BASIC NEUROSCIENCES, GENETICS AND IMMUNOLOGY - ORIGINAL ARTICLE

Regulation of the immediate-early genes arc and zif268 in a mouseoperant model of cocaine seeking reinstatement

Barbara Ziołkowska • Michał Kiełbinski •

Agnieszka Gieryk • Guadalupe Soria •

Rafael Maldonado • Ryszard Przewłocki

Received: 7 September 2010 / Accepted: 9 January 2011

� Springer-Verlag 2011

Abstract Reinstatement of extinguished operant res-

ponding for drug is an appropriate model of relapse to drug

abuse. Due to the difficulty of implementing in mice the

procedure of instrumental intravenous self-administration,

mechanisms of reinstatement have so far been studied

almost exclusively in rats. A mouse model of reinstatement

of cocaine seeking has recently been characterized (Soria

et al. 2008). The aim of the present study was to assess

regional brain activation, as measured by induction of the

immediate early genes (IEG) arc and zif268, during

priming- or cue-elicited reinstatement of cocaine seeking

using this new mouse model and the in situ hybridization

technique. We have demonstrated that cue-elicited rein-

statement of cocaine seeking was associated with induction

of the IEG in the medial prefrontal cortex (prelimbic and

infralimbic) and basolateral amygdala. Priming-induced

reinstatement produced a more widespread up-regulation

of those genes in forebrain regions including medial pre-

frontal, orbitofrontal and motor cortex, dorsal striatum and

basolateral amygdala. These patterns of IEG expression are

in agreement with previous results obtained in rats and thus

indicate that the new mouse model of reinstatement is

functionally equivalent to rat models. That comparability

adds to the usefulness of the mouse model as a tool for

addressing neurobiological mechanisms of addiction.

Keywords Cocaine � Drug seeking � Reinstatement �Immediate early gene � Brain activity mapping

Introduction

Drug addiction is a chronic and relapsing brain disorder.

Cessation of chronic taking of a drug of abuse is usually

accompanied by episodes of craving for the drug, which

often leads to relapse into the drug use even after a long

abstinence (Anton 1999; Franken 2003; O’Brien et al.

1998). Craving may be elicited by several factors, which

include stress, as well as re-exposure to a priming dose of

the drug or to environmental stimuli previously associated

with drug taking (O’Brien et al. 1998; Shaham et al. 2003;

Shalev et al. 2002). From both theoretical and therapeutical

standpoints, understanding neural mechanisms underlying

craving is essential because they actually represent the

main driving force of addiction.

Intravenous operant self-administration is considered

the most relevant, reliable and useful animal model to

study these processes (Shaham et al. 2003; Shalev et al.

2002). In addition to its similarity to human scenarios (i.e.

the fact that the drug use is controlled by the animal rather

than the experimenter), the self-administration model per-

mits the separate study of several aspects and stages of

addiction: acquisition and maintenance of operant

responding for the drug, as well as extinction and rein-

statement of the drug-directed behaviour. Similar instru-

mental self-administration paradigms (e.g. oral) can also be

B. Ziołkowska � M. Kiełbinski � A. Gieryk � R. Przewłocki (&)

Department of Molecular Neuropharmacology,

Institute of Pharmacology, Polish Academy of Sciences,

Smetna 12, 31-343 Krakow, Poland

e-mail: nfprzewl@cyf-kr.edu.pl

G. Soria � R. Maldonado

Laboratorio de Neurofarmacologıa, Universitat Pompeu Fabra,

Parc de Recerca Biomedica de Barcelona, C/Dr. Aiguader 88,

08003 Barcelona, Spain

Present Address:G. Soria

Institute for Biomedical Research (IIBB-CSIC), Experimental

MRI Unit (IDIBAPS), Rossello 161, 08036 Barcelona, Spain

123

J Neural Transm

DOI 10.1007/s00702-011-0583-z

used to study effects of natural reinforcers, such as palatable

and/or energy-dense food, which may as well produce

addiction-like behavioural disturbances and recruit partly the

same neural mechanisms as drugs of abuse (Nair et al. 2009).

