Mapping of c-fos gene expression in the brain during morphine dependence and precipitated...

Mapping of c-fos gene expression in the brain duringmorphine dependence and precipitated withdrawal, andphenotypic identi®cation of the striatal neurons involved

FrancËois Georges, Luis Stinus and Catherine Le MoineCentre National de la Recherche Scienti®que, UniteÂ Mixte de Recherche 5541, BP28, Laboratoire d'Histologie-Embryologie et

de Neuropsychobiologie de DeÂsadaptations, UniversiteÂ Victor Segalen Bordeaux 2, 146 rue LeÂo Saignat, 33076 Bordeaux

Cedex, France

Keywords: dopamine±opioid interactions, in situ hybridization, mesostriatal system, opiate dependence, rat

Abstract

The c-fos gene is expressed in the central nervous system in response to various neuronal stimuli. Using in situ hybridization, weexamined the effects of chronic morphine treatment and withdrawal on c-fos mRNA in the rat brain, and particularly within

identi®ed striatal neurons. Morphine dependence was induced by subcutaneous implantation of two pellets of morphine for 6 days

and withdrawal was precipitated by administration of naltrexone. Placebo animals and morphine-dependent rats showed a very

weak c-fos mRNA expression in all the structures studied. Our study emphasized the spatial variations in c-fos mRNAexpression, and also revealed a peak expression of c-fos mRNA at 1 h after naltrexone-precipitated withdrawal in the projection

areas of dopaminergic neurons, noradrenergic neurons and in several regions expressing opiate receptors. Interestingly,

morphine withdrawal induces c-fos mRNA expression in the two efferent populations of the striatum (i.e. striatonigral andstriatopallidal neurons) both in the caudate putamen and nucleus accumbens. Moreover, the proportions of activated neurons

during morphine withdrawal are different in the caudate putamen (mostly in striatopallidal neurons) and in the shell and core parts

of the nucleus accumbens (mostly in striatonigral neurons). The activation of striatopallidal neurons suggests a predominantdopaminergic regulation on c-fos gene expression in the striatum during withdrawal. On the contrary, c-fos induction in

striatonigral neurons during withdrawal seems to involve a more complex regulation like opioid±dopamine interactions via the mopioid receptor and the D1 dopamine receptor coexpressed on this neuronal population or the implication of other

neurotransmitter systems.

Introduction

Prolonged exposure to morphine leads to tolerance and dependence.

In the dependent state, continuous exposure to a drug is required to

avoid withdrawal symptoms (Koob & Bloom, 1988; Nestler, 1992).

The mesolimbic dopaminergic system participates in the reinforcing

effects of various drugs of abuse (Wise & Bozarth, 1987; Koob &

Nestler, 1997). The reinforcing effects of opiates and the motivational

and physical aspects of withdrawal have been associated with

changes in mesolimbic dopaminergic transmission (Wise & Rompre,

1989; Koob, 1992b; Harris & Aston-Jones, 1994). Indeed, adminis-

tration of morphine increases the activity of dopamine neurons in

substantia nigra pars compacta (SNc) and ventral tegmental area

(VTA), and increases dopamine release in caudate putamen (CPu)

and nucleus accumbens (NAc) (Matthews & German, 1984; Spanagel

et al., 1990; Di Chiara & North, 1992). It is commonly accepted that

this increase is related to a disinhibition of dopamine neurons through

the action of morphine on mu (m) opioid receptors located on GABA

interneurons in substantia nigra pars reticulata (SNr) and VTA

(Johnson & North, 1992; Bontempi & Sharp, 1997).

One way to determine the anatomical and cellular effects of

various drug treatments is to study the regulation of immediate early

genes (IEGs) like c-fos (for reviews, see Curran et al., 1996). Acute

administrations of amphetamine, cocaine or morphine increase c-fos

expression within the striatum (Graybiel, 1990; Moratalla et al.,

1993; Liu et al., 1994). Although induction of IEGs in CPu and NAc

produced by cocaine and amphetamine might be explained by their

direct effects on dopamine release and reuptake (Cooper, 1991), it is

less clear how morphine induces IEGs in CPu and NAc. Nevertheless,

it has been shown that morphine withdrawal induces neuronal

activation through activation of several secondary messenger systems

(Hayward et al., 1990; Nestler & Aghajanian, 1997). However, no

systematic spatial study of the effects of chronic morphine treatment

or withdrawal on the induction of c-fos mRNA expression throughout

the brain has been reported.

