Neuron, Vol. 48, 303–314, October 20, 2005, Copyright ª2005 by Elsevier Inc.DOI 10.1016/j.neuron.2005.09.023
Chromatin Remodeling Is a Key Mechanism
Underlying Cocaine-Induced Plasticity in Striatum
Arvind Kumar,1Kwang-Ho Choi,1,3William Renthal,1,3
Nadia M. Tsankova,1David E.H. Theobald,1
Hoang-Trang Truong,1Scott J. Russo,1
Quincey LaPlant,1Teresa S. Sasaki,1
Kimberly N. Whistler,1Rachael L. Neve,2
David W. Self,1and Eric J. Nestler1,*
1Department of Psychiatry
and Center for Basic Neuroscience
The University of Texas Southwestern Medical Center
5323 Harry Hines Boulevard
Dallas, Texas 75390
2Department of Psychiatry
Harvard Medical School
and McLean Hospital
115 Mill Street
Belmont, Massachusetts 02178
Given that cocaine induces neuroadaptations through
chromatin remodeling at specific gene promoters may
be a key mechanism. We show that cocaine induces
specific histone modifications at different gene pro-
moters in striatum, a major neural substrate for co-
caine’s behavioral effects. At the cFos promoter, H4
hyperacetylation is seen within 30 min of a single co-
caine injection, whereas no histone modifications
were seen with chronic cocaine, consistent with co-
caine’s ability to induce cFos acutely, but not chroni-
cally. In contrast, at the BDNF and Cdk5 promoters,
genes that are induced by chronic, but not acute, co-
caine, H3 hyperacetylation was observed with chronic
cocaine only. DFosB, a cocaine-induced transcription
factor, appears to mediate this regulation of the Cdk5
gene. Furthermore, modulating histone deacetylase
activity alters locomotor and rewarding responses to
cocaine. Thus, chromatin remodeling is an important
regulatory mechanism underlying cocaine-induced
neural and behavioral plasticity.
Acute and chronic exposure to cocaine alters gene ex-
pression in striatum, a major neural substrate for the
rewarding and locomotor-activating effects of psychos-
timulants and other drugs of abuse (Hyman and Mal-
enka, 2001; Nestler, 2001; Yuferov et al., 2003; Kalivas,
2004; Yao et al., 2004). The Fos family of transcription
factors has been implicated as important mediators of
some of these drug-induced changes in gene expres-
sion. Drugs of abuse rapidly but transiently induce
cFos and several other Fos family proteins in striatum
after acute administration (Graybiel et al., 1990; Young
etal., 1991). In contrast, chronic drug exposure desensi-
tizes the induction of most of these genes (Hope et al.,
1992; Daunais et al., 1993; Persico et al., 1993) and leads
instead to the selective accumulation of DFosB, a trun-
cated splice variant of the FosB gene (Hope et al.,
1994; Moratalla et al., 1996; Hiroi et al., 1997). Induction
of DFosB appears to mediate enhanced sensitivity and
drive for cocaine and other drugs of abuse in animal
models of drug addiction (Kelz et al., 1999; Nestler
et al., 2001; Colby et al., 2003; Peakman et al., 2003),
and progress has been made in identifying some of the
ioral plasticity (Ang et al., 2001; Bibb et al., 2001;
McClung and Nestler, 2003). However, the molecular
mechanisms underlying the in vivo regulation of specific
target genes by DFosB and other drug-induced tran-
scription factors remain unexplored.
of transcriptional mechanisms underlying chronic co-
caine action. Increasing evidence from other fields sug-
gests that transcription factors regulate target genes by
recruiting to the gene promoters two different classes of
enzymes: (1) enzymes that modify the core histones and
tion, phosphorylation, methylation, etc. (e.g., histone
acetyl transferases [HATs] and histone deacetylases
[HDACs]), and (2) enzymes that alter the structure of
chromatin by disrupting the nucleosome in an ATP-
and sucrose nonfermenting] complex) (for review, see
Jenuwein and Allis, 2001; Turner, 2002; Felsenfeld and
Groudine, 2003). These enzymes act on a target pro-
moter and thereby increase or decrease expression of
that gene. For example, the acetylation of histones H3
and H4, as well as phosphoacetylation of H3, at gene
promoters is linked to increased gene activity, while de-
acetylation is linked with suppression and silencing of
gene activity. Recent evidence indicates that histone
acetylation-deacetylation is regulated in adult neurons
in association with learning and memory (Guan et al.,
2002; Alarcon et al., 2004; Korzus et al., 2004; Levenson
et al., 2004) and in response to seizures (Huang et al.,
2002; Tsankova et al., 2004) and psychotropic drugs (Li
et al., 2004). Moreover, HDAC inhibitors have shown
promise as potential therapeutic agents in animal mod-
els of Huntington’s disease (Steffan et al., 2001; Hockly
et al., 2003; Mattson, 2003) and spinal muscular atrophy
support the view that the acetylation of core histones
may be a critical determinant of gene expression in
utilized chromatin immunoprecipitation (ChIP) assays to
investigate mechanisms of chromatin remodeling asso-
ciated with drug action. We chose gene promoters that
represent distinct drug mechanisms: cFos is activated
by acute cocaine, but desensitized by chronic cocaine,
while BDNF and Cdk5 are activated chronically but not
acutely. In addition, induction of the Cdk5 gene appears
to be mediated by DFosB, whereas BDNF is regulated
independently of DFosB by still unknown mechanisms.
3These authors contributed equally to this work.
Findings from this study describe prominent alterations
in histone modifications and other mechanisms of chro-
matin remodeling in striatal neurons that are associated
with cocaine regulation of specific genes and directly
implicate these mechanisms in cocaine-induced neural
and behavioral plasticity.
Quantification of Cocaine-Induced Histone
Modifications by Chromatin Immunoprecipitation
To better understand the molecular actions of cocaine,
we studied histone modifications at the promoter re-
gions of the cFos, FosB, BDNF, and Cdk5 genes in rat
striatum after acute or repeated cocaine administration.
To clearly differentiate between acute and chronic co-
caine effects, we analyzed histone modifications at sev-
eral different time points after cocaine administration.