In order to fully elucidate the mechanisms of addiction,

it is necessary to combine behavioural studies with com-

plementary molecular and genomic approaches. Recent

advances in generation of genetically modified mouse lines

have yielded powerful tools for studying multiple genes

and pathways that could be involved in drug addiction

(Changeux 2010; Kieffer and Gaveriaux-Ruff 2002; Sora

et al. 2010; Stephens et al. 2002). However, due to the

technical difficulty of implementing the self-administration

procedure in mice (Thomsen and Caine 2007), most studies

in operant models of drug self-administration have so far

been conducted in rats (Shaham et al. 2003; Shalev et al.

2002; Schmidt et al. 2005). It is therefore necessary to

develop reliable mouse models in order to take advantage

of genetic modification technologies in addiction research.

Such an operant mouse model of acquisition, extinction

and reinstatement of cocaine seeking behaviour, in which all

factors known to precipitate relapse in humans (i.e. drug

priming dose, discrete drug-associated cues and stress) rein-

stated extinguished responding for the drug, has recently been

described for the first time by Soria et al. (2008). Whereas the

model has been characterized in behavioural terms, where its

similarity to the existing rat models has been demonstrated, it

still remains to be established if neural mechanisms underly-

ing reinstatement of operant responding for cocaine after

extinction in mice are the same as in rats. This can be primarily

assessed by comparing patterns of brain activation during

reinstatement of operant responding in the different models.

Although such an approach does not give an insight into

intracellular processes, it does enable to locate brain regions

and neuronal circuits, activation of which is associated with

behavioural responses leading to reinstatement of drug seek-

ing. Thereby, brain regions and circuits can be identified, in

which some specific changes had occurred because of the

previous self-administration training.

Immediate early genes (IEG), such as c-fos, arc and

zif268 (also known as egr-1, krox-24, and NGFI-A), have

been commonly used as markers of brain activation in

laboratory animals (Herdegen and Leah 1998; Kaczmarek

and Chaudhuri 1997; Ziołkowska and Przewłocki 2002).

Their suitability results from the fact that IEG are rapidly

upregulated in neurons in a highly localized, cell-specific,

activity- and time-dependent manner in response to various

cell-excitatory stimuli (Herdegen and Leah 1998; Morgan

et al. 1987; Sagar et al. 1988). IEG expression in several

experimental models of drug addiction has been studied

extensively and has been characterized in rat models of

reinstatement of operant responding for psychostimulants

(Ciccocioppo et al. 2001; Harlan and Garcia 1998; Hearing

et al. 2008a; Kufahl et al. 2009; Neisewander et al. 2000;

Thomas et al. 2003). Several neural systems can be con-

sidered critical to the processes of reinstatement of cocaine

seeking behaviour based on IEGs being consistently

upregulated in these brain regions in the rat paradigms.

These include prefrontal cortical regions (anterior cingulate

and prelimbic cortex) and basolateral amygdala (Ciccocioppo

et al. 2001; Hearing et al. 2008a; Kufahl et al. 2009; Neise-

wander et al. 2000; Thomas et al. 2003). Some studies also

demonstrated IEG induction in additional regions, e.g.

nucleus accumbens, dorsal striatum, hippocampus and ventral

tegmental area (Hearing et al. 2008b; Kufahl et al. 2009;

Neisewander et al. 2000; Thomas et al. 2003).

In order to get insight into specific patterns of brain

activation during reinstatement of cocaine seeking in mice,

we studied regional expression of two IEG, zif268 and arc,

using in situ hybridization, in the behavioural paradigm

recently designed by Soria et al. (2008). The effect of two

relapse-triggering factors was assessed, a drug-associated

cue and drug priming, which may produce reinstatement of

responding by activation of different neuronal pathways

(Shaham et al. 2003; Shalev et al. 2002).