Changes in mesolimbic dopaminergic activity constitute, at least

in part, the neural substrate for opioid addiction and somatic

expression of abstinence (Acquas et al., 1991; Koob et al., 1992a;

Harris & Aston-Jones, 1994). Dopamine differentially regulates

the two major striatal populations, the striatonigral neurons via

D1-dopamine (D1) receptors and the striatopallidal neurons via

D2-dopamine (D2) receptors (Gerfen et al., 1990; Le Moine &

Bloch, 1995). These output pathways express different opioid

peptides, dynorphin and enkephalin, respectively (Gerfen &

Young, 1988). To better understand the neuroanatomical pathways

involved in opiate withdrawal and to characterize the regulation

Correspondence: Dr Catherine Le Moine, as above.E-mail: [email protected]

Received 25 May 2000, revised 28 September 2000, accepted 3 October 2000

European Journal of Neuroscience, Vol. 12, pp. 4475±4486, 2000 ã Federation of European Neuroscience Societies

of striatonigral and striatopallidal neurons during chronic mor-

phine exposure and naltrexone-precipitated withdrawal, detailed

analysis of the neuronal population activated was examined with

regard to the expression of c-fos mRNA. We used in situ

hybridization with riboprobes to map the brain regions which are

activated by naltrexone-precipitated withdrawal, as compared to

chronic morphine treatment. In CPu and NAc, double labelling

experiments were used to identify the neurons activated in

response to chronic morphine and during withdrawal.

Materials and methods

Animals

Twenty-eight male Sprague±Dawley rats (CERJ, Le Genest Saint-

Isle, France; 150±175 g, at the beginning of the experiment) were

used. Rats were housed in individual cages under controlled

conditions (22±23 °C, 40% relative humidity, 12 h light : 12 h dark

illumination cycle (lights from 06.00 to 18.00 h) for at least 1 week

before use. Rats were allowed free access to commercial chow and

tap water. After the adaptation period, rats were individually housed

and randomly assigned into ®ve groups (n = 4±6 per group) for in situ

hybridization experiments (Fig. 1).

Induction of morphine dependence

Rats were made morphine dependent by subcutaneous implantation

of two morphine (M) pellets, each containing 75 mg of morphine-free

base (National Institute of Drug Abuse, NIH, Rockville, MD, USA)

(Gold et al., 1994). All surgical and experimental procedures were

performed in accordance with the European Communities Council

Directive of 24 November 1986 (86/609/EEC). Control rats received

placebo (P) pellets containing the excipient without morphine.

Implantation procedure was performed under deep halothane anaes-

thesia for 1 min. To eliminate the effects of stress, all the animals

were manipulated everyday from the day of implantation until the

time of injections, and until the rats were killed (6 days after the

implantation of morphine or placebo pellets). Withdrawal (W) was

induced on day 6 by intraperitoneal injection of naltrexone

hydrochloride (RBI, 20 mg/kg dissolved in isotonic saline), and the

rats were killed by decapitation 60 min after the injection (MW). This

dose was chosen because it produced sustained maximal decrease in

dopamine levels in the NAc accompanied by typical withdrawal

physical signs (Pothos et al., 1991). Effects of naltrexone injection on

c-fos expression were studied as control, on placebo-treated rats

decapitated 60 min following injections of naltrexone at 20 mg/kg

(PW). Effects of saline injection on c-fos expression were studied as

control, on morphine-dependent rats decapitated 60 min following

injections of saline (MS). The experimental schedule for these groups

is summarized in Fig. 1. Additional doses of naltrexone, 1 mg/kg and

30 mg/kg, were also tested since they produced somatic and aversive

signs of opiate withdrawal, respectively (CailleÂ et al., 1999). For the

time-course induction of c-fos, the rats were killed by decapitation 30,

60, 90 or 120 min after the injection of naltrexone (n = 2). All rats

were killed between 10.00 and 12.00 a.m. The brains were then

dissected and frozen with liquid nitrogen without prior ®xation.

