# Differential regulation of MeCP2 and PP1 in passive or voluntary administration of cocaine or food

Sarah Pol Bodetto, Pascal Romieu, Maxime Sartori, Carolina Tesone-Coelho, Monique Majchrzak, Alexandra Barbelivien, Jean Zwiller, Patrick Anglard

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Sarah Pol Bodetto, Pascal Romieu, Maxime Sartori, Carolina Tesone-Coelho, Monique Majchrzak, et al.. Differential regulation of MeCP2 and PP1 in passive or voluntary administration of cocaine or food. International Journal of Neuropsychopharmacology, 2014, 17 (12), pp.2031-2044. 10.1017/S1461145714000972. hal-05258405

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# Differential regulation of MeCP2 and PP1 in passive or voluntary administration of cocaine or food

Sarah Pol Bodetto, Pascal Romieu, Maxime Sartori, Carolina Tesone-Coelho, Monique Majchrzak, Alexandra Barbelivien, Jean Zwiller and Patrick Anglard

Laboratoire de Neurosciences Cognitives et Adaptatives (LNCA), UMR 7364 CNRS, Université de Strasbourg, Strasbourg, France

## Abstract

Cocaine exposure induces changes in the expression of numerous genes, in part through epigenetic modifications. We have initially shown that cocaine increases the expression of the chromatin remodeling protein methyl-CpG binding protein 2 (MeCP2) and characterized the protein phosphatase-1Cβ (PP1Cβ) gene, as repressed by passive i.p. cocaine injections through a Mecp2-mediated mechanism involving *de novo* DNA methylation. Both proteins being involved in learning and memory processes, we investigated whether voluntary cocaine administration would similarly affect their expression using an operant self-administration paradigm. Passive and voluntary i.v. cocaine intake was found to induce Mecp2 and to repress PP1Cβ in the prefrontal cortex and the caudate putamen. This observation is consistent with the role of Mecp2 acting as a transcriptional repressor of PP1Cβ and shows that passive intake was sufficient to alter their expression. Surprisingly, striking differences were observed under the same conditions in food-restricted rats tested for food pellet delivery. In the prefrontal cortex and throughout the striatum, both proteins were induced by food operant conditioning, but remained unaffected by passive food delivery. Although cocaine and food activate a common reward circuit, changes observed in the expression of other genes such as *reelin* and *GAD67* provide new insights into molecular mechanisms differentiating neuroadaptations triggered by each reinforcer. The identification of hitherto unknown genes differentially regulated by drugs of abuse and a natural reinforcer should improve our understanding of how two rewarding stimuli differ in their ability to drive behavior.

Received 1 November 2013; Reviewed 16 December 2013; Revised 12 May 2014; Accepted 19 May 2014;
First published online 17 June 2014

**Key words:** Cocaine self-administration, Food reward, Memory, Methyl-CpG binding protein MeCP2, Protein phosphatase-1.

## Introduction

Cocaine exposure is known to induce changes in gene expression in brain structures related to the reward system (Maze and Nestler, 2011). Persistent transcriptional modifications are likely to contribute to neural and behavioral adaptations triggered by the drug. We and others have shown that epigenetic regulators of gene expression are involved in cocaine-altered gene expression as well as in cocaine-related behavior (Cassel et al., 2006; Romieu et al., 2008; Host et al., 2011; Robison and Nestler, 2011). Among epigenetic factors involved, the methyl-CpG binding protein 2 (MeCP2) and histone deacetylase 2 (HDAC2) were identified as two interacting proteins implicated in transcriptional repression through

chromatin compaction (Jones et al., 1998; Nan et al., 1998). We have also demonstrated that cocaine triggers changes in DNA methylation by identifying the *Cdkl5* gene as the first gene methylated in adult brain in response to cocaine leading to its repression by Mecp2 (Carouge et al., 2010).

The critical involvement of Mecp2 and DNA methylation in the regulation of behavioral responses to psycho-stimulants was further illustrated by studies showing that: (i) decreased cocaine intake was observed following Mecp2 knockdown in the caudate putamen (CPu) (Im et al., 2010); (ii) amphetamine-conditioned place preference (CPP) and enhanced locomotion were not observed in Mecp2 mutant mice (Deng et al., 2010); (iii) overexpression of the DNA methyltransferase 3A (DNMT3A) in the nucleus accumbens (NAc) reduced cocaine CPP and enhanced depression-like behavior, and conversely its knockdown enhanced cocaine CPP and had anti-depressive effects (LaPlant et al., 2010). We have recently characterized the *β* catalytic subunit of protein phosphatase-type 1 (PP1Cβ), as an additional gene

<footnote>Address for correspondence: P. Anglard, Laboratoire de Neurosciences Cognitives et Adaptatives (LNCA), UMR 7364 CNRS, Université de Strasbourg, Strasbourg, France.
Tel.: 33 3 68 85 53 80 Fax: 33 3 68 85 19 58
Email: anglard@neuro-cnrs.unistra.fr, patrick.anglard@inserm.fr</footnote>
repressed by repeated passive cocaine administration in-
volving DNA hypermethylation and increased-Mecp2
binding at its promoter region *in vivo* (Pol Bodetto et al.,
2013). PP1 is involved in synaptic and structural plas-
ticity, mediates various signaling pathways (Ceulemans
and Bollen, 2004; Mansuy and Shenolikar, 2006) and
numerous studies have demonstrated that PP1 and its en-
dogenous inhibitor DARPP-32 (dopamine- and cyclic
amphetamine-regulated phosphoprotein) are key actors
in cocaine effects (Svenningsson et al., 2004). However,
although decrease in PP1 activity is required for cocaine
behavioral effects (Hiroi et al., 1999; Zachariou et al.,
2006), the role attributed to PP1 mainly results from the
use of PP1 endogenous inhibitors such as DARPP-32 or
Inhibitor-1 that do not discriminate between PP1C
isoforms.

PP1 being implicated in cocaine effects and in learning
and memory processes as a negative regulator of memory
(Genoux et al., 2002; Miller and Sweatt, 2007; Graff et al.,
2010; Koshibu et al., 2011), we investigated in the present
study the regulation of PP1Cβ expression by voluntary
drug intake using a cocaine self-administration paradigm,
as compared to passive cocaine administration. As drugs
of abuse and natural rewards activate common brain
regions and reward circuitry, we also investigated
whether food reinforcement could differentially regulate
the expression of PP1Cβ Mecp2 and HDAC2 compared
to cocaine. The aim of the study was to find out whether
these genes playing important roles in common cognitive
functions could respond and therefore contribute differ-
ently to specific neuroadaptations in order to better
understand the molecular mechanisms triggered by
each reward.

**Materials and methods**

Animals

Male Wistar rats (Janvier, France), weighing 160-180 g,
were housed individually in standard home cages, in a
temperature- and humidity-controlled room, under
an inverted 12 h/12 h light/dark cycle (lights on at
19:00 hours). Animals were acclimated to laboratory con-
ditions and handled one week before experimental proce-
dures. Each behavioral experiment started 3 wk after their
arrival in the laboratory and was conducted during
the dark period. Animals used for the cocaine operant
self-administration experiment (n=28) had *ad libitum*
access to food (12 mm pellets, 3.95 cal/mg, 4RF21 GLP,
Mucedola srl, Italy) and water, whereas animals used
for the food pellets operant self-administration exper-
iment (n=31) were food restricted. Their body weight
was progressively reduced to 85% and was maintained
at this level throughout the experiment. All procedures
involving animal care were conducted in compliance
with national laws and policies (Council directive
87848, 1987, Service Vétérinaire de la Santé et de la

Protection animale, permission 67-165 to JZ) and with in-
ternational guidelines (NIH publication 5586-23, 1985).