Materials and methods

Animals

The experiment was carried out on male CD1 mice (Charles

River, France) weighing 20–25 g upon arrival at the labora-

tory. The animals were housed individually in plastic cages,

kept on a 12-h light/dark cycle, at constant temperature

(21 ± 1�C) and air humidity (55 ± 10%). Food and water

were provided ad libitum, except during the experimental

sessions. Mice were handled daily in order to minimize stress

during the experiments. Animal care and experimental pro-

cedures were in accordance with institutional and interna-

tional standards (the European Communities Council

Directive 86/609/EEC, 24 November 1986) and were

approved by the local Ethics Committee (CEEA-PRBB).

Control injections of cocaine and saline

Control experiments were carried out in intact, behavio-

urally non-trained animals in order to assess the pharma-

cological effect of cocaine and the impact of injection

stress on arc and zif268 gene expression. Mice were

injected i. p. with saline or cocaine (10 mg/kg), while naive

animals served as a control. First, the time-course of IEG

induction due to cocaine and stress was estimated quali-

tatively, the animals being sacrificed at 30 min or 2 h after

the injection (n = 3 to 4). Quantitative measurements of

IEG expression were then performed in an independent

B. Ziołkowska et al.

123

experiment, in mice decapitated 2 h post-injection, i.e. the

time equivalent to the duration of a reinstatement session

(saline: n = 7; cocaine: n = 7; naive: n = 5).

Behavioural training and reinstatement procedures

The present in situ hybridization experiments were performed

on the brains of the mice which had been behaviourally

characterized in the previous paper by Soria et al. (2008).

Thus, all details concerning the animal treatment and the

behavioural procedure are to be found in the aforementioned

paper. Briefly, mice were implanted with intravenous silastic

catheters and, after a recovery period, trained to self-admin-

ister cocaine hydrochloride (Ministerio de Sanidad y Con-

sumo, Spain) at the dose of 1 mg/kg per infusion. The training

consisted of daily 2-h sessions and was conducted in MED

Associates (Georgia, VT, USA) operant chambers equipped

with two retractable levers. Whenever the animal pressed one

of the levers (designated as ‘active’), it obtained one infusion

of cocaine and was presented a light cue. The training was

carried out until the mice met the criteria of self-administra-

tion acquisition (cf Soria et al. 2008). They were then sub-

jected to extinction procedure which consisted of two daily

2-h sessions in the Skinner box, during which lever presses

were not reinforced. The extinction training took place 6 days

per week until the mice reached the extinction criteria (cf Soria

et al. 2008).

One or two days after the extinction training was com-

pleted, the animals were tested for reinstatement of lever

pressing behaviour elicited either by an injection of a

priming cocaine dose (10 mg/kg, i.p.) given immediately

before placement of the animal in the operant chamber or

by presentation of the conditioned light cue previously

associated with cocaine infusions. During reinstatement

sessions, cocaine was not available, and responding was

reinforced by presentation of the drug-associated cue light

only in the cue (but not priming) reinstatement group. A

control group for the priming cocaine dose effect was

injected i.p. with saline and tested for responding in the

Skinner box. All the reinstatement sessions lasted 2 h. An

animal was considered a responder in the reinstatement test

if its behaviour met the following criteria: (1) the number

of responses on the ‘active’ lever was at least double of that

during the last extinction session and (2) the number of

responses on the ‘active’ lever equalled ten or more.

Animal groups analyzed by in situ hybridization

Four animal groups were used for the biochemical analysis

described herein: (a) sham-operated animals, which were

placed in Skinner boxes for 20 daily 2-h sessions without

receiving any infusions and without receiving any other

type of reinforcers for pressing the lever (sham; n = 5);

(b) mice subjected to the self-administration and then to the

extinction training which met the extinction criteria

(extinction; n = 5); (c) mice after self-administration and

extinction training which then underwent testing for rein-

statement triggered by the drug priming dose (priming-

induced reinstatement; n = 10) or (d) by the conditioned

stimulus (cue-induced reinstatement; n = 5). All the mice

were sacrificed by decapitation immediately after com-

pletion of the last 2-h session in the operant chamber.