Striatal punches were fixed in 1% formaldehyde to
chromatin was then sheared to fragments of w500 bp in
length via sonication. We then performed ChIP assays
with antibodies against acetylated H4, acetylated H3,
or phosphoacetylated H3 and quantified the amount of
DNA associated with the modified histones using real-
Several controls were performed to confirm the spec-
ificity and validity of the assay (Figure 1). To control for
the specificity of antibody binding, we immunoprecipi-
tated chromatin samples with nonimmune IgG, which
precipitated negligible levels of the various genes stud-
us to measure acetylation of histones only at trans-
criptionally active regions in the genome where acetyla-
tion is present in vivo, we confirmed that acetylation is
absent in striatum at transcriptionally silenced genes,
such as the b-globin gene (Figure 1A), which is inacti-
vated within neurons early in development, and on
genes (e.g., tyrosine hydroxylase) whose expression is
Figure 1. Use of Chromatin Immunoprecipitation and Real-Time PCR to Measure Histone Modifications in Striatum of Cocaine-Treated and
(A) Levels of acetylated H4 (acH4) at the cFos and several other gene promoters in striatum of rats treated acutely with saline or cocaine and
analyzed 30 min later were quantified using real-time PCR, by comparing relative Ct values for the various genes of interest. Parallel experi-
ments, not shown, were carried out for acetylated and phosphoacetylated H3. Ct values of immunoprecipitated samples (saline control versus
cocaine) were normalized to Ct values obtained from ‘‘input’’ or nonimmunoprecipitated DNA. The figure shows increased levels of the cFos
promoter in anti-acetylated H4 immunoprecipitates after cocaine treatment (Ct values of 28.08 for control, and 26.68 for cocaine, indicating
a w2.5-fold increase with cocaine). Immunoprecipitation with nonimmune IgG yielded much higher Ct values (w37) indicating almost no pre-
cipitation of the cFos promoter in the absence of a specific antibody. In contrast to the cFos promoter, acute cocaine, chronic cocaine, or self-
administered cocaine (Self Adm), had no effect on levels of several other gene promoters in anti-acH4 or anti-acH3 immunoprecipitates, in-
cluding the b-tubulin promoter (A and B), the b-globin promoter (A), the tyrosine hydroxylase (TH) promoter (C), and the H4 promoter (D).
Data are expressed as mean 6 SEM (n = 6–8).
highinother brainregionsbut absentfrom striatum(Fig-
ure 1C). Finally, we measured levels of histone acetyla-
tion in striatum at the promoters of the b-tubulin (Figure
1B)andcorehistone H4genes(Figure 1D),whichareex-
pressed inadult striatumbut have expression levelsthat
are unchanged by acute and chronic cocaine (McClung
els of histone modifications at these two promoters did
not differ between control and acute or chronic cocaine-
treated animals. These findings indicate that the ob-
served changes in histone modifications at the cFos,
FosB, BDNF, and Cdk5 genes, presented below, are
not global, but are limited to genes with expression
that varies as a result of cocaine exposure.
Cocaine Regulation of Histone Modifications
at the cFos and FosB Promoters
We first assayed histone modifications at the cFos pro-
moter after acute and chronic cocaine. As shown in Fig-
ures 2A–2C, we found that acute cocaine causes the
robust induction of both H4 acetylation and H3 phos-
phoacetylation, but not of H3 acetylation alone, at the
cFos promoter as early as 30 min after drug administra-
tion. All of these effects reverted to control levels within
3 hr and remained unchanged 24 hr after drug adminis-
tration. Acute administration of amphetamine, a related
psychostimulant, caused a similar pattern of histone
modifications at the cFos promoter (data not shown).
These findings of rapid and dramatic histone modifica-
tions at the cFos gene in striatum in response to acute
psychostimulants are consistent with the fact that
cFos is an immediate early gene, which is known to be
activatedveryrapidly afteracuteadministration ofthese
drugs of abuse (Graybiel et al., 1990; Young et al., 1991;
Hope et al., 1992).
Very different results were obtained after a course of
chronic cocaine administration. Under these conditions,
levels of H4 and H3 acetylation (Figure 2D) and of H3
phosphoacetylation (data not shown) were unchanged
compared to saline controls, both 1 hr and 24 hr after
the last cocaine dose. The lack of induction of histone
modifications at the cFos promoter at the 1 hr time point
represents the complete desensitization of histone
acetylation after chronic exposure to cocaine and is
consistent with earlier observations that induction of
cFos mRNA is similarly desensitized after chronic psy-
chostimulant exposure (Hope et al., 1992; Daunais et al.,
1993; Persico et al., 1993).
We next examined cocaine regulation of histone mod-
ifications at the FosB gene promoter. Like cFos, full-
length FosB mRNA is induced rapidly and transiently
by acute cocaine, but unlike cFos shows only partial—
ministration (Hope et al., 1992, 1994; Chen et al., 1995).
Figure 2. Histone Modifications at the cFos Promoter in Striatum after Acute or Chronic Cocaine Administration
ChIP was performed on striata of cocaine-treated and control rats with anti-acetylated H4 (acH4), anti-acetylated H3 (acH3), or anti-phosphoa-
cetylated H3 (pacH3) antibodies, and levels of the cFos promoter in the immunoprecipitates were measured with real-time PCR. (A)–(C) show
acH4, acH3, and pacH3 at 30, 90, or 180 min after a single injection of cocaine or saline. (D) shows these histone modifications 1 or 24 hr after
a chronic course of cocaine or saline. Data are expressed as mean 6 SEM (n = 6–8). *p < 0.05, Student’s t test.
Chromatin Remodeling and Cocaine Action
The FosB gene was also of interest, because it encodes
DFosB, whose mRNA is induced rapidly and transiently
by acute cocaine and shows only partial desensitization
with chronic cocaine, but DFosB protein accumulates
uniquely during a course of chronic cocaine administra-
tion due to the stability of the protein (Chen et al., 1995,
1997; Nestler et al., 2001). Analysis of histone modifica-
tions at the FosB promoter showed similarities to those
observed at the cFos promoter after acute cocaine (Fig-
ure 3A), but the modifications were strikingly different
after chronic cocaine (Figure 3B). Thus, we observed in-
duction of H4 acetylation, but no change in H3 acetyla-
tion or phosphoacetylation (data not shown), at the
FosB promoter after acute cocaine administration. In
contrast, the FosB promoter showed significant induc-
tion of H3 acetylation, but not H3 phosphoacetylation
or H4 acetylation, at 1 hr after the last dose of a chronic
cocaine regimen (Figure 3B). These data are consistent
with earlier observations that induction of FosB and
DFosB mRNA, in contrast to cFos mRNA, only partially
desensitizes with chronic cocaine administration (Chen
et al., 1995).