Surgery, apparatus and cocaine operant
self-administration

For technical details, see Supplement 1 (available online).

Apparatus and food pellets operant self-administration
procedure

Dark five-choice operant chambers (25.2×28×24 cm,
Bioseb, BP 89 92370 Chaville, France) placed in
sound-attenuated and ventilated enclosures were used
to test food pellets operant self-delivery. The curved
rear wall comprised nine contiguous 2.3 cm square hole,
located 2.2 cm above the grid floor. Each hole was
equipped with an infrared photocell beam to detect
nose pokes (NPs). The two farthest holes were used as
the active and the inactive holes, the other holes being
obstructed by a metal cover. NPs into both holes were
recorded. NPa and NPi were counterbalanced between
right and left position in each experimental group and
in the various groups of rats. The active hole was associ-
ated with the delivery of a food pellet (Precision-Pellets
45 mg, 3.62 cal/mg, 259901-PEL, Bioserv purified formula,
TSE systems, Germany) into a magazine located at the
opposite side of the chamber and equidistant from each
hole. Each chamber was automatically controlled by
Packwin software (Panlab S.P., Spain).

As for the cocaine operant self-administration exper-
iment, rats were divided into three different experimental
groups: 'SA pellets', 'Yoked pellets' and 'Yoked control'.
'SA pellets' and 'Yoked pellets' rats were initially given
access to food pellets in their home cage (10 pellets per
day during 5 consecutive days) to get used to the reinfor-
cer. In a first training phase, rats from each group were
placed in the chamber for 15 min and the panel of
the magazine in open position. During this phase, for
the 'SA pellets' and the 'Yoked pellets' groups, the maga-
zine was filled with 15 food pellets to familiarize rats to
eat the reinforcer, but it remained empty for the 'Yoked
control' rats. In a second training phase, rats received
two food magazine training sessions (20 min/session,
1 session/d), in which 20 food pellets were delivered ac-
cording to a variable time schedule (mean=60 s) for the
'SA pellets' and the 'Yoked pellets' groups. No pellet
was delivered for the animals of the 'Yoked control'
group. The house-light was turned off during this
phase. On the first session, the magazine was blocked
in order to maintain the food magazine open. For the
second session, as for the sessions of the food pellets
operant self-administration procedure, rats had to push
the panel to retrieve the food pellet.

A procedure similar to that used for the cocaine oper-
ant self-administration experiment was carried out. 'SA
pellets' rats were first submitted to a FR1 schedule of re-
inforcement during daily 2 h sessions for 4 d, and then to
<table><caption>**Table 1.** Primer sequences</caption><thead><tr><th>Gene</th><th>Primer sequence (5'-3')</th><th>Primer names</th><th>GenBank Acc. #</th></tr></thead><tbody><tr><td rowspan="2">PP1β</td><td>TGGTGGTATGATGAGTGTGGA</td><td>FA</td><td>BC062033.1</td></tr><tr><td>ACCTTTTCTTCGGTGGATTAG</td><td>RB2</td><td></td></tr><tr><td rowspan="2">HDAC2</td><td>CCCTCAAACATGACAAACCA</td><td>F</td><td>NM_053447.1</td></tr><tr><td>TGTCAGGGTCTTCTCCATCC</td><td>R</td><td></td></tr><tr><td rowspan="2">Mecp2</td><td>AAGTCTGGTCGCTCTGCTG</td><td>FP</td><td>DQ897369.1</td></tr><tr><td>TCTCCCAGTTACAGTGAAGTC</td><td>RQ</td><td></td></tr><tr><td rowspan="2">36B4</td><td>GTGCCTCACTCCATCATCAA</td><td>FA</td><td>NM_022402</td></tr><tr><td>TCCGACTCTTCCTTTGCTTC</td><td>RB</td><td></td></tr><tr><td rowspan="2">Reelin</td><td>GCAATCCATCCTTCCACCTC</td><td>RLN E57F</td><td>NM_080394</td></tr><tr><td>GCTTCACAACCCACCACAA</td><td>RLN E58R</td><td></td></tr><tr><td rowspan="2">GAD67</td><td>CAAGTTCTGGCTGATGTGGA</td><td>GAD67F</td><td>GQ468528</td></tr><tr><td>TCGGAGGCTTTGTGGTATGT</td><td>GAD67R</td><td></td></tr></tbody></table>

Primer sequences used for real-time qPCR are indicated with gene names and GenBank/NCBI accession numbers. F and R indicate forward and reverse primers, respectively.

a FR5 schedule during daily 2 h sessions for 6 d. When the required number of NPs into the active hole was reached, a food pellet was delivered. NPs into the inactive hole had no programmed consequence. No cutoff was applied concerning the number of food pellets delivered the rat was able to induce during each session. Two yoked groups were exposed to the same associated cues based on the 'SA pellets' group. Each rat from these groups was paired with a rat from the 'SA pellets' group. Rats from the 'Yoked pellets' group received passively the same amount of food pellets as that of 'SA pellets' paired rats, whereas no food pellet was delivered to rats from the 'Yoked control' group. Rats subjected to cocaine and food self-administration experiments were randomly distributed for mRNA and immunohistochemistry analyses, based on their homogeneous score in respect to self-administration.

### Reverse transcription-quantitative PCR analysis

RNA extraction, cDNA synthesis and real-time polymerase chain reaction (PCR) were performed as previously described (Ludwig et al., 2000; Carouge et al., 2010). Primers were from Sigma-Aldrich Co. and are listed in Table 1. For the detailed protocol of release experiments, see Supplement 2 (available online).

### Immunohistochemistry

Immunostaining for Mecp2 and PP1Cβ was performed using standard protocols described earlier (Host et al., 2011; Pol Bodetto et al., 2013). For the detailed protocol of immunohistochemistry, see Supplement 3 (available online).

### Statistical analysis

In behavioral experiments, the preference for the active vs. inactive hole was evaluated by comparing the percentage of NPs performed into the active hole (%NPa) to 50% considered as random. Daily and mean performances realized during the FR1 and FR5 periods were compared to 50%. The task acquisition was evaluated by comparing the %NPa to 80%, considered as the acquisition criterion. Daily and mean performances during both FR1 and FR5 periods) were compared to 80%. The number of NPs into the active (NPa) and the inactive hole (NPI) was evaluated with two-way analysis of variance (ANOVA). Reward self-delivery was assessed by one-way ANOVA with repeated measures in order to evaluate 'Day' effect. 'FR' effect was assessed by one-way ANOVA. In RT-qPCR and immunohistochemistry analyses, one-way ANOVA was performed to evaluate 'Group' effect. Newman-Keuls post-hoc was performed, if required. Significance was set at $p \le 0.05$. Data are expressed as means±S.E.M.