In situ hybridization

After sacrifice, the brains were removed and frozen on dry

ice. They were then cut into 12 lm thick coronal sections

on a cryostat microtome (Leica Microsystems, Nussloch,

Germany), the sections were thaw-mounted on gelatin-

chrom-alum-coated slides and processed for in situ

hybridization according to the method of Young et al.

(1986). Briefly, the sections were fixed with 4% parafor-

maldehyde, washed with PBS and acetylated by incubation

with 0.25% acetic anhydrite (in 0.1 M triethanolamine and

0.9% sodium chloride). The sections were then dehydrated

using increasing concentrations of ethanol (70–100%),

treated with chloroform for 5 min, and rehydrated with

decreasing concentrations of ethanol.

The sections were hybridized for approximately 15 h at

37�C with arc and zif268 oligonucleotide probes. The arc

probe 50 TGC CCA CCG ACC TGT GCA ACC CTT TCA

GCT CTC GCT CCA 30 (accession No. NM_018790;

probe complementary to nucleotides 368–406) had been

designed by Ammon et al. (2003). The zif268 probe 50

CCG TTG CTC AGC AGC ATC ATC TCC TCC AGT

TTG GGG TAG TTG TCC 30 (accession No. NM_007913;

probe complementary to nucleotides 369–413) had been

designed by Wisden et al. (1990). The probes were labeled

with 35S-dATP by the 30-tailing reaction using terminal

transferase (MBI Fermentas, Vilnius, Lithuania).

After hybridization, the slices were washed three times

for 20 min with 1xSSC/50% formamide at 40�C, and twice

for 50 min with 19 SSC at room temperature. Then, the

slices were dried and exposed to Fujifilm phosphorimager

imaging plates for 5 days. The hybridization signal was

digitized using the Fujifilm BAS-5000 phosphorimager and

the ImageReader software.

Image analysis

In situ hybridization signal was analyzed using the MCID

Elite system (Imaging Research, St. Catharines, ON,

Canada). Signal density was measured bilaterally in

selected forebrain regions in the Fujifilm BAS-5000 ima-

ges. The regions were chosen based on their reported

activation in rat models and included: the anterior

Regulation of the immediate-early genes arc and zif268

123

cingulate, primary motor cortex, ventrolateral orbital cor-

tex, prelimbic and infralimbic cortical areas, dorsal stria-

tum, nucleus accumbens, hippocampus and the basolateral

amygdala (Fig. 1). In addition, the pharmacological effect

of cocaine injection was assessed in the medial dorsal

striatum (Fig. 1), which had previously been demonstrated

to be the main site of cocaine-elicited IEG induction

(Berke et al. 1998; Fosnaugh et al. 1995; Graybiel et al.

1990; Moratalla et al. 1992).

In those regions of interest, the arc and zif268 hybrid-

ization signal was distributed heterogeneously, i.e. patches

or layers of very intense signal were interspersed with areas

of very low expression, both in basal and stimulated condi-

tions. In order to exclude areas of very low (near background)

expression from the analysis, a thresholding method was

used, in which only those pixels were quantified whose

signal density exceeded a preselected threshold value. For

the spots of the signal exceeding the threshold, area and mean

signal density were measured. Tissue background signal,

measured over the corpus callosum, was subtracted from all

density measurements. The final result was calculated

according to the following formula:

Expression ¼ ðD� bgÞ � AS

AT;

where D represents mean optical density of the above-threshold

pixels, bg represents tissue background (corpus callosum), AS

represents area occupied by pixels with above-threshold den-

sity, and AT represents total sampled area. The data were then

expressed as percentage of the mean in the extinction group.