Coronal cryostat-sections (10 mm) were collected on gelatine-coated

slides and stored at ±80 °C until use.

Probe synthesis and labelling for in situ hybridization35S-labelled cRNA probes were prepared by in vitro transcription

from cDNA clones corresponding to fragments of the rat c-fos cDNA

(Curran et al., 1987), and rat preproenkephalin A (PPA), D1 and D2

dopamine receptor cDNAs (Yoshikawa et al., 1984; Monsma et al.,

1989; Monsma et al., 1990). Transcriptions were performed from

50 ng linearized plasmid using either 35S-UTP (> 1000 Ci/mmol,

NEN Life Science, France) or digoxigenin-11-UTP (Roche

Diagnostic, France) and SP6 or T3 RNA polymerases, as described

by Le Moine & Bloch (1995). After alkaline hydrolysis to obtain 250-

bp cRNA fragments, the 35S-labelled probes were puri®ed on G50-

Sephadex. The 35S-labelled probes and the digoxigenin-labelled

probes were precipitated in 3 M sodium acetate pH 5 (0.1 : 2.5, v/v in

absolute ethanol).

Single in situ hybridization experiments

Sections were post®xed in 4% paraformaldehyde for 5 min at

room temperature, rinsed twice in 4 3 sodium chloride sodium

citrate buffer (SSC) and placed into 0.25% acetic anhydride in

0.1 M triethanolamine/4 3 SSC (pH 8) for 10 min at room

temperature. After dehydration in graded alcohols, the sections

were hybridized overnight at 55 °C with 106 c.p.m. of 35S-

labelled probe for c-fos mRNAs in 50 mL hybridization solution

(20 mM Tris-HCl, 1 mM EDTA, 300 mM NaCl, 50% formamide,

10% dextran sulphate, 1 3 Denhardt's 250 mg/mL yeast tRNA/

100 mg/mL salmon sperm DNA/0.1% SDS/0.1% sodium thiosul-

phate). The slides were washed in 4 3 SSC (5 min, four times),

RNAse A (20 mg/mL) (20 min, at 37 °C), 2 3 SSC (5 min,

twice), 1 3 SSC (5 min), 0.5 3 SSC (5 min) at room tempera-

ture, and rinsed in 0.1 3 SSC at 65 °C (30 min, twice) before

being dehydrated in graded alcohols (all the SSC washes

contained 1 mM dithiothreitol). The slides were then apposed to

a Biomax ®lm (Kodak) for 1 month and thereafter dipped into

Ilford K5 emulsion (diluted 1 : 3 in 1 3 SSC) and exposed for

3 months, developed and stained with Mayer's haemalun.

Double in situ hybridization experiments

In these experiments, the c-fos cRNA probe was labelled with

[35S]UTP, whereas cRNA probes against preproenkephalin A

(PPA), D1 and D2 receptors were labelled with digoxigenin-11-

FIG. 1. Experimental schedule. P indicates the placebo treatment. Mcorresponds to rats treated during 6 days with morphine. MW indicatesmorphine withdrawal state, 60 min after injection of naltrexone tomorphine-dependent rats. PW and MS are two control groups whichcorrespond to the effect of an injection of naltrexone to placebo rats, or tothe effect of an injection of saline to morphine-dependent rats, respectively.D corresponds to the time the rat was killed.

4476 F. Georges et al.

ã 2000 Federation of European Neuroscience Societies, European Journal of Neuroscience, 12, 4475±4486

UTP. Cryostat sections were pretreated as mentioned above. The

sections were hybridized overnight at 55 °C with a combination

of 35S-and digoxigenin-labelled probes (106 c.p.m. of 35S-labelled

probe and approximately 20 ng of digoxigenin-labelled probe in

TABLE 1. Relative c-fos mRNA expression in rat brain of control animals (P) or following chronic morphine (M), naltrexone (PW, 20 mg/kg), and naltrexone-

precipitated morphine withdrawal (MW, 20 mg/kg)