To determine whether these effects of chronic investi-
gator-administered cocaine on histone modifications at
diction, we determined whether similar modifications
occur after chronic self-administration of the drug. In-
deed, chronic self-administration of cocaine in rats was
associated with no histone modifications at the cFos
promoter (data not shown), but with dramatic induction
moter (Figure 3C).
Cocaine Regulation of Histone Modifications
at the BDNF and Cdk5 Promoters
Unlike cFos, which is induced by acute, but not chronic,
cocaine, BDNF and Cdk5 are examples of genes that
are induced by chronic, but not acute, cocaine adminis-
tration (Bibb et al., 2001; Grimm et al., 2003; McClung
and Nestler, 2003). Whereas a single dose of cocaine
did not produce any significant histone modifications
at the BDNF or Cdk5 promoters (data not shown), we
found dramatic regulation at these promoters after
chronic cocaine (Figure 4). Chronic cocaine robustly in-
duced levels of H3 acetylation at the BDNF and Cdk5
promoters in striatum, with no changes seen in H4 acet-
ylation. Strikingly, this induction of H3 acetylation was
observed 24 hr after the last dose of cocaine (Figures
Figure 4. Histone Modifications at the BDNF
and Cdk5 Promoters
Chronic Cocaine Administration
ChIP was performed on striata of chronic co-
caine- and saline-treated rats (used 24 hr af-
ter the last injection) with anti-acetylated H4
(acH4), anti-acetylated H3 (acH3), or anti-
phosphoacetylated H3 (pacH3) antibodies,
and levels of the BDNF (A) or Cdk5 (B) pro-
moters in the immunoprecipitates were mea-
sured using real-time PCR. Results are not
shown for pacH3, since Ct values were virtu-
ally as high as those seen for nonimmune IgG
and were unaffected by cocaine exposure.
There was also no effect of acute cocaine on
association of the BDNF or Cdk5 promoters
with these various histone modifications
(data not shown). Data are expressed as
mean 6 SEM (n = 6–8). *p < 0.05, Student’s
Figure 3. Histone Modifications at the FosB Promoter in Striatum after Acute or Chronic Cocaine Administration
ChIP was performed on striata of cocaine-treated and control rats with anti-acetylated H4 (acH4), anti-acetylated H3 (acH3), or anti-phosphoa-
cetylated H3 (pacH3) antibodies, and levels of the FosB promoter in the immunoprecipitates were measured with real-time PCR. (A) shows
histone modifications 1 hr after acute cocaine or saline; (B) shows histone modifications 1 hr after the last injection of chronic cocaine or saline;
and (C) shows histone modifications 24 hr after the last cocaine or saline self administration (Self Adm). Results are not shown for pacH3, since
Ct values were virtually as high as those seen for nonimmune IgG and were unaffected by cocaine exposure. Data are expressed as mean 6
SEM (n = 6–8). *p < 0.05, Student’s t test.
4A and 4B), suggesting relatively long-lived adaptations
at these promoters in contrast to what was seen with
acute cocaine at the cFos promoter. An even more dra-
matic selective increase in H3 acetylation levels was
seen at the BDNF and Cdk5 promoters 24 hr after the
last session of chronic self-administration of cocaine
To examine the longevity of increased H3 acetylation
at the BDNF and Cdk5 gene promoters, we analyzed
striatal samples after 1 week of withdrawal. Interest-
ingly, the level of acetylated H3 at the BDNF promoter
was strikingly high at this time point (14.1 6 3.6-fold;
p < 0.05) compared to the w3-fold increase seen after
1 day of withdrawal. Thus, histone modifications at the
BDNF promoter are very long-lasting and even build
over the course of withdrawal. This pattern of H3 acety-
lation at the BDNF gene is interesting in light of findings
thatlevelsofBDNF proteininthisregion increasefurther
as a function of withdrawal (Grimm et al., 2003). In con-
trast, the level of acetylated H3 at the Cdk5 promoter
remained elevated after 1 week of withdrawal (1.9 6
icant. This suggests that histone modifications at the
Cdk5 gene are beginning to return to normal at this
Role for DFosB in Cocaine Regulation
of the Cdk5 Gene
Previous work has suggested that Cdk5 is one target
gene through which DFosB produces its long-lasting ef-
fects on hippocampal and striatal function (Bibb et al.,
2001; Chen et al., 2000; Peakman et al., 2003). This is
based partly on the observations that DFosB overex-
pression induces Cdk5 in striatum, while a dominant
negative antagonist blocks cocaine induction of Cdk5.
Cdk5 is a particularly interesting target gene, since re-
cent evidence has implicated it in mediating the ability
of chronic cocaine to induce the outgrowth of dendritic
arborizations of striatal neurons (Norrholm et al., 2003).
However, direct evidence that DFosB’s regulation of
Cdk5 in striatum represents a direct effect on the
Cdk5 gene promoter has been lacking. Indeed, there re-
mains some controversy in the field as to whether
DFosB regulates gene expression directly or, rather, in-
directly by interfering with the actions of other transcrip-
tion factors (see Andersson et al., 2001; Nestler et al.,
2001). It was, therefore, important to use ChIP assays
to gain further insight into mechanisms governing regu-
lation of the Cdk5 gene in vivo. First, we carried out ChIP
assays using an anti-DFosB antibody to immunoprecip-
itate chromatin fragments from striata of control and
chronic cocaine-treated rats. As predicted, we found
a w3-fold increase in the association of DFosB with
the Cdk5 gene promoter after chronic cocaine exposure
(Figure 5A). In contrast, there was no evidence of
increased association of DFosB with the BDNF gene
(data not shown), consistent with reports that BDNF ex-
pression is not affected by DFosB in striatum (McClung
and Nestler, 2003). We know that DFosB, and not full-
length FosB, is the protein associating with the Cdk5
gene promoter, since no effect was seen using an anti-
body that recognizes full-length FosB but not DFosB.