Data analysis

Data are presented as group means, with error bars showing

the standard error of the mean. The effects of treatment on

IEG mRNA levels were analyzed with one-way analysis of

variance (ANOVA) followed by the post-hoc Newman–

Keuls test to detect differences between groups.

Results

Regulation of IEG expression by cocaine and injection

stress (Figs. 2, 3)

Saline injection produced a widespread induction of the arc

and zif268 genes at 30 min post-injection in the cortex,

striatum and hippocampus, which may be attributed to the

stress of injection and animal handling (Figs. 2a, 3a).

Administration of cocaine (10 mg/kg, i.p.), in addition to

the stress-related IEG activation, up-regulated arc and

zif268 genes in the medial dorsal striatum at 30 min, which

probably reflects the direct pharmacological action of the

drug (Figs. 2a, 3a). Such a cocaine effect has long been

described in the literature (Berke et al. 1998; Fosnaugh

et al. 1995; Graybiel et al. 1990; Moratalla et al. 1992).

Both stress- and cocaine-related IEG inductions dissipated

by 2 h post-injection (Figs. 2, 3). A quantitative analysis of

arc and zif268 mRNA hybridization signal in eight brain

regions (selected for analysis in self-administration rein-

statement paradigms) demonstrated that the IEG mRNA

levels were not significantly elevated in any region at 2 h.

Actually, they were decreased in some areas, especially in

the striatum and motor cortex, as compared to naive ani-

mals, which might be a rebound effect following the earlier

IEG induction in the same brain regions (Figs. 2b, 3b).

Regulation of IEG expression by priming-induced

reinstatement (Figs. 4, 5)

Of the two reinstatement paradigms studied, cocaine priming

produced greater changes in arc and zif268 gene expression.

Induction of both genes during priming reinstatement was

most pronounced in cortical regions, especially the anterior

cingulate and motor cortex, but significant increases were also

detected in the prelimbic/infralimbic (for both arc and zif268),

Fig. 1 Outlines of the regions of interest in which optical density

measurements were performed. AC anterior cingulated, BLA basolat-

eral amygdale, DStr dorsal striatum, HIP hippocampus, m medial

dorsal striatum; M1/2 primary motor cortex, NAc nucleus accumbens,

PRL ? IL prelimbic and infralimbic cortex, VLO ventrolateral orbital

cortex. The section schemes were taken from the atlas by Paxinos and

Franklin (2001)

B. Ziołkowska et al.

123

as well as ventrolateral orbitofrontal cortex (only arc). A large

increase in zif268, but not arc expression, was also observed in

the dorsal striatum. In addition, both genes were significantly

upregulated in the basolateral amygdala. No effect of priming

reinstatement was found in the nucleus accumbens and

hippocampus.

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Fig. 2 Regulation of arc mRNA in the mouse brain by injection

stress and cocaine. a Upper panel Representative in situ hybridization

images showing the arc expression in the striatum and cortex 30 min

and 2 h after an i.p. injection of saline or cocaine (10 mg/kg). Lowerpanel Quantitative results of in situ hybridization analysis showing

arc mRNA levels in the medial dorsal striatum, i.e. the main site of

cocaine-elicited induction. The results are expressed as percentage of

the mean in the naive group. Asterisks denote statistically significant

differences from the respective saline group (***p \ 0.001), and

hashes denote significant differences from the naive group

(###p \ 0.001) by ANOVA followed by the Newman–Keuls test.

b Quantitative results of in situ hybridization analysis showing arcmRNA levels at 2 h after the saline or cocaine injection. The results

are expressed as percentage of the mean in the naive control group.