### Results

#### Acquisition of the operant conditioning task by cocaine or food pellets self-administering rats

For cocaine or food pellets self-administration, rats were submitted to a FR1 schedule of reinforcement for 4 d, and then to a FR5 schedule of reinforcement for 6 d. They self-administered about 110 cocaine injections per day during the whole experiment (Fig. 1a) and no significant changes were observed from one session to another whatsoever the FR schedule considered (FR1 vs. FR5). Similarly, rats self-delivered about 150 food pellets per day (Fig. 1e), except for the first day of each schedule of
<figure><img src="image_3.png" /><figcaption>Fig. 1. Cocaine (a–d) and food pellets (e–h) operant self-administration. The white and gray areas materialize the FR1 and the FR5 schedules of reinforcement, respectively. Number of cocaine injections self-administered by ‘SA cocaine’ rats (a) and number of food pellets self-delivered by ‘SA pellets’ rats (e). (b, f) Percentage of nose pokes (NPs) in the active hole (NPa). Chance level is represented by a dotted line at 50% NPa. (c, g) Number of NPs in the active hole (NPa) and in the inactive hole (NPI) is represented by circles and triangles, respectively. (d, h) Percentage of NPs in the active hole (NPa) performed during the valid period. Circles and triangles refer to NPa and NPI, respectively. Black circles and triangles represent rats self-administering cocaine or self-delivering food pellets, while gray ones represent yoked rats receiving passively the same amount of cocaine or food pellets. White circles and triangles correspond to yoked rats receiving either NaCl instead of cocaine or no food pellets. Data represent the mean±s.e.m., *n*=8–10 per group for the cocaine experiment (ABCD) and *n*=8–12 per group for the food pellets one (EFGH).</figcaption></figure>

reinforcement, and this number was not altered by the FR
schedule. The performance came from the rapid prefer-
ence for the active hole, as the percentage of NP in the ac-
tive hole (NPa) associated with cocaine (Fig. 1b) or food
(Fig. 1f) self-delivery was above 80%, which is highly dif-
ferent from 50%. In cocaine self-administering rats, the ac-
quisition criterion of 80% was achieved after the switch
from FR1 to FR5 schedule of reinforcement at day 6
(Fig. 1b). In rats self-delivering food pellets, it was already
observed during the FR1 schedule (Fig. 1f). In contrast,
rats from all yoked groups (Fig. 1b, f) displayed no prefer-
ence for the active or the inactive hole in none of the ses-
sions, as the percentage of NPa did not significantly differ
from 50%. Accordingly, no significant difference in the
number of NPa and NPI performed by yoked rats was
noticed (Fig. 1c, g). Surprisingly, yoked rats from the
food pellets experiment made almost no NPs at all
(Fig. 1g), as compared to yoked rats from the cocaine ex-
periment (Fig. 1c). This discrepancy may be attributed to
the nature of the reinforcer either administered or deliv-
ered, or from environmental factors such as enclosure
background. Contrary to yoked animals, rats self-
administering cocaine made much more NPa than NPI es-
pecially when submitted to the FR5 schedule (Fig. 1c), as
those performing food operant conditioning during both
schedules of reinforcement (Fig. 1g). Taken together, the
data show that only self-administering rats differentiate

the hole associated with cocaine or food pellets delivery
from the inactive hole, and therefore fulfill behavioral ac-
quisition rules. Rats self-administering cocaine (Fig. 1d)
made much more NPa during the time-out period without
any reinforcement than those self-delivering food pellets
(Fig. 1h) during both FR schedules of reinforcement.
Indeed, at the end of the experiment, only 30% NPa of
rats self-administering cocaine were made during the
valid period, whereas about 75% NPa of rats self-delivering
food pellets were made during the valid period.

Passive and voluntary cocaine intake similarly regulate
Mecp2 expression in brain structures of the reward
system

To evaluate the effect of cocaine and its associated cues
on *Mecp2* expression, we first compared rats receiving
passive cocaine injections to rats receiving passive saline
injections. Cocaine operant conditioning effect was also
addressed by comparing rats receiving passive cocaine
injections to rats self-administering cocaine. *Mecp2* ex-
pression was investigated by RT-qPCR (Fig. 2a) and by
immunohistochemistry (Fig. 3a–e) 24 h after the beginning
of the last session of cocaine self-administration. No dif-
ference was observed in *Mecp2* mRNA levels in none of
the experimental groups neither in the PFCx, nor in the
CPu (Fig. 2a). However, the number of Mecp2 positive
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

**Fig. 2.** Methyl-CpG binding protein 2 (*Mecp2*) mRNA expression in the prefrontal cortex (PFCx) and the caudate putamen (CPu) in response to passive vs. voluntary cocaine or food pellets delivery. The effects of cocaine or food reward and that of operant conditioning on *Mecp2* mRNA level were evaluated by quantitative RT-PCR 24 h after the beginning of the last session of cocaine (a) or food pellets administration (b) in the medial part of the PFCx and in the CPu, as indicated. The effect of passive cocaine or food pellets intake was investigated by comparing either ‘Yoked cocaine’ (n=5) to ‘Yoked control’ rats (n=4) (a) or ‘Yoked pellets’ (n=6) to ‘Yoked control’ rats (n=4) (b), whereas the effect of voluntary intake was evaluated by comparing either ‘SA cocaine’ (n=5) to ‘Yoked cocaine’ rats (a) or ‘SA pellets’ (n=5) to ‘Yoked pellets’ rats (b). The amount of *Mecp2* transcript was normalized to that of 36B4 mRNA. Data represent the mean±S.E.M. Statistical analysis performed was one-way analysis of variance (ANOVA).

cells was increased in these two brain structures in re-
sponse to passive cocaine injection, as illustrated in
the PFCx (Fig. 3b) and in the CPu (Fig. 3c), but not in
the NAc subregions (Fig. 3d). There was no difference
in the number of Mecp2 immunoreactive cells between
rats receiving cocaine passively and self-administering
rats, in none of the brain structures analyzed (Fig. 3b-e).
Our data are consistent with the induction of Mecp2 pro-
tein by cocaine reported in the dorsal striatum and not in
the NAc 24 h after the last session of self-administration
(Cassel et al., 2006; Im et al., 2010; Host et al., 2011) and
highlight the effect of cocaine reward together with its
associated cues, rather than that of the operant condition-
ing associated with drug self-administration.

Enhanced *Mecp2* expression by food in reward-related brain structures requires learning and memory processes involved in operant conditioning

Mecp2 mRNA expression was next investigated in re-
sponse to food pellets administration in the same brain
structures. The expression was not affected by food intake
when evaluated 24 h after the last session in none of the
experimental groups (Fig. 2b) neither in the PFCx, nor
in the CPu. In contrast, Mecp2 protein expression showed
a quite different pattern. Although the number of Mecp2
positive cells was increased by both passive and volun-
tary cocaine intake, this number evaluated following
passive food pellets delivery did not differ from that
observed in control rats receiving no food in all structures
examined (Fig. 3g-j). Unlike cocaine, food reward by it-
self was therefore not sufficient to alter Mecp2 expression.
However, the number of Mecp2 immunoreactive cells
was increased in food pellets self-delivering rats com-
pared to rats receiving food pellets passively, not only
in the PFCx (Fig. 3g) and the CPu (Fig. 3h), but also in
the NAcC (Fig. 3i) and the NAcS (Fig. 3j). Thus, unlike
cocaine reward and its associated cues that were sufficient
to induce Mecp2 expression, learning and memory
mechanisms involved in operant conditioning are
required to induce Mecp2 expression when a natural re-
ward is used.

HDAC2 expression is not altered by cocaine or food
self-administration

As Mecp2 interacts with HDAC2 in a complex inducing chromatin remodeling and transcriptional repression, we evaluated HDAC2 expression by RT-qPCR and by immunohistochemistry. In the CPu, the amount of HDAC2 transcript was not affected neither by cocaine (Fig. 4a), nor by food intake (Fig. 4d) and the number of immunoreactive cells and their staining intensity remained unaffected by cocaine (Fig. 4b) and by food intake (Fig. 4e, f). Similar observations were made in the PFCx and the NAc (Supplemental Figs 1 and 2), although cocaine was previously shown to induce HDAC2 expression in various brain structures including the CPu, when evaluated after a shorter time following the last cocaine administration (Cassel et al., 2006; Host et al., 2011). Taken together, the data suggest a transient dynamic HDAC2 regulation in response to both passive and voluntary cocaine intake.