These findings demonstrate that, after chronic cocaine
administration, the Cdk5 gene in striatum shows signs
of transcriptional activation (i.e., increased H3 acetyla-
tion) as well as increased association with DFosB.
Second, we analyzed striatum from a line of bitrans-
genic mouse that inducibly overexpresses DFosB with
usesthetetracyclinegene regulation systemtomaintain
DFosB so that it is turned off during development and to
turn it on in striatum of adult animals. We found that in-
duction of DFosB in these mice, which induces Cdk5
mRNA and protein expression (Bibb et al., 2001), was
associated with a w3-fold increase in association of
DFosB with the Cdk5 promoter in striatum (Figure 5B).
In contrast, no effect was seen at the BDNF promoter
(data not shown). This further supports the notion that
DFosB interacts directly with the Cdk5 gene in striatum
Having established evidence that the Cdk5 gene is
activated in striatum in vivo by chronic cocaine and
that this effect may be mediated, at least in part, via
DFosB, we examined whether another class of chroma-
tin remodeling protein, SWI-SNF, is also involved. Brg1
is the core ATPase of this multisubunit chromatin
Figure 5. Role of DFosB in Regulation of the Cdk5 Gene Promoter in Striatum
(A) ChIP was performed on striata of chronic cocaine-and saline-treated rats (used 24 hr after the last injection) with anti-DFosB, anti-full length
FosB, or anti-Brg1 antibodies, and levels of the Cdk5 promoter in the immunoprecipitates were measured using real-time PCR. (B) ChIP was
performed on striata of inducible bitransgenic mice either expressing (+DFosB) or not expressing (2DFosB) DFosB with anti-DFosB, anti-full
length FosB, or anti-Brg1 antibody, and levels of the Cdk5 promoter in the immunoprecipitates were measured with real-time PCR. Data are
expressed as mean 6 SEM; n = 6–8. *p < 0.05, Student’s t test.
Chromatin Remodeling and Cocaine Action
remodeling complex, which is implicated in the regula-
tion of many mammalian genes (Neely and Workman,
2002; Kadam and Emerson, 2003). We found that immu-
noprecipitation of chromatin fragments from striatum
with anti-Brg1 antibody revealed a several-fold greater
association of Brg1 with the Cdk5 gene promoter in
striatum of mice expressing DFosB compared with lit-
termate control mice (Figure 5B). A similar effect was
seen in striatum of chronic cocaine-treated rats, where
the association of Brg1 with the Cdk5 promoter was
several-fold higher compared with saline-treated con-
trols (Figure 5A). Together, these findings provide sev-
eral lines of important evidence that DFosB induces
the Cdk5 gene in the brain in vivo via direct activation
of the gene promoter.
In contrast to Cdk5, there was no change in the asso-
ciation of Brg1 with the BDNF promoter after chronic
cocaine (data not shown). These findings further indi-
cate that different genes activated in striatum by
chronic cocaine are activated via fundamentally distinct
mechanisms. These in vivo findings are consistent with
a recent report in cell culture, which shows distinct
SWI-SNF complexes, some containing Brg1 and others
containing different factors, directing promoter-specific
regulation of target genes (Kadam and Emerson, 2003).
Regulation of the Biochemical and Behavioral
Actions of Cocaine by the HDAC Inhibitor,
Levels of histone acetylation in intact cells can be
increased by use of HDAC inhibitors, which are being
developed as pharmaceutical treatments of diverse
disorders (see Discussion). We used sodium butyrate,
a widely used—albeit nonspecific—HDAC inhibitor,
validated in cultured cells and peripheral tissues, to de-
termine whether it can influence levels of histone acety-
lation inbrain.Wemeasured levelsofH3phosphoacety-
lation using immunohistochemical analysis of brain
sections in response to cocaine with and without the
co-administration of sodium butyrate. (We could not
use the antibodies to acetylated H4 or H3 because
they do not work for immunohistochemistry [data not
shown].) Systemic administration of sodium butyrate at
a dose of 200 mg/kg did not influence overall levels of
H3 phosphoacetylation in striatum (Figure 6). Cocaine
alone (at a dose of 15 mg/kg i.p.) caused a significant
Figure 6. Effect of the HDAC Inhibitor, Sodium Butyrate, on Biochemical Responses to Cocaine
Rats were treated with two i.p. injections, administered 15 min apart, and were used 30 min after the second injection. Rats received two saline
injections (D), sodium butyrate (200 mg/kg) followed by saline (B), saline followed by cocaine ([C], 15 mg/kg), or sodium butyrate (200 mg/kg)
followed by cocaine (A). For comparison, two additional groups of rats, administered saline followed by caffeine (10 mg/kg) or sodium butyrate
followed by caffeine, were analyzed. Brain sections were stained immunohistochemically with anti-phosphoacetylated H3 antibody followed
by a cy3-labeled secondary antibody (A–D) or HRP-tagged secondary antibody analyzed with DAB staining for quantitation (E). Results shown
in the figure are representative of multiple sections obtained from four rats in each treatment group. Bar graphs represent the mean 6 SEM.
*p < 0.05 compared to Sal/Sal, **p < 0.05 compared to Sal/Coc, Student’s t test. (F) and (G) show the levels of cFos mRNA quantitated by RT-
PCR and levels of phosphoacetylated H3 at the cFos promoter, by ChIP and real-time PCR, respectively. Sodium butyrate (200 mg/kg), given
in conjunction with cocaine (15 mg/kg), induced significantly higher levels of cFos mRNA (F) and phosphoacetylated H3 at the cFos gene (G) in
striatum compared to levels in rats given cocaine alone. Sodium butyrate by itself had no effect on cFos mRNA or H3 phosphoacetylation at
the cFos gene. Data are expressed as mean 6 SEM (n = 4–6 in each treatment group). *p < 0.05 compared to Sal/Sal, **p < 0.05 compared to
Sal/Coc, Student’s t test.
increase in H3 phosphoacetylation. Strikingly, adminis-
tration of sodium butyrate followed by cocaine caused
a more dramatic induction of H3 phosphoacetylation in
striatum (Figure 6). This synergistic interaction between
sodium butyrate and cocaine was apparent not only by
the increased number of cells positive for H3 phosphoa-
cetylation (Figure 6), but also by the increased intensity
onstrate that sodium butyrate augments the ability of
cocaine to induce histone modifications in striatum.