Asterisks denote statistically significant differences versus the naive

group by ANOVA followed by the Newman–Keuls test (p \ 0.05)

Regulation of the immediate-early genes arc and zif268

123

Regulation of IEG expression by cue-induced

reinstatement (Figs. 4, 5)

At the end of the cue-induced reinstatement session, a signifi-

cant induction of the arc gene was observed only in the pre-

limbic/infralimbic cortex, whereas zif268 was upregulated only

in the basolateral amygdala. Expression of those IEG tended to

increase in the anterior cingulate and motor cortex, but the

changes did not reach the level of statistical significance. Like

in the case of priming reinstatement, the IEG expression in the

nucleus accumbens and hippocampus remained completely

unaffected by cue-induced reinstatement.

Discussion

Our results demonstrate that in the mouse model of rein-

statement of cocaine seeking behaviour, significant

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Fig. 3 Regulation of zif268 mRNA in the mouse brain by injection

stress and cocaine. a Upper panel Representative in situ hybridization

images showing the zif268 expression in the striatum and cortex

30 min and 2 h after an i.p. injection of saline or cocaine (10 mg/kg).

Lower panel Quantitative results of in situ hybridization analysis

showing zif268 mRNA levels in the medial dorsal striatum, i.e. the

main site of cocaine-elicited induction. The results are expressed as

percentage of the mean in the naive group. Asterisks denote

statistically significant differences from the respective saline group

(*p \ 0.05), and hashes denote significant differences from the naive

group (##p \ 0.01) by ANOVA followed by the Newman–Keuls test.

b Quantitative results of in situ hybridization analysis showing zif268mRNA levels at 2 h after the saline or cocaine injection. The results

are expressed as percentage of the mean in the naive control group.

No statistically significant differences between groups were detected

by one-way ANOVA

B. Ziołkowska et al.

123

induction of IEG associated with a cue-elicited responding

takes place in the prelimbic/infralimbic cortex (PRL/IL)

and the basolateral amygdala (BLA). In contrast, rein-

statement produced by a cocaine priming injection resulted

in IEG up-regulation in the ventrolateral orbitofrontal

(VLO) and motor (M1/M2) cortex, anterior cingulate (AC)

and dorsal striatum (DStr), in addition to the PRL/IL and

BLA. It is important to underline that the overall brain IEG

arc

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cingulate dorsal striatum

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Sham Extinction Cue relapse Priming relapse

Fig. 4 Induction of arc mRNA in the mouse brain elicited by cue-

and priming-induced reinstatement of cocaine seeking. a Quantitative

results of in situ hybridization analysis. The results are expressed as

percentage of the mean in the extinction (ext) group. Asterisks denote

statistically significant differences versus the extinction group

(*p \ 0.05; **p \ 0.01) by ANOVA followed by the Newman–

Keuls test. No significant differences between the sham and extinction

groups were detected in any brain region analyzed. b Representative

in situ hybridization images showing expression levels of arc in the

forebrain

Regulation of the immediate-early genes arc and zif268

123

response to cocaine priming was considerably stronger than

to a cocaine-associated cue. This phenomenon likely

reflects a greater reactivity of the mice to cocaine priming

than to the cocaine-associated cue, illustrated at the

behavioural level by a significantly lower number of active

lever presses during the test in the cue reinstatement group

(Soria et al. 2008).

The present findings in the cue-elicited reinstatement

model are in line with earlier reports using cues to induce

reinstatement in rat paradigms. In these paradigms, discrete

zif268

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Fig. 5 Induction of zif268 mRNA in the mouse brain elicited by cue-

and priming-induced reinstatement of cocaine seeking. a Quantitative

results of in situ hybridization analysis. The results are expressed as

percentage of the mean in the extinction (ext) group. Asterisks denote

statistically significant differences vs. the extinction group

(*p \ 0.05; **p \ 0.01) by ANOVA followed by the Newman–

Keuls test. No significant differences between the sham and extinction

groups were detected in any brain region analyzed. b Representative

in situ hybridization images showing expression levels of zif268 in the

forebrain

B. Ziołkowska et al.

123

cues, previously associated with cocaine infusions or pre-

dictive of the drug availability were shown to induce c-fos

mRNA and protein in the rat prelimbic and infralimbic

cortex, as well as in the BLA (Ciccocioppo et al. 2001;