PP1Cβ repression by passive and voluntary cocaine
administration correlates with Mecp2 expression in
specific brain structures of the reward system

We next checked whether enhanced levels of Mecp2
observed in the PFCx and the CPu in response to cocaine
intake translated into decreased expression of PP1Cβ.
PP1Cβ mRNA level was found to be strongly repressed
in both the PFCx (Fig. 5a) and the CPu (Fig. 5b) of rats
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Methyl-CpG binding protein 2 (Mecp2) protein expression in major brain structures of the reward system of rats self-administering cocaine or self-delivering food pellets. The expression of Mecp2 protein was evaluated by immunohistochemistry in brain structures of rats self-administering cocaine (a–e) or self-delivering food pellets (f–j) 24 h after the beginning of the last session of administration. Representative Mecp2 immunostaining performed in the caudate putamen (CPu) is shown for rats self-administering cocaine (A) and rats self-delivering food pellets (f). Scale bar applicable to all micrographs, 100 µM. Quantification of Mecp2 immunoreactivity was carried in the PFCx (b, g), the CPu (c, h), the nucleus accumbens core (NAcC) (d, i) and the nucleus accumbens shell (NAcS) (e, j). Data represent the mean±s.e.m., Yoked control, *n*=4; Yoked cocaine, *n*=5; SA cocaine, *n*=5 (b–e), Yoked control, *n*=4; Yoked pellet, *n*=6, SA pellet, *n*=6 (g–j). Statistical analysis performed was one-way analysis of variance (ANOVA) followed by Newman-Keuls *post-hoc*, when required. \*p<0.05, \*\*p<0.01, \*\*\*p<0.001. PCFx: prefrontal cortex.</figcaption></figure>

receiving cocaine compared to saline-treated rats. No difference in *PP1Cβ* mRNA level was observed in rats receiving cocaine voluntarily or passively neither in the PFCx, nor in the CPu (Fig. 5a, b). We then verified that protein expression reflected the corresponding mRNA expression. A decrease in the number of *PP1Cβ* immunoreactive cells was observed in the PFCx (Fig. 6b) and the CPu (Fig. 6c) of rats receiving cocaine, compared to rats treated with the vehicle. As for Mecp2 (Fig. 3d, e), no difference was noticed in *PP1Cβ* protein expression in the NAc subregions, neither in the NAcC, nor in the NAcS of these two groups of rats (Fig. 6d). As for *PP1Cβ* mRNA level, there was no effect of cocaine operant conditioning, as the number of *PP1Cβ* immunoreactive cells did not differ between the two groups of rats receiving cocaine, in none of the brain structures evaluated (Fig. 6b–e). The data therefore underline, as for Mecp2, the effect of cocaine reward and its associated cues on *PP1Cβ* expression and not that of cocaine operant conditioning. They also reveal an inverse correlation between

the levels of the two proteins in the PFCx and the CPu consistent with the role of Mecp2 acting as a repressor of *PP1β* (Pol Bodetto et al., 2013).

*PP1Cβ is only induced by voluntary food delivery in the PFCx, the CPu and the NAc*

We next investigated whether *PP1Cβ* expression could be differentially regulated by yoked food pellets delivery vs. food pellets delivered voluntarily. *PP1Cβ* mRNA level was induced in the CPu of food pellets self-delivering rats compared to rats with passive food delivery (Fig. 5d). A similar pattern was observed in the PFCx, even if significance was not reached (Fig. 5c). Contrary to cocaine self-administration experiments, we did not observe any difference in *PP1Cβ* mRNA level between the two yoked groups, neither in the PFCx, nor in the CPu, which shows that *PP1Cβ* expression is not regulated by natural reward alone (Fig. 5a, b vs. Fig. 5c, d). When measuring protein expression, we found an increase in
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** HDAC2 expression in the caudate putamen in response to passive *vs.* voluntary cocaine or food pellets delivery. The effect of cocaine (*a*–*c*) and food pellets (*d*–*f*) on HDAC2 expression was evaluated in the CPu 24 h after the beginning of the last session of administration by quantitative RT-PCR (*a*, *d*) and by immunohistochemistry (*b*, *c*, *e*, *f*). The amount of HDAC2 transcript was normalized to that of 36B4 mRNA. For ‘Yoked control’, ‘Yoked cocaine’ and ‘SA cocaine’ rats, data represent the mean±S.E.M., *n*=4–5 per group (AB), and for ‘Yoked control’, ‘Yoked pellets’ and ‘SA pellets’ rats, data represent the mean±S.E.M., *n*=4–6 per group (DE). Statistical analysis performed was one-way ANOVA. Immunohistochemistry of HDAC2 in the CPu (*c*, *f*), scale bar applicable to all micrographs, 100 µM.</figcaption></figure>

the number of PP1Cβ immunoreactive cells in the PFCx (Fig. 6g), the CPu (Fig. 6h), the NAcC (Fig. 6i) and the NAcS (Fig. 6j) of food pellets self-delivering rats compared to rats receiving food pellets passively. In agreement with the expression of PP1Cβ mRNA, food pellets reward alone did not alter the number of PP1Cβ immunoreactive cells in none of the brain structures evaluated, as their number did not differ between rats receiving food pellets passively and rats receiving no food pellet. Thus, although cocaine reward and its associated cues are sufficient to repress PP1Cβ, food reward alone does not affect its expression that is only induced upon food operant conditioning. Such an opposite regulation by cocaine and food underlines a striking different involvement of PP1Cβ in mediating the response to the two rewarding stimuli.

## Reelin and GAD67 genes are differentially regulated by cocaine or food

We next checked whether other direct Mecp2 target genes are also differentially regulated by cocaine and food. As Mecp2 is widely expressed in post-mitotic GABAergic neurons in the CPu and the frontal cortex (Cassel et al., 2006), the expression of *reelin* and glutamic acid decarboxylase 67 (GAD67) genes that are negatively regulated by DNA methylation-dependent mechanisms (Zhubi et al., 2014) was evaluated in these structures. Reelin is preferentially synthesized in GABAergic neurons in the cortex and the CPu (Costa et al., 2009) and GAD67 is a marker for GABAergic neurons (Rasmussen et al., 2007). The

expression of both genes was not affected by cocaine, independently of the administration mode (Fig. 7a, c). In contrast, a 2- to 3- fold increase was observed for both genes in response to passive food pellet delivery in the PFCx, but not following contingent delivery (Fig. 7b, d). In the CPu, a 2-fold decrease was observed in *reelin* expression in rats self-delivering food pellets relative to rats with passive food delivery or yoked control rats (Fig. 7b). *GAD67* expression remained unaffected by food pellet delivery (Fig. 7d). Thus, although *reelin* and *GAD67* expression is not affected by cocaine, their regulation by food appears to depend on the brain structure considered and the mode of administration and provides evidence for additional changes in gene expression occurring in response to each reinforcer.