The effects seen in striatum were not apparent through-
in frontal cortex, septum, or other neighboring brain re-
gions (data not shown). The lack of regulation seen in
these other regions presumably represents a limit of de-
tection, since cocaine is known to alter gene expression
in some of these other brain regions (see Kalivas, 2004),
albeit to a lesser extent than instriatum, due to the lower
levels of the dopamine transporter (target of cocaine) in
these various regions.
The cocaine-induced increase in H3 phosphoacetyla-
tion in striatum was selective for this psychostimulant.
Caffeine, at a dose (10 mg/kg) that causes the same
level of locomotor activation as cocaine (Self et al.,
1996), failed to activate H3 phosphoacetylation in stria-
tum, even when it was injected in conjunction with so-
dium butyrate (Figure 6E). This finding also indicates
that increased phosphoacetylation of H3 in striatum is
not a consequence of increased locomotor activity
Having established that sodium butyrate promotes
cocaine-induced histone modifications in striatum, it
was of interest to study the effect of this HDAC inhibitor
on cocaine regulation of the cFos gene. We found that
administration of sodium butyrate 15 min prior to co-
caine administration increased the degree of induction
of cFos mRNA expression in striatum (Figure 6F). So-
dium butyrate alone had no effect. Similarly, sodium bu-
tyrate potentiated the ability of cocaine to induce asso-
ciation of phosphoacetylated H3 with the cFos gene
promoter (Figure 6G).
We next investigated whether the enhancement of co-
caine’s effects on histone modifications and gene regu-
lation in striatum by sodium butyrate is associated with
alterations in cocaine’s behavioral effects. To address
this question, animals were given sodium butyrate at
the lower dose of 100 mg/kg followed by cocaine
15 min later. This dose of sodium butyrate by itself
had no effect on the animals’ locomotor activity, but
enhanced the locomotor-activating effects of cocaine
(Figure 7). This effect was more apparent on the second
day of cocaine exposure, when sodium butyrate nearly
doubled the locomotor activation induced by cocaine
(Figures 7C and 7D).
Regulation of Cocaine Reward by HDAC
Inhibition or Overexpression
We used the conditioned place preference (CPP) para-
digm, in which animals learn to prefer an environment
paired with cocaine, to study the influence of histone
Figure 7. Effect of the HDAC Inhibitor, Sodium Butyrate, on Cocaine-Induced Locomotor Activity
Rats were habituated to the locomotor chambers for 30 min, were given sodium butyrate (NaBt, 100 mg/kg i.p.), and 20 min later were given
cocaine (15 mg/kg i.p.). This regimen was repeated the following day. Line graphs represent the number of ambulatory counts recorded in 5
min bins (mean 6 SEM). Bar graphs represent total activity. *p < 0.05 compared to saline/saline, #p < 0.05 compared to saline/cocaine. In
saline-cocaine and butyrate-cocaine groups, n = 16; n = 8 in saline-saline and butyrate-saline groups. Error bars indicate the mean 6 SEM.
Chromatin Remodeling and Cocaine Action
acetylation on cocaine reward. We first used the HDAC
inhibitor, trichostatin A (TSA), which is structurally dis-
tinct from butyrate and has been widely validated as
an HDAC inhibitor. As with butyrate, we found that sys-
temic administration of TSA enhances H3 phosphoace-
tylation seen in striatum in response to cocaine (data
not shown). As shown in Figure 8B, TSA administration
also increased the rewarding effects of cocaine, as indi-
cated by enhanced place conditioning to a moderate
cocaine dose. These findings further substantiate our
observations that HDAC inhibition promotes the behav-
ioral actions of cocaine.
To gain further insight into these phenomena, we
used a Herpes Simplex Virus (HSV) vector to overex-
press a particular subtype of HDAC, HDAC4, directly
in striatum. GFP was expressed as a control. We tar-
geted the ventral portion of striatum, the nucleus ac-
cumbens, which is most closely associated with the re-
warding effects ofcocaine. We chose HDAC4 because it
is highly expressed in brain and because we found in
preliminary experiments that it is highly expressed with-
in striatum. We found that overexpression of HDAC4 in
striatum dramatically decreased the rewarding effects
of cocaine in the place conditioning assay in compari-
son with control animals that received injections of
HSV-GFP (Figure 8A). We also showed that HDAC4
overexpression reduced levels of H3 acetylation in the
tissue, as would be expected (data not shown). As
with several other HSV vectors (e.g., see Carlezon et al.,
1998; Barrot et al., 2002), we found high levels of
HDAC4 overexpression in a w1 mm3area around the in-
jection site. HDAC4 overexpression was not associated
with any detectable toxicity, assessed by Nissl staining
and GFAP immunohistochemistry, beyond that seen
with injections of vehicle alone (data not shown).
Cocaine Induces Acetylation
and Phosphoacetylation of Histones at Specific
In this study we demonstrate that acute and repeated
cocaine administration induces prominent alterations
in the N-terminal tails of H3 and H4 associated with
specific gene promoters. The clinical relevance of the
chronic cocaine effects on histone modifications is
supported by our observations that the chronic self-
administration of cocaine results in equivalent regula-
tion. Our data reveal an interesting phenomenon: the
acute effects of cocaine are associated predominantly
with acetylation of H4 (cFos and FosB), whereas chronic
effects of cocaine are restricted to acetylation of H3
(FosB, Cdk5, and BDNF). In addition, phosphoacetyla-
tion of H3 was seen for the acute induction of the
cFos promoter only. This switch from H4 acetylation
acutely to H3 acetylation chronically holds even for indi-
vidual genes that are induced under both conditions: for
example, acute induction of FosB involves H4 acetyla-
tion, whereas chronic induction of the gene involves
H3 acetylation. These patterns of histone modifications
are generally consistent with those observed recently in
hippocampus in response to acute and chronic seiz-
ures: acute regulation is associated with H4 acetylation
and, for cFos, with phosphoacetylation of H3 as well,
while chronic regulation is associated with H3 acetyla-
tion (Crosio et al., 2003; Tsankova et al., 2004). To-
gether, these findings begin to provide a general
scheme of how histones are covalently modified in brain
in response to acute and chronic perturbation.
