each of which was crossed with NSE-tTA lines A and B generated by our
group (Chen et al., 1998). Previous studies demonstrate that these
lines induce overexpression of a reporter gene
(luciferase) and another target gene
(FosB) under the TetOp promoter to varying degrees in
striatum, nucleus accumbens, cortex, and hippocampus (Chen et al.,
1998). Immunohistochemical analysis demonstrated significant levels of
CREB overexpression in three lines of bitransgenic mice: TetOp-CREB 6 line × NSE-tTA B line (CREB6-B line); TetOp-CREB 6 line × NSE-tTA A line (CREB6-A line); and TetOp-CREB 3 line × NSE-tTA A
line (CREB3-A line).
, +/)
(Fig. 2). Higher magnification (400×)
demonstrates that CREB overexpression is restricted to the nucleus of
cells. CREB was also overexpressed in nucleus accumbens, primarily in
the core subdivision (Fig. 2, lower). In the CREB6-A line, CREB was
overexpressed in striatum and nucleus accumbens, similar to the
expression pattern in the CREB6-B line (summarized in Table
1). In addition, CREB was sparsely, but
significantly, overexpressed in deep and superficial layers of
cingulate cortex (Fig. 3). CREB
overexpression in deep layers of parietal cortex was also observed
(data not shown). No CREB overexpression was observed in hippocampus in
either the CREB6-A or -B lines (Table 1). However, low levels of CREB
overexpression were observed in hippocampus of the CREB3-A line (Fig.
3, lower trace). CREB was sparsely but clearly overexpressed in CA1
pyramidal cells and granular cells of dentate gyrus (Fig. 3, lower
trace). CREB overexpression was also observed in the dorsal striatum
and nucleus accumbens but at lower levels than in the CREB6-A or
CREB6-B lines (summarized in Table 1). There was no expression of CREB observed in monotransgenic TetOp-CREB mice (i.e., without the NSE-tTA),
demonstrating that there is no leak of the TetOp-CREB transgene (not
shown).
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) (data not shown).
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mice was examined. In preliminary time course experiments, the induction of phospho-CREB by
forskolin was found to peak after 15 min and then gradually decrease
with longer periods of incubation. Therefore, a 15-min time point was
chosen for further studies. Stimulation with forskolin increased
phospho-CREB immunoreactivity relative to control, and this effect was
greater in the CREB6-B +/+ mice relative to the +/ mice (Fig. 4B).
Two phospho-CREB immunoreactive bands, which were just above an intense
nonspecific band, were observed in response to forskolin. The
phospho-CREB bands in the bitransgenic mice comigrate with the
phospho-CREB bands prepared from forskolin-treated CATH.a cells (Fig.
4B), which are also detected as a doublet upon shorter exposure (data
not shown). The expression of CREB is relatively high in the CATH.a
cells, and we have used them routinely as markers for CREB and
phospho-CREB (Widnell et al., 1994, 1996). The relatively lower level
of CREB and phospho-CREB immunoreactivity in the mouse brain compared
with CATH.a cells is consistent with the low levels observed in our
previous studies (Widnell et al., 1994, 1996). Figure 4B also
demonstrates that levels of total CREB immunoreactivity are higher in
the +/+ mice relative to the +/ mice.
The influence of doxycycline, a derivative of tetracycline, on the
expression of CREB in the bitransgenic mice was examined next. This was
necessary to confirm that the tTA-TetOp system was functioning
correctly in vivo. Application of doxycycline (50 µg/ml in water) for
25 days completely blocked CREB overexpression in the 6-B line (Fig.
5). Removal of doxycycline for 2 weeks
allowed for a nearly complete recovery of CREB overexpression (Fig. 5, right). These findings demonstrate that the expression of CREB can be
turned off and then back on upon addition and removal of doxycycline.
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Regulation of CREB, ATF, and CREM Isoforms in the Bitransgenic
Mice.
Studies were conducted to determine whether overexpression
of CREB alters levels of endogenous CREB or CREB-related transcription factors, including CREM and ATF1. Expression of three major isoforms of
CREB (i.e., , , and ) (Ruppert et al., 1992; Blendy et al., 1996), were analyzed by RPA in NSE-tTA × TetOp-CREB bitransgenic mice. A riboprobe that can distinguish the CREB isoforms by the size of
protected fragments was prepared by RT-PCR (Fig.