Brain areas Fig. 2 P M PW MW

Cortical areasAI, agranular insular area A±H + + ++ ++++CA1, Field CA1, Ammon's horn H±L +/± +/± +/± +++CA3, Field CA3, Ammon's horn H±L +/± +/± +/± +++cb, cerebellum P±U +/± +/± + ++++cg, cingulate cortex C±M + + +++ ++++cl, claustrum A±C + + ++ ++++DGlb-sg, Dentate gyrus lateral blade-granule cell layer H±L ± ± + ++++Ed, endoperiform nucleus A±C + + ++ ++++ENT, entorhinal K±P ± ± + ++++M, motor cortex C±M ± ± + ++++P, parietal cortex C±M ± + + ++++PIR, piriform area A±H ++ ++ ++++ ++++pl, prelimbic cortex A, B + + ++ ++++SUBv, subiculum ventral K, L ± ± + ++++

StriatumACb, nucleus accumbens A, B ± ± + ++++Cpu, caudoputamen nucleus B±F ± ± + ++++

Septal areasBNST, bed nuclei stria terminalis E ± ± + ++++LS, lateral septal nucleus D±E ± + ++ ++++MS, medial septal nucleus D±E ± ± ± ++NDB, nucleus of the diagonal band E ± ± ± ++

AmygdalaAAA, anterior amygdaloid area G ± ± +/± ++++CEA, central nucleus amygdala G ± ± ++ ++++MEA, medial nucleus amygdala H ± ± +/± ++++HypothalamusARH, arcuate nucleus hypothalamus H ± ± +/± +++DMH, dorsomedial nucleus hypothalamus I ± ± ± +++LHA, lateral hypothalamic area I ± ± + +++LPO, lateral preoptic area D±F ± ± +/± +++MPO, medial preoptic area F ± ± +/± +++PVH, paraventricular nucleus hypothalamus I ± ± ± +++PH, posterior hypothalamic nucleus I±K ± ± ± ++++SC, suprachiasmatic nucleus G ± ± +/± ++++SO, supraoptic nucleus G ± ± +/± ++++

ThalamusAV, anteroventral nucleus thalamus G ± ± +/± +++LG, lateral geniculate complex I±J ± ± + +++LH, lateral habenula H, I ± ± + ++++LD, lateral dorsal nucleus thalamus H±J ± + + +++LP, lateral posterior nucleus thalamus J, K ± ± +/± +++MD, mediodorsal nucleus thalamus H±J ± ± + +++MG, medial geniculate complex K ± ± + +++MH, medial habenula H, I ± ± + ++++PVT, paraventricular nucleus of the thalamus G±I ± ± +/± ++++VM, ventro medial thalamic area I, J ± ± +/± +++

MidbrainCLI, central linear nucleus rapheÂ M±O ± ± ± +++MRN, mesencephalon reticular nucleus O ± ± ± +++PAG, periaqueductal grey J±O ± ± ± ++Pbm, parabrachial nucleus, medial part P ± ± +/± +++PG, pontine grey M±O ± ++ ++ ++++PN, Pretectal nucleus K ± ± +/± +++SC, superior colliculus K±M ± ± + +++SNc, substantia nigra pars compacta K ± ± ± +SNr, substantia nigra pars reticulata K ± ± ± +VTA, ventral tegmental area K, L ± ± ± +

HindbrainA1, A1 T, U ± ± +/± ++++LC, locus coeruleus Q, R ± ± + ++++NTS, nucleus of the solitary tract S±U ± ± +/± ++++

Rats were implanted with placebo or morphine pellets for a 6-day period. Withdrawal was induced by injection of 20 mg/kg of naltrexone. Relative levels of signalwere analysed on ®lm autoradiograms after in situ hybridization and indicated in the table from ± to ++++.