The phosphoacetylation of histone H3 (i.e., phosphor-
ylation at Ser10 and acetylation at Lys14) was seen only
for the cFos promoter. As stated above, this was the
case for cocaine in striatum, as well as for seizures
and for activation of several signaling pathways in hip-
pocampus (Crosio et al., 2003; Tsankova et al., 2004).
For all other promoters studied here and previously,
the Ct values from samples pulled down by anti-
phosphoacetylated H3 were very high, close to the Ct
values obtained with nonimmune IgG. This is true even
for other genes (e.g., FosB and BDNF), which, like cFos,
are considered immediate early genes on the basis of
their rapid induction. However, these other genes, while
induced rapidly, are not induced to nearly the same de-
gree or with the same rapidity as cFos. This suggests
that phosphoacetylation of H3 may be relatively specific
for the cFos gene and related to the unique rapidity and
magnitude of its induction. Earlier work has shown de-
sensitization of cFos after chronic administration of co-
caine or other drugs of abuse (Hope et al., 1992; Dau-
nais et al., 1993; Persico et al., 1993). We found that
this desensitization corresponds to the loss of H4 acet-
ylation and H3 phosphoacetylation seen at the cFos
promoter after the first cocaine exposure. In contrast,
at another Fos family promoter, FosB, cocaine contin-
ues to induce histone modifications (H3 acetylation)
even after prior chronic exposure to the drug, indicating
that the FosB gene still shows evidence of gene activa-
tion. This is consistent with our proposed model for
DFosB: that each drug exposure continues to induce
the FosB gene, which allows its stable gene product,
Figure 8. Effect of HDAC Overexpression or Inhibition on Reward-
ing Responses to Cocaine
Mice were injected with HSV-HDAC4 or HSV-GFP into the nucleus
accumbens and then assessed for place conditioning to cocaine
(5 mg/kg i.p.). In parallel experiments, mice were treated with TSA
(0.2 mg/kg i.p.) or DMSO vehicle prior to each cocaine dose during
place conditioning to cocaine (5 mg/kg). CPP score represents the
time (in seconds) that animals spend in the cocaine-paired chamber
posttraining versus pretraining. Overexpression of HDAC4 (n = 8)
dramatically blocked cocaine place conditioning compared to find-
ings in GFP-expressing control animals (n = 8). Conversely, TSA in-
creased cocaine place conditioning compared to vehicle-treated
controls (n = 12 in each group). Bar graphs represent the mean 6
SEM. *p < 0.05, Student’s t test. Note that the same dose of cocaine
in control animals; this is due to the known sensitizing effect of intra-
cranial surgery on this measure (see Carlezon et al., 1998).
DFosB, to accumulate in striatal neurons, where it be-
comes a key mediator of the drug-addicted state (Nes-
tler et al., 2001). The molecular switch from H4 acetyla-
tion at the FosB promoter acutely to H3 acetylation
basis of this switch is not known. We found no evidence
that acute or chronic cocaine administration alters
levels of specific HDACs in striatum (data not shown),
which suggests that the underlying mechanisms may
be quite complex.
Chromatin Remodeling at the BDNF and Cdk5
Promoters: Two Target Genes for Chronic Cocaine
The BDNF and Cdk5 genes show evidence of activation
after chronic cocaine administration, with no regulation
seen after acute cocaine. These observations are con-
sistent with the induction of these genes in striatum af-
ter chronic, but not acute, cocaine exposure (Bibb et al.,
2001; Grimm et al., 2003; McClung and Nestler, 2003).
Moreover, in contrast to FosB, where the increased lev-
els of H3 acetylation that occur with chronic cocaine in-
jections revert to control within a few hours, this same
histone modification at the BDNF and Cdk5 promoters
persists for at least 24 hr after the last cocaine injection.
This histone modification increases significantly further
at the BDNF gene after 1 week of withdrawal from co-
caine, whereas it begins to recover toward normal levels
at the Cdk5 gene. To our knowledge, these are the most
long-lived examples of drug-induced chromatin remod-
eling in brain published to date. The data show that
changes induced in chromatin structure persist long af-
ter cocaine is removed from the system and raise the
critical question of the underlying mechanisms in-
volved. We provide evidence here that, at least for the
Cdk5 gene, DFosB is one critical factor that may drive
some of these changes in chromatin remodeling. Such
long-lived changes in chromatin remodeling might
be one of the crucial mechanisms for cocaine-induced
neuroadaptations in striatum, which mediate the neu-
ral and behavioral plasticity that underlies cocaine
Direct Evidence that Cdk5 Is a Target Gene
for DFosB in Striatum In Vivo
Using ChIP, we provide direct evidence that, after
a course of chronic, but not acute, cocaine administra-
tion, there is the selective association of the Cdk5
gene promoter with DFosB. We show further that the in-
ducible overexpression of DFosB in striatum of adult bi-
expression in vivo (Bibb et al., 2001), causes increased
association of DFosB with the Cdk5 gene. Moreover, as-
sociation of DFosB with the Cdk5 gene is associated
with increased binding of Brg1-containing SWI-SNF
chromatin remodeling proteins to the Cdk5 promoter.
Together, these data support a scheme whereby the
gradual accumulation of DFosB, in response to chronic
cocaine administration, recruits chromatin remodeling
factors to its target genes like Cdk5. Such factors would
include the HATs that lead to H3 acetylation as well as
Brg1 and other components of the SWI-SNF complex.