6A). The relative levels of the three
isoforms are similar to what has been reported (Blendy et al., 1996).
In wild-type animals, levels of CREB are highest, with approximately
equal amounts of CREB and CREB. In striatum from either CREB6-A
or -B bitransgenic (+/+) mice, CREB mRNA, the isoform used to
develop the transgenic lines, is significantly increased as expected
(Fig. 6, B and C). In addition, levels of CREB and isoforms are
significantly up-regulated. The induction of the , as well as and , isoforms in the CREB6-A line was reversed by addition of
doxycycline to the drinking water (Fig. 6C).
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; Ziff,
1990; Hai and Curran, 1991). Within the CREB/ATF family of
transcription factors, CREB, CREM, and ATF1 are known to preferentially
form heterodimers with one another in vitro, indicating that these
three transcription factors function as partners for regulation of
CRE-mediated transcription in vivo (Hai and Curran, 1991). To examine
the influence of CREB overexpression on these related proteins, RPA
analysis of CREM and ATF1 was performed.
A riboprobe was generated for CREM, an activator of CRE-mediated
trans-activation that can discriminate between this isoform and CREM and , two repressors of CRE trans-activation,
by the size of the protected fragments (Fig.
7A). Both repressor and activator types
of CREM (CREM, , and , respectively) were down-regulated by
approximately 20% in striatum and cerebellum of CREB6-B bitransgenic mice (Fig. 7). Down-regulation of CREM was not observed in CREB6-A bitransgenic mice (data not shown). This may be related to the greater
induction of CREB in the CREB6-B line relative to the -A line, as
demonstrated in Fig. 6B.
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, -, and - (Molina et al., 1993). Basal levels of ICER mRNA were not
significantly influenced in striatum of CREB6-B bitransgenic mice (Fig.
7). Because ICER is rapidly induced by activation of the CREB,
induction of this isoform in response to amphetamine was also examined
in control and CREB-6B bitransgenic mice. Induction of ICER in response to amphetamine was observed in both single (NSE-tTA) and bitransgenic (NSE-tTA × TetOp-CREB) mice, but there was no significant
difference between these two groups [273 ± 22.6 and 228 ± 14.6% of saline, for single and bitransgenic CREB6-B mice,
respectively (mean ± S.E.M., n = 10 per group)].
There was also no significant difference in levels of mRNA for ATF1,
another member of the CREB/ATF family, in striatum or cerebellum of the
single CREB6-B bitransgenic line (Fig. 7).
Regulation of Dynorphin Expression in NSE-tTA × TetOp-CREB
Bitransgenic Mice.
Previous reports demonstrate that chronic
administration of a psychostimulant (e.g., cocaine or amphetamine)
up-regulates the cAMP pathway, which leads to activation of
CRE/CREB-mediated transcription in nucleus accumbens and dorsal
striatum (Terwilliger et al., 1991; Konradi et al., 1994; Cole et al.,
1995; Hyman, 1996; Unterwald et al., 1996; Nestler, 1997; Nestler and
Aghajanian, 1997; Turgeon et al., 1997). In addition, dynorphin has
been shown to be a target of CREB-mediated trans-activation
in these regions after chronic cocaine or amphetamine treatment (Hurd
et al., 1992; Daunais et al., 1993; Spangler et al., 1993; Cole et al.,
1995; Carlezon et al., 1998). The promoter region of the rat dynorphin gene has three predicted CRE sites, and promoter activity is increased by stimulation of receptors that activate the cAMP pathway in vitro
(Collins-Hicok et al., 1994). These findings led us to examine the
regulation of dynorphin in the dorsal striatum and nucleus accumbens of
NSE-tTA × TetOp-CREB bitransgenic mice.
) were used as a control. Using the first
paradigm, in which CREB overexpression is on throughout development, we
found that basal dynorphin expression was depressed in striatum of
CREB6-B bitransgenic mice relative to the +/ control mice (Fig.
8). In NSE-tTA × TetOp-CREB
bitransgenic (+/+) mice, dynorphin mRNA was decreased by 40 to 60%,
determined by either RPA or in situ hybridization, compared with
control +/ mice (Fig. 8A). Basal expression of dynorphin was also
significantly reduced in the striatum of CREB6-A bitransgenic mice
(data not shown). Induction of dynorphin mRNA in response to a
relatively high dose of amphetamine (15 mg/kg, i.p.) was also examined.