c-fos induction during morphine withdrawal 4477


50 mL of hybridization solution). As described earlier, the slides

were washed in RNAse A and various concentrations of SSC, but

without dithiothreitol. At the end of the washes, the slides were

put in 0.1 3 SSC at room temperature to cool. The sections were

rinsed twice for 5 min in buffer A (1 M NaCl, 0.1 M Tris, 2 mM

MgCl2; pH 7.5), and then for 30 min in buffer A containing 3%

normal goat serum and 0.3% Triton X-100. After 5 h of

incubation at room temperature with alkaline phosphatase-conju-

gated antidigoxigenin antiserum (Roche Diagnostic, 1 : 1000 in

buffer A/3% normal goat serum, 0.3% Triton X-100), the sections

were rinsed in buffer A (5 min, twice), then for 10 min twice in

buffer B (1 M NaCl, 0.1 M Tris, 5 mM MgCl2; pH 9.5), and

10 min twice in 0.1 M buffer B (containing 0.1 M NaCl). The

sections were then incubated overnight in the dark at room

temperature in 0.1 M buffer B (pH 9.5) containing 0.34 mg/mL

nitroblue tetrazolium and 0.18 mg/mL bromochloroindolylpho-

sphate. The sections were thereafter rinsed in 0.1 M buffer B

(pH 9.5), then in 1 3 SSC, dried and dipped into Ilford K5

emulsion (diluted 1 : 3 in 1 3 SSC). After being exposed for

3 months, the sections were developed and mounted without

further counterstaining.

Data analysis

Quanti®cation for c-fos mRNA expression was performed by

densitometry on X-ray ®lms, as well as by neuronal counting,

depending on the material examined, as described by Le Moine

(2000). Labelled neurons from in situ hybridization experiments

were counted as previously described on similar material (Le

Moine & Bloch, 1995; Le Moine et al., 1997). Accordingly, a

labelled neuron corresponded to a density of silver grains at least

twofold higher than background. One section per animal was

analysed for counting in double in situ hybridization. The

FIG. 2. Film autoradiograms after in situ hybridization showing the expression of c-fos mRNA 1 h after naltrexone-precipitated morphine withdrawal atdifferent levels along a rostrocaudal axis (A±U). Negative images of X-ray ®lms. The regions expressing c-fos mRNA appear in white. Naltrexone-precipitated withdrawal produces an intense induction of c-fos mRNA, especially in CPu, NAc, septal areas, several nuclei of the thalamus or hypothalamus,amygdala and extended amygdala areas, locus coeruleus and cortical areas. All c-fos-positive areas after naltrexone precipitated withdrawal are summarized inTable 1. Controls (B¢, G¢, O¢ representing the same anatomical levels of B, G and O) correspond to placebo rats killed 1 h after naltrexone injection (20 mg/kg). The injection of naltrexone to placebo rats produces an induction of c-fos mRNA in the piriform cortex (B¢) and the central amygdala (G¢). Scale bar,3 mm.



densities of c-fos mRNA-containing neurons were studied in the

dorsolateral CPu as well as in the NAc (+1.2 mm from bregma)

according to Paxinos & Watson (1982). The labelled neurons

were counted using an image analyser system for cartography

(HISTO 200, Biocom, Les Ulis, France) over a surface area of 1±

2 mm2 for the dorsolateral CPu and in the entire NAc (2.5±

3 mm2). The densities of c-fos mRNA-expressing neurons (num-

ber of c-fos- mRNA-positive neurons per mm2) were pooled and

averaged for each group. Statistical analysis was performed using

analysis of variance (ANOVA) followed by post hoc Newman±

Keuls test.

Results

Mapping of c-fos induction in the brain during morphinedependence and withdrawal

Compared with placebo (P), animals implanted for 6 days with

morphine pellets (M) showed a very weak expression of c-fos mRNA

in the brain (Table 1).

Morphine withdrawal (MW) was associated with an intense

c-fos mRNA expression within numerous brain regions 1 h after

naltrexone injection (Fig. 2, Table 1). However, the induction of

c-fos mRNA in morphine-withdrawn rats (MW) was not identical

in all the structures. In the basal ganglia, both the CPu and the

NAc showed an intense expression of c-fos mRNA, whereas c-fos

mRNA was not induced in the globus pallidus or in the

subthalamic nucleus. The CPu showed a rostrocaudal gradient of

labelling with an intense expression of c-fos mRNA in the rostral

CPu which decreased toward the caudal CPu. Within the VTA

and the medial third of the SNr, only a few neurons demonstrated

an intense expression of c-fos mRNA (data not shown). A number

of septal areas, thalamic nuclei (ventral and dorsal nuclei,

posterior nuclei, parafascicular nucleus, reticular nucleus), hypo-

thalamic nuclei (arcuate nucleus, dorsomedial nucleus, paraven-

tricular nucleus, supraoptic nucleus), extended amygdala areas

(bed nuclei stria terminalis [BNST], mediocentral amygdala),

mesencephalic areas (superior colliculus, periaqueductal grey, red

nucleus) and cerebella exhibited intense c-fos mRNA signal. A

strong expression of c-fos mRNA was also detected in

noradrenergic nuclei, especially locus coeruleus (LC), and A1,

FIG. 2. (Continued)



A2 nuclei. Also note that in morphine-withdrawn rats, c-fos

mRNA was highly expressed in most cortical areas including

prelimbic cortex, agranular insular cortex, cingulate cortex, motor

and parietal cortices, ventral subiculum and entorhinal cortex.