These factors then act to reorganize histone-DNA inter-
actions to facilitate transcription (see Narlikar et al.,
2002). In contrast to Cdk5, we found no evidence for
increased association of DFosB or Brg1 with the BDNF
gene. This is consistent with the evidence that DFosB
does not regulate BDNF gene expression in striatum
(McClung and Nestler, 2003). Presumably, chronic co-
caineleads tothe long-term induction of BDNF via adis-
tinctmechanism. Onepossibility isthetranscriptionfac-
tor CREB, which is activated in a sustained fashion by
chronic cocaine and, upon its inducible overexpression
in striatum, can induce BDNF (McClung and Nestler,
It is worth emphasizing the technical advances made
possible by ChIP assays. Before the advent of this tech-
nology, studies of gene regulation in the brain in vivo
were severely limited. For example, it was possible to
demonstrate that levels of Cdk5 mRNA expression
could be increased by chronic cocaine and that this ef-
fect could be mimicked upon overexpression of DFosB
(Bibb et al., 2001). However, all studies of underlying
mechanisms would have to be performed in vitro, where
it was possible to show that the Cdk5 gene promoter
contains an AP1 site that binds DFosB and that DFosB
could activate the Cdk5 promoter in cell culture (Chen
et al., 2000). Now, with ChIP, it is possible for the first
time to study such transcriptional mechanisms in the
brain in vivo and understand, with increasing complex-
ity, how chronic cocaine administration leads to the
long-term regulation of its target genes.
HDAC Modulation Affects Biochemical
and Behavioral Responses to Cocaine
HDACinhibitors causeincreased acetylation ofhistones
in intact cells and are being explored as treatments for
several diseases, such as certain cancers (Marks et al.,
2001), Huntington’s disease (Steffan et al., 2001; Fer-
rante et al., 2003; Hockly et al., 2003), and spinal muscu-
lar atrophy (Chang et al., 2001; Minamiyama et al., 2004).
Weusedsodium butyrate, anonspecificHDACinhibitor,
to explore whether the changes in histone acetylation
observed in this study are relevant for cocaine’s bio-
chemicaland behavioral effects.Wefound thatadminis-
trationofsodium butyrate itselfdoesnot increaseglobal
levels of histone acetylation in striatum as measured im-
munohistochemically. Furthermore, while cocaine ad-
ministration alone increases H3 phosphoacetylation in
striatum, coadministration of sodium butyrate and co-
was observed for cocaine induction of both cFos mRNA
and increased association of acetylated H3 with the
following the coadministration of sodium butyrate and
cocaine. This establishes a very clear correlation be-
tween changes in histone acetylation and regulation of
gene expression in striatum. A similar correlation was
also evident for the behavioral effects of cocaine: ad-
ministration of sodium butyrate, at a dose that did not
alter locomotor activity, almost doubled the locomotor
response to cocaine.
The potentiation of cocaine’s behavioral effects by
HDAC inhibition was also seen in the conditioned place
preference assay, which provides a measure of cocaine
reward. Here, we showed that TSA, a more potent and
structurally distinct HDAC inhibitor compared to buty-
Importantly, overexpression of HDAC4, achieved via
Chromatin Remodeling and Cocaine Action
viral-mediated gene transfer, had the opposite effect:
a dramatic reduction in cocaine place conditioning.
cleus accumbens selectively. This experiment thereby
provides a critical causal connection between the regu-
lation of histone acetylation in this brain region specifi-
cally and an animal’s sensitivity to the behavioral effects
of cocaine. The fact that HDAC inhibition increases sen-
sitivity to cocaine, and HDAC overexpression produces
the opposite effect, is not an obvious finding, since co-
caine causes complex effects on gene expression,
with gene induction and repression both observed
(e.g., see McClung and Nestler, 2003). Our findings are
therefore interesting, because they suggest that stimu-
lation of gene transcription may be the predominant
mechanism for cocaine-induced behavioral plasticity.
In summary, we show here that individual histone
modifications are associated with different transcrip-
tional regulatory mechanisms after acute or chronic co-
eral histone modifications can take place in concert,
ton et al., 2000) or on tails of different histones (Turner
et al., 1992; Zeitlin et al., 2001; Sun and Allis, 2002).
Thus, specific combinations of histone modifications
could correspond to various states of remodeled chro-
matin and to the activation or repression of distinct
sets of genes. This has been termed the ‘‘histone
code’’ (Jenuwein and Allis, 2001; Turner, 2002). Results
of the present study raise the interesting possibility
distinct patterns of histone modifications, which, to-
getherwith ATP-dependent chromatinremodeling com-
plexes, induce unique patterns of chromatin remodeling
at the promoters of certain genes to regulate their tran-
scription. Such regulation provides a new layer of com-
plexity, at the molecular level, through which cocaine
produces neural and behavioral plasticity, and reveals
mechanisms for the treatment of cocaine addiction
that involve interfering with this plasticity.
All animal procedures were in accordance with the NIH Guide for the
Care and Use of Laboratory Animals and were approved by our IA-
CUC. Male Sprague-Dawley rats (initial weight, 250 g; Charles River,
Kingston, RI), Bl6/C57 mice (10–12 weeks old; JAX, Bar Harbor, ME),
and DFosB mutant mice off and on doxycycline (10–14 weeks old)—
under conditions that maximally induce DFosB (Kelz et al., 1999)—
were used in these studies. The animal colonies were kept on a 12 hr
light/dark cycle. Mice were housed in groups (four per cage), while
rats were housed in pairs; food and water were made available ad
Acute: rats were given a single i.p. injection of 20 mg/kg cocaine or
saline in equal volumes and killed 30 min to 24 hr later. Chronic:
rats were given an i.p. injection of 20 mg/kg cocaine or saline
once daily for 7 days and were killed 1 hr, 24 hr, or 7 days later. Self-
Administration: rats were trained for cocaine self-administration ac-
cording to published procedures for 15 days (Sutton et al., 2003).
Control rats could press the same levers, but they administered
only saline. The average intake of cocaine was 40 mg/kg/day. Ani-
mals were killed 24 hr after the last self-administration session.
Brains were removed rapidly from decapitated animals and pro-
cessed immediately for ChIP.