Kufahl et al. 2009). zif268 mRNA was induced in the BLA,

and arc mRNA in the prelimbic cortex of rats undergoing

cue-elicited reinstatement of cocaine seeking (Thomas

et al. 2003; Zavala et al. 2008), in agreement with the

results obtained in the present mouse model. The rat

studies also reported arc and c-fos induction in the AC

(Cg1 or Cg2) and VLO, where only slight increase ten-

dencies in arc mRNA levels were observed in our exper-

iment. Kufahl et al. (2009) described c-fos activation in

several additional brain regions, which was not consistent

with previous observations in the rat or our mouse model

(Ciccocioppo et al. 2001; Thomas et al. 2003; Zavala et al.

2008), perhaps due to minor procedural differences and

variability in signal detection sensitivity.

Thus, while spatiotemporal patterns of induction may

slightly differ for specific IEG (see below), both rat and

mouse IEG studies provide concurring evidence for

activation of medial prefrontal cortical subregions and

BLA during cue-elicited reinstatement of cocaine seek-

ing. The predictive value of such mapping is indicated by

the fact that activation of the corresponding human brain

regions was demonstrated by in vivo imaging techniques

in drug addicts undergoing craving after presentation of

cocaine-associated stimuli (Bonson et al. 2002; Childress

et al. 1999; Grant et al. 1996; Kilts et al. 2001). The

causative role of dorsomedial prefrontal cortex (PRL and

AC) and BLA in cue-induced reinstatement of cocaine

seeking was confirmed in experimental rat models, where

their lesion or temporary functional inactivation pre-

vented reinstatement of instrumental responding for

cocaine (Kantak et al. 2002; McLaughlin and See 2003;

Meil and See 1997).

The influence of priming-induced reinstatement of

cocaine seeking on IEG expression has so far been assessed

in only one study in rats to our knowledge (Neisewander

et al. 2000). The authors reported a specific effect of

cocaine priming on Fos protein expression only in the AC,

while, in addition to AC, we found arc and/or zif268

mRNA induction in many other brain regions (VLO, PRL/

IL, M1/M2, DStr and BLA). Our control experiments

excluded a contribution of IEG inductions related to

injection stress and pharmacological cocaine actions to the

arc and zif268 activation observed at the end of the rein-

statement session, i.e. 2 h after the priming injection, thus

indicating that the observed gene up-regulation was spe-

cifically associated with animals’ behaviour during the

reinstatement test. Both Neisewander et al. (2000) and our

study are consistent in showing the lack of IEG expression

changes in the nucleus accumbens and hippocampus.

Whereas the prefrontal cortical subregions VLO and

PRL/IL were not subjected to examination by Neisewander

et al. (2000), there is substantial evidence from functional

inactivation and pharmacological studies indicating that

activation of the rat PRL/IL (along with the adjacent AC) is

absolutely necessary for a cocaine prime to trigger rein-

statement responding (Capriles et al. 2003; McFarland and

Kalivas 2001; Schmidt et al. 2005). Involvement of the

VLO has also been suggested by effects of its lesioning

(Fuchs et al. 2004), although not confirmed by other

methods (cf. Schmidt et al. 2005). Thus, our finding in the

mouse model of IEG activation in all prefrontal cortical areas,

i.e. VLO, PRL/IL and AC provides new information about the

neurobiological substrate involved in priming-induced rein-

statement of cocaine seeking and is in agreement with previ-

ous pharmacological and lesion studies in rats.

The discrepancy between our data and those by Neise-

wander et al. (2000) consists in the finding of IEG induc-

tion in the DStr, M1/M2 and BLA of mice but not rats.