As food restriction was used to motivate rats in operant-conditioning experiments, its effect on gene expression was addressed by comparing yoked control rats from the food pellet experiments that were food restricted, to yoked control rats from the cocaine experiments that were not. Baseline expression of *Mecp2*, *PP1Cβ*, *HDAC2*, *reelin* and *GAD67* genes was simultaneously evaluated in the PFCx and the CPu in both groups. In the PFCx, a 2.5-fold repression was observed for *reelin* upon food restriction, whereas no significant change was noticed for the other genes (Fig. 8a). In the CPu, except for *reelin*, the expression of all genes was affected by food restriction (Fig. 8b). A 2.4- to 3.8-fold induction was observed for *Mecp2*, *HDAC2* and *GAD67* genes, whereas a 2-fold repression was observed for *PP1Cβ*. That *Mecp2* was increased by food restriction
<figure><img src="image_8.png" /><figcaption>Fig. 5. PP1Cβ mRNA expression in the pre-frontal cortex (PFCx) and the caudate putamen (CPu) in response to passive vs. voluntary cocaine or food pellets delivery. The effects of cocaine and food reward and that of related operant conditioning on PP1Cβ mRNA level were evaluated by quantitative RT-PCR 24 h after the beginning of the last session of cocaine (a, b) and food pellets (c, d) administration in the medial part of the PFCx and in the CPu, as indicated. The amount of PP1Cβ transcript was normalized to that of 36B4 mRNA. Data represent the mean±S.E.M., n=4-6 per group (ABCD). Statistical analysis performed was one-way analysis of variance (ANOVA) followed by Newman-Keuls post-hoc, when required. \*p<0.05, \*\*p<0.001.</figcaption></figure>

may reflect a reward for food that has been reported in some Mecp2 mutant mice or in human males with hypo-morphic mutations overexpressing the gene or in females with milder variants of Rett syndrome that often become obese (Fyffe et al., 2008). HDAC2 induction by food restriction appears consistent with enhanced HDAC2 labeling intensity observed in food-restricted control animals (Fig 4c, f). This induction in the CPu suggests that it could participate to the 2-fold repression of PP1Cβ resulting from food restriction (Fig. 8b), consistent with a decrease in PP1Cβ immunoreactive cells in control food-restricted rats (Fig. 6f, h) relative to rats that were not food restricted (Fig. 6a, c).

Discussion

In the present report, we examined the effect of passive
vs. voluntary cocaine intake on Mecp2 and PP1Cβ ex-
pression. Cocaine-induced repression of PP1Cβ in the
PFCx and the CPu was inversely correlated with
Mecp2 levels and was similar, independently of the

administration mode, further supporting the role of
Mecp2 as a repressor of PP1Cβ. Accordingly, no change
in PP1Cβ expression was observed in the NAc in which
Mecp2 levels remained unaffected by cocaine adminis-
tration. When comparing cocaine effects to those of
food, a striking different expression pattern was observed
for both proteins. Despite food rewarding effects, their
levels remained unaffected by passive delivery in all
brain structures examined, as both proteins were induced
by food operant conditioning in the PFCx and throughout
the striatum. Their regulation differed depending the
brain structures considered, the nature of the reinforcer
and operant conditioning-related memory processes. As
the mesostriatal dopamine system is activated by food
and by drugs of abuse, the data provide new insights
into the mechanism by which two proteins involved in
cocaine reward (Zachariou et al., 2002; Romieu et al.,
2008; Feng and Nestler, 2010), or in food consumption
(Fyffe et al., 2008; Danielli et al., 2010; Cui et al., 2012)
are differentially regulated.

Food restriction induced significant changes in the
baseline gene expression of Mecp2, PP1Cβ, HDAC2 and
GAD67 in the CPu (Fig. 8b), indicating that the compari-
son between rats exposed to cocaine administration vs.
food delivery in this brain structure has to be taken
with caution, notably in respect to the regulation of
PP1Cβ by Mecp2. In contrast, food restriction did not af-
fect the basal expression of most genes in the PFCx
(Fig. 8a) and therefore allows a comparison between
their relative expressions in response to cocaine vs. food.
Mecp2 and PP1Cβ proteins being differentially regulated
by cocaine and food in the CPu and the PFCx, their regu-
lation in these structures is likely to result from different
molecular mechanisms.

Our data comfort the cocaine-induced increase in Mecp2
protein expression previously reported (Cassel et al., 2006;
Im et al., 2010; Host et al., 2011). However, this increase
observed 24 h after the last self-administration session
was not accompanied by any changes in Mecp2 mRNA ex-
pression. As Mecp2 transcriptional activation was pre-
viously observed when evaluated 2 h after the last
cocaine self-administration session (Host et al., 2011),
such a rapid activation is likely to be transient and whether
it could account for a delayed increase in protein ex-
pression remains to be determined. Alternatively, cocaine
may regulate Mecp2 protein by controlling either posi-
tively its translation or negatively its degradation after 24
h without affecting its transcription.

HDAC2 response to cocaine might also be transient, as
cocaine-induced HDAC2 mRNA and protein 2 h after the
last session of cocaine self-administration (Host et al.,
2011), whereas no increase was observed 24 h after the
last cocaine intake. The mechanism may contribute to
the delayed repression of PP1Cβ, consistent with
HDAC2 recruitment by Mecp2 in a complex silencing
transcriptional activity (Nan et al., 1998; Harikrishnan
et al., 2005). Alternatively, an HDAC-independent
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** PP1Cβ protein expression in major brain structures of the reward system of rats self-administering cocaine or self-delivering food pellets PP1Cβ protein expression was evaluated by immunohistochemistry 24 h after the beginning of the last session of cocaine (*a*–*e*) or food pellets (*f*–*j*) administration. Representative PP1Cβ immunostaining performed in the caudate putamen (CPu) is shown for rats self-administering cocaine (*a*) and rats self-delivering food pellets (*f*) together with control yoked experiments, as indicated. Scale bar of 100 µM is applicable to all micrographs. Quantification of PP1Cβ immunoreactivity was carried in pre-frontal cortex (PFCx) (*b*, *g*), the CPu (*c*, *h*), the nucleus accumbens core (NAcC) (*d*, *i*) and the nucleus accumbens shell (NAcS) (*e*, *j*), as indicated. Data represent the mean±S.E.M., *n*=4–5 per group (*b*–*e*), *n*=4–6 per group (*g*–*j*). Statistical analysis performed was one-way analysis of variance (ANOVA) followed by Newman-Keuls *post-hoc*, when required. \*p<0.05, \*\*p<0.001.</figcaption></figure>

mechanism of MeCP2-mediated repression has been reported to be particularly relevant in neurons that have high levels of methylated CpG and MeCP2 (Theisen et al., 2013).

Interestingly, the cocaine-induced Mecp2 expression was in phase with a decrease in PP1Cβ expression independently of the cocaine administration mode. As demonstrated previously, repeated passive cocaine administration repressed PP1Cβ and PP1Cγ expression through DNA hypermethylation and increased Mecp2 binding (Anier et al., 2010; Pol Bodetto et al., 2013). The down-regulation of PP1Cβ observed in cocaine self-administering rats, is therefore likely to result from DNA hypermethylation at the *PP1Cβ* gene followed by increased Mecp2 binding. Gene regulation by DNA methylation involves MBDs, DNMTs and TET proteins, the two latter controlling the balance between methylated CpG, hydroxymethylated CpG and non-methylated CpG (Guo et al., 2011). Drugs of abuse alter the expression of some MBD and DNMT proteins in the brain (Cassel et al., 2006; Anier et al., 2010; LaPlant et al., 2010;

Pol Bodetto et al., 2013), underlining their effects on DNA methylation mechanisms that has been actually reported for few genes. These genes are either repressed by DNA methylation and Mecp2, like *CDKL5* (Carouge et al., 2010), *PP1Cβ* (Pol Bodetto et al., 2013) and *PP1Cγ* or induced by DNA demethylation like *fosB* or *TACR3* (Anier et al., 2010; Barros et al., 2011). In contrast to *PP1Cβ*, the expression of *reelin* and *GAD67* genes was not affected by cocaine neither in the PFCx, nor in the CPu, independently of the administration mode (Fig. 7a, c). Although Mecp2 has been shown to bind to them, cocaine may trigger their demethylation, as a crucial role of global PFCx DNA hypomethylation in the rewarding effect of cocaine has been reported (Tian et al., 2012).