Chromatin Immunoprecipitation Assays
ChIP assays were performed on the basis of a protocol from Up-
state Biotechnology ChIP kit (Upstate Biotechnology, Lake Placid,
NY) and modified using published methods (Johnson and Bresnick,
2002; Wells and Farnham, 2002; Tsankova et al., 2004). The follow-
ing antibodies were used: anti-acetylated H3 raised against acety-
lated K9 and 14 H3; anti-acetylated H4 raised against acetylated
K5, 8, 12, and 16 H4; phosphoacetylated H3 raised against phos-
phorylated S10 and acetylated K14 H3; anti-Brg1; and nonimmune
rabbit and mouse IgG (all from Upstate Biotechnology). For DFosB
ChIP pulldown, first, the chromatin was incubated overnight with
mouse anti-FosB (C-terminal antibody; Center for Biomedical In-
ventions, University of Texas Southwestern Medical School), which
recognizes full-length FosB only, and then with by rabbit anti-FosB
(SC048; Santa Cruz Biotechnology, Santa Cruz, CA), which recog-
nizes the middle region of both full-length FosB and DFosB. How-
ever, since virtually all of the full-length FosB is pulled down by
the C-terminal antibody, the second immunoprecipitation brings
down DFosB only.
Quantification of Chromatin Immunoprecipitation
by Real-Time PCR
Levels of specific histone modifications, Brg1, or DFosB at each
gene promoter of interest were determined by measuring the
amount of that gene in chromatin immunoprecipitates by use of
real-time PCR (ABI Prism 7700; Applied Biosystems, CA) (see Table
S1 for primers used, in the Supplemental Data available with this ar-
ticle online). Input or total DNA (nonimmunoprecipitated) and immu-
noprecipitated DNA were PCR amplified in triplicate in the presence
of SYBR Green (Applied Biosystems, CA). Ct values from each sam-
ple were obtained using the Sequence Detector 1.1 software. Rela-
tive quantification of amplified template was performed as de-
scribed earlier by (Chakrabarti et al., 2002) and by the ABI manual
and with some modifications (see Tsankova et al., 2004 for details).
Each real-time PCR reaction, run in triplicate for each brain sample,
was repeated at least twice independently.
RNA Isolation and RT-PCR Detection of cFos mRNA
Rats were treated with saline-saline, saline-cocaine, butyrate-
saline, and butyrate-cocaine using 15 mg/kg cocaine and 200 mg/kg
sodium butyrate, both administered i.p. Striatal dissections were
obtained from decapitated rats, and one punch from each rat
from each of the four groups was used for isolating total RNA (re-
maining punches were used for ChIP assays). RNA was processed,
reverse-transcribed to cDNA using a first-strand synthesis kit (Invi-
trogen, Carlsbad, CA), and quantified using real-time PCR as de-
scribed (Tsankova et al., 2004). PCR reactions were repeated at
least two times independently.
Rats, randomized into four groups as outlined above (saline-saline,
sodium butyrate-saline, saline-cocaine, and sodium butyrate-
cocaine) were perfused with 4% paraformaldehyde 30 min after the
last injection, fixed brains were sectioned, and coronal sections
through striatum were stained immunohistochemically with an
anti-phosphoacetylated H3 polyclonal rabbit antibody (Upstate Bio-
technology) and a fluorescent Cy3 secondary antibody. Brain sec-
tions were also stained immunohistochemically using HRP-tagged
secondary antibody and diaminobenzidine (DAB), which yielded
equivalent results. DAB-stained sections were used to quantify
the number of phosphoacetylated H3-positive nuclei. Several sec-
tions from each animal were used for quantification. Only the anti-
phosphoacetylated H3 antibody was used, since the anti-acetylated
H3 or H4 antibodies did not work satisfactorily in immunohisto-
Locomotor Activity Assay
Spontaneous locomotor activity was monitored in a circular corridor
as described (Rahman et al., 2003). Animals were randomized into
four groups (saline/saline, sodium butyrate/saline, saline/cocaine,
and sodium butyrate/cocaine) and habituated to the locomotor ac-
tivity apparatus for 60 min. The next day, animals were habituated to
the apparatus for 30 min; then they received their first injection (ve-
hicle or sodium butyrate 100 mg/kg i.p.),and remained in the activity
apparatus for 20 min. They were then given the second injection (ve-
hicle or cocaine 15 mg/kg) and placed in the apparatus for an addi-
tional 60 min. The process was repeated for a second day. Data
were analyzed by two-way ANOVA (sodium butyrate 3 cocaine)
for main effects and interaction effect. For multiple comparisons,
one-way ANOVA with post hoc tests was performed.
Virus-Mediated Gene Transfer
HDAC4 cDNA, a gift from Dr. Eric Olson at UT Southwestern, was
sub-cloned and packaged into a published bicistronic HSV-GFP vi-
ral vector (Clark et al., 2002; Barrot et al., 2002). Activity of the vector
was verified by demonstrating HDAC4 expression in PC12 cells by
immunoblotting (data not shown). HSV-HDAC4 or HSV-GFP (used
as a control) vectors were injected into the mouse nucleus accum-
bens by stereotaxic surgery. Mice were used because a single injec-
tion of virus can infect a much larger portion of the nucleus accum-
bens and thereby induce a greater behavioral effect. The following
coordinates were used: +1.6 mm A/P, +1.5 mm lateral, and 24.4
mm D/V from bregma (relative to dura). Mice were anesthetized, vi-
rus was delivered bilaterally using a pump (at a rate of 0.1 ml/min for
a total volume of 0.5 ml on each side), and behavior was tested 3–5
days after viral injection, when transgene expression is maximal
(Barrot et al., 2002).
Conditioned Place Preference Behavior
Place conditioning was conducted as described (Rahman et al.,
2003). For the TSA experiments, mice received either TSA (2 mg/
kg of body weight i.p.) or DMSO 30 min prior to the saline or cocaine
(5 mg/kg) injection. Conditioning involved one pairing a day for
4 days with the test conducted on day 5. TSA was formulated as
2 mg/ml in DMSO and then diluted 1:5 with phosphate-buffered sa-
line. DMSO was diluted similarly for the vehicle control group. For
the HSV experiments, HSV-HDAC4- or HSV-GFP-injected mice
were paired four times on two consecutive days, followed by testing
on days 3–5 of viral injection.
Supplemental Data include a table and can be found with this article
online at http://www.neuron.org/cgi/content/full/48/2/303/DC1/.
We would like to thank Dr. Devanjan Sikder (UT Southwestern), for
technical advice and a critical reading of the manuscript, and
Dr. Eric Olson (UT Southwestern), for providing HDAC4 cDNA.
This work was supported by grants from the NIDA and NIMH.
Received: February 3, 2005
Revised: August 4, 2005
Accepted: September 19, 2005
Published: October 19, 2005
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