Levels of dynorphin mRNA were determined 3 h after amphetamine
administration. In both CREB6-B +/+ and +/ mice, amphetamine
increased dynorphin expression, but the induction relative to the basal
state was greater in the CREB6-B +/+ mice than in the control +/ mice
(Fig. 8B).
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Regulation of Cocaine-Induced Locomotor Activity in CREB
Bitransgenic Mice.
Because of the high level of CREB
overexpression in the dorsal striatum and nucleus accumbens and the
regulation of dynorphin expression by cocaine, we examined
cocaine-induced locomotor activity in the CREB bitransgenic mice. The
responses to a single dose of cocaine, as well as to repeated
administration of cocaine, which is known to produce locomotor
activation, were examined. For these studies, bitransgenic mice
maintained on doxycycline in the drinking water (CREB off) were
compared with bitransgenic littermates withdrawn from doxycycline at
weaning (CREB on). This paradigm was chosen for the behavioral studies
because all animals have exactly the same genotype. First, locomotor
activity after administration of saline was determined on 5 consecutive
days. This allows for analysis of baseline locomotor activity and
provides a period of time when the animals can habituate to the test
chambers. There was no significant difference between the CREB-on and
CREB-off bitransgenic mice when they were first exposed to the chamber (day 1) or upon subsequent exposures (Fig.
10). Administration of cocaine (10 mg/kg) increased locomotor activity on day 1 and this effect was
similar in both the CREB-on and -off groups. Repeated cocaine
administration on subsequent days resulted in a greater increase in
locomotor activity as expected in both groups. However, this effect was
significantly lower in the CREB-on group relative to the CREB-off
animals (Fig. 10). These findings are consistent with previous reports
demonstrating that viral-mediated CREB expression in the nucleus
accumbens decreases behavioral responses to cocaine (Carlezon et al.,
1998).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Characterization of NSE-tTA × TetOp-CREB Bitransgenic
Mice.
In the present study, we characterized three lines of
NSE-tTA × TetOp-CREB bitransgenic mice (CREB6-A, CREB6-B, and
CREB3-A lines). In all three lines, CREB was overexpressed in dorsal
striatum and nucleus accumbens (Table 1). In the CREB6-A line, CREB
overexpression was also observed in deep layers of cortex, whereas in
the CREB3-A line, CREB was overexpressed, albeit sparsely, in
hippocampal CA1 pyramidal and dentate gyrus granule cell layers (Figs.
2 and 3, Table 1). In the CREB6-B line, immunoblotting also
demonstrated overexpression of CREB in striatum and in cerebellum (Fig.
4A). Analysis of CREB mRNA and protein revealed that levels of
expression in dorsal striatum and nucleus accumbens are more prominent
in CREB6-B than in CREB6-A mice. These patterns of CREB expression in A
and B NSE-tTA lines are similar to those seen in TetOp-luciferase reporter mice crossed with these lines (Chen et al., 1998). This indicates that the expression pattern of the TetOp-driven target gene
is determined largely by the expression pattern of NSE-tTA. The results
also demonstrate that CREB overexpression can be turned off and on by
addition or removal, respectively, of doxycycline, an analog of
tetracycline. It is notable that a relatively low dose of doxycycline
(50 µg/ml in water) can inhibit CREB overexpression. The low dose
used could also explain the relatively short washout period required
for CREB expression after the removal of doxycycline. These findings
demonstrate that the tTA-TetOP system functions correctly in the CREB
overexpressing bitransgenic mice.
Regulation of CREB/ATF-Like Transcription Factors in CREB
Bitransgenic Mice.
Within the CREB/ATF family of transcription
factors, CREB, CREM, and ATF1 are known to heterodimerize with one
another in vitro (Hai and Curran, 1991). In addition, these
transcription factors are regulated by the cAMP pathway and share
sequence homology (Brindle and Montominy, 1992; Lee and Masson, 1993).