As *PP1Cβ* and *γ* genes are repressed by cocaine, the overexpression of Mecp2 protein in all brain structures examined (Fig. 3g–j), together with that of *PP1Cβ* mRNA (Fig. 5c, *d*) or protein (Fig. 6g–j) in food self-delivering rats was not expected. Similarly, *reelin* modulation also needs operant conditioning in the CPu, *PP1Cβ* being induced (Figs 5d and 6h) and *reelin* being
<figure><img src="image_10.png" /><figcaption>**Fig. 7.** *Reelin* and *GAD67* mRNA expression in the pre-frontal cortex (PFCx) and the caudate putamen (CPu) in response to passive vs. voluntary cocaine or food pellets delivery. The effects of cocaine and food reward and that of related operant conditioning on *reelin* and *GAD67* mRNA level were evaluated by quantitative RT-PCR 24 h after the beginning of the last session of cocaine (a, c) and food pellets (b, d) administration in the medial part of the PFCx and in the CPu, as indicated. The amount of *reelin* and *GAD67* transcripts was normalized to that of 36B4 mRNA. Data represent the mean±S.E.M., *n*=4–6 per group (ABCD). Statistical analysis performed was one-way analysis of variance (ANOVA) followed by Newman-Keuls *post-hoc*, when required. \*p<0.05, \*\*p<0.01, \*\*\*p<0.001.</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>**Fig. 8.** Comparison of *Mecp2*, *PP1Cβ*, *HDAC2*, *reelin* and *GAD67* gene expression in the pre-frontal cortex (PFCx) and the caudate putamen (CPu) in response to food restriction. Yoked control rats from the cocaine experiment that are not food restricted were compared to those from the food pellet experiment that are food restricted. Expression of the five genes was measured in the PFCx (a) and the CPu (b) by quantitative RT-PCR and normalized to the expression of 36B4. Data represent the mean±S.E.M. of the control group (n=4) and that of the food-restricted rats (n=4). Statistical analysis performed was one-way analysis of variance (ANOVA). \*p<0.05, \*\*p<0.01, \*\*\*p<0.001.</figcaption></figure>
repressed (Fig. 7b), revealing an opposite regulation pre-
viously reported in response to contextual fear condition-
ing (Miller and Sweatt, 2007). Considering the repression
of *PP1Cβ* by food restriction (Fig. 8b), this effect was
reversed by contingent food delivery (Figs. 5d and 6h).
In contrast, *Mecp2* mRNA was induced by food restriction
(Fig. 8b) and the percentage of Mecp2 immunoreactive
cells was by contingent food delivery (Fig. 3h), observa-
tions that appear consistent with the documented role
of Mecp2 in reward and memory. On the other hand,
most genes were not affected by food restriction in the
PFCx except *reelin* that was repressed (Fig. 8a). That
food delivery to food-restricted animals reverses its re-
pression (Fig. 7b) seems to make sense, but that it only
occurs in response to passive food delivery is intriguing
raising concerns about the mechanism by which a gene
usually repressed by Mecp2 and by DNA methylation
is induced together with Mecp2. On one hand, Mecp2
may act as a transcriptional repressor or activator
depending on its interaction with either a corepressor
such as SIN3A, or a coactivator such as CREB (Yasui
et al., 2007; Chahrour et al., 2008; Cortes-Mendoza
et al., 2013). On the other hand, food, unlike cocaine,
may not alter DNA methylation. In line with this hypoth-
esis, DNMT inhibitors (Anier et al., 2010; Han et al., 2010),
or Mecp2 repression (Im et al., 2010) or dysfunction
(Deng et al., 2010) negatively regulate cocaine or amph-
tamine intake, whereas administration of DNMT inhibi-
tors does not change sucrose intake (Warnault et al.,
2013). These data support the idea that a learning process
associated with natural reinforcer may not require de novo
DNA methylation that can block access to transcription
factors.

In learning and memory processes, the contribution of Mecp2 (Moretti et al., 2006; Pelka et al., 2006; Stearns et al., 2007; Na et al., 2012) and PP1 (Genoux et al., 2002; Miller and Sweatt, 2007; Graff et al., 2010; Koshibu et al., 2011) has been well documented and their control by cocaine may therefore alter these functions. As operant learning processes are involved in self-administration, it is rather surprising that neither Mecp2, nor PP1Cβ protein levels were modified by cocaine operant conditioning compared to passive cocaine injections. However, the effect of cocaine with its associated cues could hide that of operant conditioning. Thus, alteration in Mecp2 and PP1Cβ levels following passive cocaine intake may be sufficient to trigger the rewarding effect of the drug and to contribute to learning and memory processes related to the drug. Despite the rewarding and motivational effects of food resulting from restriction, food reward had no effect on Mecp2 and PP1Cβ protein expression without operant conditioning. Although cocaine operant conditioning did not affect their expression relative to passive cocaine intake, the expression of both proteins was increased in all examined brain structures of food pellets self-delivering rats. These results indicate that the mechanisms underlying operant conditioning

differ between drugs of abuse and natural rewards. It
therefore supports the concept that addiction may be, at
least in part, a learning disorder, consistent with previous
reports (Di Chiara, 1999; Hyman, 2005) and raises ques-
tions about how gene expression changes occurring in
the PFCx and CPu affect learning and memory. Old or re-
mote memory is stored in a cortical network, whereas the
striatum has a well-documented role in procedural learn-
ing and memory resulting in the formation of stimulus-
response habits. In addition, working memory has been
associated with striato-frontal brain regions. Changes in
gene expression in the PFCx and CPu may therefore be
involved in processing these types of memory.

Although *Mecp2* mRNA expression was not affected neither by cocaine, nor by food (Fig. 2), Mecp2 protein was induced by both contingent and non-contingent cocaine self-administration (Fig. 3a-c), but only by contingent food delivery in the PFCx and the CPu (Fig. 3f-h). In these structures, expression of *PP1Cβ* mRNA and protein was repressed by both contingent and non-contingent cocaine administration (Figs 5a, b and 6a-c), but was in contrast induced by contingent food delivery (Figs 5c, d and 6f-h). On the other hand, expression of *reelin* and *GAD67* mRNA was altered by food with changes depending on the brain structures and the type of food delivery (Fig. 7b, d), but no change was noticed in response to cocaine (Fig. 7a, c). Such differential regulation of gene or protein expression in response to the two reinforcers was unexpected. Indeed, neural circuits implicated in drug conditioning, craving and relapse extensively overlap with those involved in natural reward and reinforcement like food (Blum et al., 2011). Most of the genes or proteins that have been compared in response to both rewarding agents are similarly regulated. This is the case of ΔfosB and CREB which are induced or activated in striatum following repeated exposure to drugs (McClung and Nestler, 2003) or by natural reward (Shiflett et al., 2009; Christiansen et al., 2011). In addition, ΔfosB overexpression in the NAc or the dorsal striatum enhances food-motivated behavior, as it does for drug rewards (Olausson et al., 2006), whereas manipulation of CREB activity affects drug rewards as well as preference for natural rewards (Barrot et al., 2002). However, studies based on HDAC and DNMT inhibitors provide evidence that drugs of abuse and natural reinforcers do not rely on similar mechanisms, consistent with specific neuroadaptations triggered by each reinforcer (Carelli et al., 2000; Koya et al., 2006; Levy et al., 2007; Chen et al., 2008; DiLeone et al., 2012). Taken together, these studies support the notion that specific hitherto unknown molecular changes in gene expression are induced by each reinforcer activating a common reward circuitry.