Whereas species-specific mechanisms may account for

some of these discrepancies, they may just as well result

from differences in the design of both studies, taking into

consideration the fact that different genes were assessed

(Fos vs. arc and zif) and that inductions were detected

either at the mRNA level (in our study) or on protein level

(by Neisewander et al. 2000), which have largely non-

overlapping time-courses. In addition, detection of a rein-

statement-related induction in the DStr in the Neisewander

et al. study might have been confounded by pharmaco-

logical effect of cocaine priming (which was excluded in

our experimental setting) (Berretta et al. 1992; Graybiel

et al. 1990). On the other hand, although Neisewander et al.

(2000) did not detect Fos induction in the BLA during

priming-induced reintatement in rats, both lesion and

pharmacological rat studies (Alleweireldt et al. 2003; Yun

and Fields 2003) suggest an involvement of BLA in

priming-triggered cocaine seeking.

Activation of the IEG transcription in the motor cortex

during the reinstatement session could reflect brain activity

associated with the motor act of instrumental responding.

However, IEG were not induced in the motor cortex in

several rat studies in which animals were allowed to lever-

press during the session of reinstatement induced by

priming, cocaine-associated cues or context (Ciccocioppo

et al. 2001; Kufahl et al. 2009; Neisewander et al. 2000;

Zavala et al. 2008). On the other hand, Hearing et al.

(2008b) reported up-regulation of c-fos, arc and zif268

genes in the motor cortex during context-elicited cocaine

seeking, which did not depend on whether the rats actually

performed the instrumental reaction or not since levers

were retracted during the test session. Thus, it seems that

both in mice and in rats, motor cortex IEG activation does

occur only in some reinstatement paradigms and may be

Regulation of the immediate-early genes arc and zif268

123

related to general motor activation during the test session

rather than being specifically associated with instrumental

responding.

Interestingly, our experiment demonstrated differences

between induction patterns of arc and zif268 genes. While

some of these differences seemed to be of quantitative nature

(one gene being significantly induced and the other one dis-

playing a non-significant tendency to up-regulation in the

same brain region), others seemed rather qualitative. In par-

ticular, only zif268 but not arc was induced in the DStr,

whereas only arc but not zif268 was induced in the VLO, both

in the priming reinstatement group. These apparently some-

what distinct spatiotemporal patterns of arc and zif268 mRNA

induction may result from (i) different intracellular mecha-

nisms of transcriptional activation for each gene (cf. e.g.

Lemberger et al. 2008; Rodriguez Parkitna et al. 2010; Worley

et al. 1993) and (ii) differences between brain regions in decay

rates of specific mRNA species (Kelly and Deadwyler 2003).

Therefore, due to the partial complementarity of IEG

expression patterns, the use of more than one IEG marker for

brain activity mapping may yield a more complete picture.

In conclusion, we have demonstrated that in the mouse

model of relapse, cue-elicited reinstatement of cocaine

seeking is associated with induction of the IEG arc and

zif268 in the medial prefrontal cortex (PRL/IL) and BLA.

Priming-induced reinstatement produced a more wide-

spread up-regulation of those genes in forebrain regions.

These patterns of IEG mRNA expression are mostly in

agreement with those described in previous studies in rats,

suggesting that no large-scale functional differences exist

between the mouse and rat models. Thus, future conclu-

sions derived from the present mouse model can be com-

bined with the existing abundant data obtained in rat

models to address further issues concerning brain mecha-

nisms of drug craving and relapse.

Acknowledgments This work was supported by the EU grant

LSHM-CT-2007-037669 (PHECOMP), the Polish Ministry of Sci-

ence and Higher Education subsidiary grant No. 478/6. PR UE/2007/

7, statutory funds from the Institute of Pharmacology, Polish Acad-

emy of Sciences, the Spanish Ministerio de Ciencia e Innovacion

(#SAF 2007-64062), the Catalan Government (S6R 2009-00131) and

the ICREA Foundation (ICREA-Academia 2008).

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