Based on homology and sequence conservation, the CREB and
CREM genes are thought to have been formed by duplication of
an ancestral gene (Ruppert et al., 1992). Previous reports have
revealed that the CREB isoform and CREM are up-regulated in mice
that have a null mutation of CREB and - isoforms (Hummler et al.,
1994; Blendy et al., 1996). These results indicate that null mutation
of CREB/CREB results in compensatory expression of other members
of this transcription factor family. Here we studied what the
compensatory responses of CREB-related proteins (CREB, CREM, ICER, and
ATF1 isoforms) might be in response to overexpression of CREB in
specific brain regions, which has not yet been examined. The results
demonstrate that CREB and CREB isoforms are up-regulated in
CREB-overexpressing mice. Levels of ATF1 and ICER were not altered
significantly in the bitransgenic mice.
and CREB is consistent with previous
reports that activation of the cAMP pathway in most cell types results
in up-regulation of CREB gene expression (Meyer et al.,
1993; Walker et al., 1995; Coven et al., 1998). Sequence analysis
demonstrates that the CREB promoter contains three CRE elements that mediate the up-regulation of CREB gene
expression in response to stimulation of the cAMP system (Coven et al.,
1998). A similar mechanism could underlie the up-regulation of CREB and CREB observed in CREB-overexpressing mice. These findings seem
to contradict the up-regulation of CREB in the CREB/ null mutant mice (Blendy et al., 1996). However, increased CREB
expression in the latter study was reported to result from alternative
splicing and increased mRNA stability, not increased CREB
gene expression.
The mechanisms underlying the down-regulation of CREM (CREM, ,
and isoforms) in CREB-overexpressing bitransgenic mice seem to
involve another mechanism. There are no CRE elements in the promoter of
the CREM gene that control the expression of these isoforms (Molina et
al., 1993). However, previous studies have reported that CREM
expression during spermatogenesis is controlled by post-transcriptional
mechanisms, including alternative splicing and increased mRNA stability
(Foulkes et al., 1993). It is possible that down-regulation of the CREM
isoforms observed in the present study results from similar mechanisms.
There is a CRE in an intronic promoter in the CREM gene that
controls the induction of ICER (Molina et al., 1993). ICER contains
only the DNA binding domain and acts as a repressor of CRE-mediated
gene expression. It is surprising that expression of ICER is not
up-regulated in the CREB-overexpressing mice. However, ICER has been
shown to rapidly counter-regulate its own induction (Molina et al.,
1993), and it is possible that a similar mechanism occurs in the
CREB-overexpressing mice. The induction of ICER in response to
amphetamine, although not different between the single and bitransgenic
CREB mice, demonstrates the relatively normal responsiveness of this
inducible isoform in the transgenic animals.
Regulation of Dynorphin in CREB Bitransgenic Mice.
In
bitransgenic mice in which CREB is overexpressed throughout
development, we found that dynorphin expression was reduced relative to
the littermate monotransgenic control mice. In contrast, in
bitransgenic mice in which CREB overexpression is turned on only after
weaning, there is an increase in basal dynorphin expression and the
induction of dynorphin by amphetamine is increased relative to
littermate control mice expressing normal levels of CREB. The repression of basal dynorphin expression could represent a compensatory response to long-term overexpression of CREB. For example,
overexpression of CREB in cultured cells is reported to decrease
dynorphin promoter activity (Collins-Hicok et al., 1994). One possible
explanation is that in the absence of phosphorylation, the
overexpressed CREB could compete with endogenous, phosphorylated CREB
for the CRE sites and could thereby act as a repressor of dynorphin
gene expression. CREB could also compete for other activator proteins
(e.g., ATF-2, AP-1) and thereby repress dynorphin expression. Yet
another possibility is that overexpressed CREB could interact with
other regulatory elements in the prodynorphin gene, such as a
downstream regulatory element (DRE) and its binding protein
(DRE-antagonist modulator), which represses basal prodynorphin
transcription (Carrion et al., 1998, 1999).
).
Studies conducted in primary striatal cultures confirm the induction of
dynorphin gene expression by activation of the cAMP pathway and
demonstrate that this effect is mediated by three CRE elements in the
prodynorphin promoter (Cole et al., 1995). Administration of
amphetamine or cocaine is reported to increase the expression of
dynorphin in the striatum and nucleus accumbens (Hurd et al., 1992;
Daunais et al., 1993; Spangler et al., 1993; Cole et al., 1995;
Carlezon et al., 1998). In this study, we demonstrate that
administration of amphetamine increases dynorphin expression and that
this effect is greater in the CREB-overexpressing mice. Moreover, the
induction of dynorphin expression was observed in an area of the
nucleus accumbens in which the expression of CREB, as well as pCREB, is
greatest in the CREB bitransgenic mice. Taken together, the results
indicate that overexpression of CREB in bitransgenic mice increases
psychostimulant-induced expression of dynorphin. Moreover, the
repression of basal levels of dynorphin expression after long-term
expression of CREB, and then psychostimulant induction is consistent
with studies of the dynorphin promoter in vitro (Collins-Hicok et al.,
1994).