In conclusion, passive and voluntary cocaine adminis-
tration, positively regulate Mecp2 and negatively PP1Cβ
protein expression in reward-related brain structures. In
contrast, the two proteins were induced by food operant
conditioning, no effect being noticed when food was
passively delivered. Most genes examined were found to
be differentially regulated by cocaine and food depending
on the brain structures and the administration mode.
Considering the role of these genes in functions such as
reward, memory, or gene transcription, their differential
regulation by cocaine vs. food provide new insights into
the mechanisms underlying the neuroadaptations trig-
gered by each type of reward. Deciphering mechanisms
controlling these changes in gene expression triggered
by drugs of abuse or natural rewards remains a major
issue to understand how they differ in their ability to
drive behavior.

**Supplementary material**

For supplementary material accompanying this paper,
visit http://dx.doi.org/10.1017/S1461145714000972

Acknowledgments

This work was supported by CNRS, by the Université
de Strasbourg and by the Association Française du
Syndrome de Rett (AFSR). S. Pol Bodetto was a recipient
of a fellowship from the ‘Ministère de l’Enseignement
Supérieur et de la Recherche’. Authors are grateful to
K. Geiger for her technical assistance in transcardial
perfusions.

Statement of Interest

The authors declare no conflict of interest.

**References**

Anier K, Malinovskaja K, Aonurm-Helm A, Zharkovsky A, Kalda A (2010) DNA methylation regulates cocaine-induced behavioral sensitization in mice. Neuropsychopharmacology 35:2450-2461.

Barros M, Dempster EL, Illott N, Chabrawi S, Maior RS, Tomaz C, Silva MA, Huston JP, Mill J, Muller CP (2011) Decreased methylation of the NK3 receptor coding gene (TACR3) after cocaine-induced place preference in marmoset monkeys. Addict Biol 18:452-454.

Barrot M, Olivier JD, Perrotti LI, DiLeone RJ, Berton O, Eisch AJ, Impey S, Storm DR, Neve RL, Yin JC, Zachariou V, Nestler EJ (2002) CREB activity in the nucleus accumbens shell controls gating of behavioral responses to emotional stimuli. Proc Natl Acad Sci USA 99:11435-11440.

Blum K, Liu Y, Shriner R, Gold MS (2011) Reward circuitry dopaminergic activation regulates food and drug craving behavior. Curr Pharm Des 17:1158-1167.

Carelli RM, Ijames SG, Crumling AJ (2000) Evidence that separate neural circuits in the nucleus accumbens encode cocaine vs. "natural" (water and food) reward. J Neurosci 20:4255-4266.

Carouge D, Host L, Aunis D, Zwiller J, Anglard P (2010) CDKL5 is a brain MeCP2 target gene regulated by DNA methylation. Neurobiol Dis 38:414-424.

Cassel S, Carouge D, Gensburger C, Anglard P, Burgun C, Dietrich JB, Aunis D, Zwiller J (2006) Fluoxetine and cocaine induce the epigenetic factors MeCP2 and MBD1 in adult rat brain. Mol Pharmacol 70:487-492.

Ceulemans H, Bollen M (2004) Functional diversity of protein phosphatase-1, a cellular economizer and reset button. Physiol Rev 84:1-39.

Chahrour M, Jung SY, Shaw C, Zhou X, Wong ST, Qin J, Zoghbi HY (2008) MeCP2, a key contributor to neurological disease, activates and represses transcription. Science 320:1224-1229.

Chen BT, Bowers MS, Martin M, Hopf FW, Guillory AM, Carelli RM, Chou JK, Bonci A (2008) Cocaine but not natural reward self-administration nor passive cocaine infusion produces persistent LTP in the VTA. Neuron 59:288-297.

Christiansen AM, Dekloet AD, Ulrich-Lai YM, Herman JP (2011) "Snacking" causes long term attenuation of HPA axis stress responses and enhancement of brain FosB/deltaFosB expression in rats. Physiol Behav 103:111-116.

Cortes-Mendoza J, Diaz de Leon-Guerrero S, Pedraza-Alva G, Perez-Martinez L (2013) Shaping synaptic plasticity: the role of activity-mediated epigenetic regulation on gene transcription. Int J Dev Neurosci 31:359-369.

Costa E, Chen Y, Dong E, Grayson DR, Kundakovic M, Maloku E, Ruzicka W, Satta R, Veldic M, Zhubi A, Guidotti A (2009) GABAergic promoter hypermethylation as a model to study the neurochemistry of schizophrenia vulnerability. Expert Rev Neurotherapeutics 9:87-98.

Cui H, Mason BL, Lee C, Nishi A, Elmquist JK, Lutter M (2012) Melanocortin 4 receptor signaling in dopamine 1 receptor neurons is required for procedural memory learning. Physiol Behav 106:201-210.

Danielli B, Scheggi S, Grappi S, Marchese G, De Montis MG, Tagliamonte A, Gambarana C (2010) Modifications in DARPP-32 phosphorylation pattern after repeated palatable food consumption undergo rapid habituation in the nucleus accumbens shell of non-food-deprived rats. J Neurochem 112:531-541.

Deng JV, Rodriguez RM, Hutchinson AN, Kim IH, Wetsel WC, West AE (2010) MeCP2 in the nucleus accumbens contributes to neural and behavioral responses to psychostimulants. Nat Neurosci 13:1128-1136.

Di Chiara G (1999) Drug addiction as dopamine-dependent associative learning disorder. Eur J Pharmacol 375:13-30.

DiLeone RJ, Taylor JR, Picciotto MR (2012) The drive to eat: comparisons and distinctions between mechanisms of food reward and drug addiction. Nat Neurosci 15:1330-1335.

Feng J, Nestler EJ (2010) MeCP2 and drug addiction. Nat Neurosci 13:1039-1041.

Fyffe SL, Neul JL, Samaco RC, Chao HT, Ben-Shachar S, Moretti P, McGill BE, Goulding EH, Sullivan E, Tecott LH, Zoghbi HY (2008) Deletion of Mecp2 in Sim1-expressing neurons reveals a critical role for MeCP2 in feeding behavior, aggression, and the response to stress. Neuron 59:947-958.

Genoux D, Haditsch U, Knobloch M, Michalon A, Storm D, Mansuy IM (2002) Protein phosphatase 1 is a molecular constraint on learning and memory. Nature 418:970-975.

Graff J, Koshibu K, Jouvenceau A, Dutar P, Mansuy IM (2010) Protein phosphatase 1-dependent transcriptional programs for long-term memory and plasticity. Learn Mem 17:355-363.

Guo JU, Su Y, Zhong C, Ming GL, Song H (2011) Emerging roles of TET proteins and 5-hydroxymethylcytosines in
active DNA demethylation and beyond. Cell Cycle
10:2662-2668.

Han J, Li Y, Wang D, Wei C, Yang X, Sui N (2010) Effect of 5-aza-2-deoxycytidine microinjecting into hippocampus and prelimbic cortex on acquisition and retrieval of cocaine-induced place preference in C57BL/6 mice. Eur J Pharmacol 642:93-98.

Harikrishnan KN, Chow MZ, Baker EK, Pal S, Bassal S, Brasacchio D, Wang L, Craig JM, Jones PL, Sif S, El-Osta A (2005) Brahma links the SWI/SNF chromatin-remodeling complex with MeCP2-dependent transcriptional silencing. Nat Genet 37:254-264.