Cocaine-Induced Locomotor Activity Is Decreased in
CREB-Overexpressing Mice.
The dorsal striatum and nucleus
accumbens are targets of the mesolimbic dopamine system and are known
to play a prominent role in mediating behavioral responses to drugs of
abuse (Nestler and Aghajanian, 1997). Moreover, adaptations of CREB and
its target genes, such as dynorphin, in these regions have been
implicated in the chronic actions of cocaine and other drugs of abuse
(Guitart et al., 1992; Hurd et al., 1992; Spangler et al., 1993;
Widnell et al., 1994; Cole et al., 1995; Nestler and Aghajanian, 1997; Carlezon et al., 1998). The results of the present study are consistent with these reports and demonstrate that behavioral responses to cocaine
are altered in CREB bitransgenic mice. We found that repeated cocaine
treatment for three days or longer caused significantly less locomotor
activation in bitransgenic mice relative to monotransgenic littermate
control mice. In contrast, there was no difference in the response to
the first day of cocaine administration, or in response to saline
administration over 5 days of exposure to the test chambers. These
finding indicate that CREB overexpression in bitransgenic mice alters
the response to repeated cocaine administration, but not to acute
cocaine or basal levels of locomotor activity.
-opioid receptors on
dopaminergic terminals in striatum to decrease dopamine release and
thereby serve as a feedback inhibitor of dopamine transmission (see
Carlezon et al., 1998). Repeated cocaine administration results in a
greater induction of dynorphin, which could lead to a greater feedback
inhibition of dopamine transmission. Thus, the reduced locomotor
response could result from a greater up-regulation of dynorphin in
response to repeated cocaine treatment in the CREB bitransgenic mice.
This hypothesis must be further tested by analysis of dynorphin peptide
expression in the CREB bitransgenic mice. The reason for the delay in
the response is not clear, but it may be related to the requirement for
up-regulation of other components of the cAMP cascade (e.g., adenylyl
cyclase or cAMP-dependent protein kinase) that are necessary for full
activation of the overexpressed CREB.
Conclusions. The results outlined in this article represent the initial characterization of inducible CREB overexpressing mice, and additional studies will be needed to further characterize the biochemical and behavioral phenotype of these animals. Studies are currently underway to identify other gene targets regulated by CREB, including glutamate and dopamine receptor subtypes, other neuropeptides, and neurotrophic factors. The behavioral phenotype of these mice will be further characterized in other models of drug abuse (e.g., place preference and self-administration), as well as in behavioral models of depression and anxiety. The results presented here indicate that the CREB over-expressing bitransgenic mice will be beneficial and unique tools to investigate the mechanisms underlying neural plasticity in response to a variety of behavioral and pharmacological stimuli.
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Acknowledgments |
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We acknowledge Rose Terwilliger and Antonia Dow for excellent histochemistry support.
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Footnotes |
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Received August 15, 2001; Accepted February 22, 2002
This work was supported by United States Public Health Service grants MH45481 and 2-PO1-MH25642, a Veterans Administration National Center for Post Traumatic Stress Disorder grant, and the Connecticut Mental Health Center.
Address correspondence to: Ronald S. Duman, Ph.D., Abraham Ribicoff Research Facilities, Yale University School of Medicine, 34 Park Street, New Haven, CT 06508. E-mail: ronald.duman{at}yale.edu
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Abbreviations |
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CRE, cAMP responsive element; CREB, cAMP response element binding protein; ATF, activation transcription factor; tTA, tetracycline transactivator; TetOp, tetracycline-operated promoter; NSE, neuron-specific enolase; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; BSA, bovine serum albumin; pCREB, phospho-cAMP response element binding protein; RPA, RNase protection analysis; RT, reverse transcriptase; bp, base pair(s); CREM, cAMP response element modulator; ICER, inducible cAMP early repressor; DRE, downstream regulatory element.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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FosB in the brain controls sensitivity to cocaine.
Nature (Lond)
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(middle) (Fitzgerald et al., 1996a