Hiroi N, Fienberg AA, Haile CN, Alburges M, Hanson GR, Greengard P, Nestler EJ (1999) Neuronal and behavioural abnormalities in striatal function in DARPP-32-mutant mice. Eur J Neurosci 11:1114-1118.

Host L, Dietrich JB, Carouge D, Aunis D, Zwiller J (2011) Cocaine self-administration alters the expression of chromatin-remodelling proteins; modulation by histone deacetylase inhibition. J Psychopharmacol 25:222-229.

Hyman SE (2005) Addiction: a disease of learning and memory. Am J Psychiatry 162:1414-1422.

Im HI, Hollander JA, Bali P, Kenny PJ (2010) MeCP2 controls BDNF expression and cocaine intake through homeostatic interactions with microRNA-212. Nat Neurosci 13:1120-1127.

Jones PL, Veenstra GJ, Wade PA, Vermaak D, Kass SU, Landsberger N, Strouboulis J, Wolffe AP (1998) Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nat Genet 19:187-191.

Koshibu K, Graff J, Mansuy IM (2011) Nuclear protein phosphatase-1: an epigenetic regulator of fear memory and amygdala long-term potentiation. Neuroscience 173:30-36.

Koya E, Spijker S, Voorn P, Binnekade R, Schmidt ED, Schoffelmeer AN, De Vries TJ, Smit AB (2006) Enhanced cortical and accumbal molecular reactivity associated with conditioned heroin, but not sucrose-seeking behaviour. J Neurochem 98:905-915.

LaPlant Q et al. (2010) Dnmt3a regulates emotional behavior and spine plasticity in the nucleus accumbens. Nat Neurosci 13:1137-1143.

Levy D, Shabat-Simon M, Shalev U, Barnea-Ygael N, Cooper A, Zangen A (2007) Repeated electrical stimulation of reward-related brain regions affects cocaine but not "natural" reinforcement. J Neurosci 27:14179-14189.

Ludwig MG, Basset P, Anglard P (2000) Multiple regulatory elements in the murine stromelysin-3 promoter. Evidence for direct control by CCAAT/enhancer-binding protein beta and thyroid and retinoid receptors. J Biol Chem 275:39981-39990.

Mansuy IM, Shenolikar S (2006) Protein serine/threonine phosphatases in neuronal plasticity and disorders of learning and memory. Trends Neurosci 29:679-686.

Maze I, Nestler EJ (2011) The epigenetic landscape of addiction. Ann NY Acad Sci 1216:99-113.

McClung CA, Nestler EJ (2003) Regulation of gene expression and cocaine reward by CREB and DeltaFosB. Nat Neurosci 6:1208-1215.

Miller CA, Sweatt JD (2007) Covalent modification of DNA regulates memory formation. Neuron 53:857-869.

Moretti P, Levenson JM, Battaglia F, Atkinson R, Teague R, Antalffy B, Armstrong D, Arancio O, Sweatt JD, Zoghbi HY

(2006) Learning and memory and synaptic plasticity are impaired in a mouse model of Rett syndrome. J Neurosci 26:319-327.

Na ES, Nelson ED, Kavalali ET, Monteggia LM (2012) The impact of MeCP2 loss- or gain-of-function on synaptic plasticity. Neuropsychopharmacology 38:212-219.

Nan X, Ng HH, Johnson CA, Laherty CD, Turner BM, Eisenman RN, Bird A (1998) Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature 393:386-389.

Olausson P, Jentsch JD, Tronson N, Neve RL, Nestler EJ, Taylor JR (2006) DeltaFosB in the nucleus accumbens regulates food-reinforced instrumental behavior and motivation. J Neurosci 26:9196-9204.

Pelka GJ, Watson CM, Radziewic T, Hayward M, Lahooti H, Christodoulou J, Tam PP (2006) Mecp2 deficiency is associated with learning and cognitive deficits and altered gene activity in the hippocampal region of mice. Brain 129:887-898.

Pol Bodetto S, Carouge D, Fonteneau M, Dietrich JB, Zwiller J, Anglard P (2013) Cocaine represses protein phosphatase-1Cbeta through DNA methylation and Methyl-CpG Binding Protein-2 recruitment in adult rat brain. Neuropharmacology 73:31-40.

Rasmussen M, Kong L, Zhang GR, Liu M, Wang X, Szabo G, Curthoys NP, Geller AI (2007) Glutamatergic or GABAergic neuron-specific, long-term expression in neocortical neurons from helper virus-free HSV-1 vectors containing the phosphate-activated glutaminase, vesicular glutamate transporter-1, or glutamic acid decarboxylase promoter. Brain Res 1144:19-32.

Robinson AJ, Nestler EJ (2011) Transcriptional and epigenetic mechanisms of addiction. Nat Rev Neurosci 12:623-637.

Romieu P, Host L, Gobaille S, Sandner G, Aunis D, Zwiller J (2008) Histone deacetylase inhibitors decrease cocaine but not sucrose self-administration in rats. J Neurosci 28:9342-9348.

Shiflett MW, Mauna JC, Chipman AM, Peet E, Thiels E (2009) Appetitive Pavlovian conditioned stimuli increase CREB phosphorylation in the nucleus accumbens. Neurobiol Learn Mem 92:451-454.

Stearns NA, Schaevitz LR, Bowling H, Nag N, Berger UV, Berger-Sweeney J (2007) Behavioral and anatomical abnormalities in Mecp2 mutant mice: a model for Rett syndrome. Neuroscience 146:907-921.

Svenningsson P, Nishi A, Fisone G, Girault JA, Nairn AC, Greengard P (2004) DARPP-32: an integrator of neurotransmission. Annu Rev Pharmacol Toxicol 44:269-296.

Theisen JW, Gucwa JS, Yusufzai T, Khuong MT, Kadonaga JT (2013) Biochemical analysis of histone deacetylase-independent transcriptional repression by MeCP2. J Biol Chem 288:7096-7104.

Tian W, Zhao M, Li M, Song T, Zhang M, Quan L, Li S, Sun ZS (2012) Reversal of cocaine-conditioned place preference through methyl supplementation in mice: altering global DNA methylation in the prefrontal cortex. PLoS ONE 7:e33435.

Warnault V, Darcq E, Levine A, Barak S, Ron D (2013) Chromatin remodeling—a novel strategy to control excessive alcohol drinking. Transl Psychiatry 3:e231, doi: 210.1038/ tp.2013.1034.
Yasui DH, Peddada S, Bieda MC, Vallero RO, Hogart A, Nagarajan RP, Thatcher KN, Farnham PJ, Lasalle JM (2007) Integrated epigenomic analyses of neuronal MeCP2 reveal a role for long-range interaction with active genes. Proc Natl Acad Sci USA 104:19416–19421.

Zachariou V, Benoit-Marand M, Allen PB, Ingrassia P, Fienberg AA, Gonon F, Greengard P, Picciotto MR (2002) Reduction of cocaine place preference in mice lacking the protein phosphatase 1 inhibitors DARPP 32 or Inhibitor 1. Biol Psychiatry 51:612–620.

Zachariou V, Sgambato-Faure V, Sasaki T, Svenningsson P, Berton O, Fienberg AA, Nairn AC, Greengard P, Nestler EJ (2006) Phosphorylation of DARPP-32 at Threonine-34 is required for cocaine action. Neuropsychopharmacology 31:555–562.

Zhubi A, Chen Y, Dong E, Cook EH, Guidotti A, Grayson DR (2014) Increased binding of MeCP2 to the GAD1 and RELN promoters may be mediated by an enrichment of 5-hmC in autism spectrum disorder (ASD) cerebellum. Transl Psychiatry 4:e349.