Most of these cortical regions showed also a detectable c-fos

labelling in morphine-dependent rats (M) (Table 1). However, the

intensity of c-fos expression in these areas was much lower than

in withdrawn animals (MW).

Scattered c-fos mRNA labelling was also found in the brain of rats

implanted with placebo pellets for 6 days and injected with

naltrexone (PW) (Table 1, Figs 2 and 5D). These areas included

many of the aforementioned cortical areas, as well as the central

nucleus of the amygdala and some thalamic nuclei. However, the

extent of the distribution, as well as the intensity of c-fos expression

in these areas, were much lower than in withdrawn animals (as

illustrated in Table 1 and Fig. 2). In morphine-treated rats injected

FIG. 3. Time-course of the expression of c-fos mRNA in the CPu (whitesquare) and in the NAc (black circle) after injection of naltrexone (20 mg/kg) to morphine-dependent rats. The induction of c-fos mRNA is maximalin both structures during the ®rst hour of withdrawal and decreases duringthe second hour following the injection of naltrexone (m corresponds to theinjection of naltrexone).

FIG. 4. Expression of c-fos mRNA in the NAc and in the CPu during morphine dependence and naltrexone-precipitated withdrawal (20 mg/kg). (A) Negativeimages of X-ray ®lms after in situ hybridization showing the speci®c expression of c-fos mRNA in the CPu and the NAc following placebo treatment (P),chronic morphine (M), naltrexone-induced morphine withdrawal (MW), naltrexone alone (PW), and saline (MS). Scale bar, 3 mm. (B) Histograms showingquanti®cation of c-fos mRNA expression in the NAc and in the CPu following the treatments mentioned above [placebo groups (white); morphine-dependentgroups (black); naltrexone-precipitated withdrawal group (grey)]. Data represent mean 6 SEM of the optical density values measured in the different regions.No change is observed in c-fos mRNA expression after chronic morphine. Naltrexone-precipitated withdrawal produces a strong induction of c-fos mRNA inCPu as well as in NAc. The control group PW shows a weak effect on c-fos expression in the NAc after naltrexone injection. Saline injection in morphine-dependent rats (MS) does not induce c-fos in these areas. An ANOVA followed by Newman±Keuls test for pairwise comparisons was performed for eachregion. sP < 0.01 vs. chronic morphine. rP < 0.01 vs. placebo. Scale bar, 2 mm.



with saline (MS), the c-fos mRNA labelling was similar to placebo-

and morphine-treated groups.

Expression of c-fos in the caudate putamen and in thenucleus accumbens after chronic morphine treatment andprecipitated withdrawal

C-fos mRNA expression during withdrawal precipitated by naltrexone

was evaluated at several time-points during the 2 h following the

injection (Fig. 3). The maximal induction of c-fos appeared during the

®rst hour of withdrawal (in the CPu and NAc) and decreased during the

second hour. No change in the level of c-fos mRNA expression was

observed within the CPu and the NAc after 6 days of morphine

treatment as compared with placebo group (Figs 4, and 5A and B).

Thus for the detailed analysis of the striatal c-fos activation, animals

were killed 1 h after saline or naltrexone injections. The CPu and the

NAc of morphine-withdrawn rats showed an intense c-fos induction

1 h after the injection of naltrexone (+900% in the CPu and +1500% in

the NAc, P < 0.001) (Figs 4 and 5C). Quanti®cation of c-fos induction

was also performed in rats treated with morphine for 6 days and killed

1 h after injection of saline (MS) and in placebo animals injected with

naltrexone (PW). The injection of naltrexone resulted in a signi®cant

induction of c-fos mRNA in the NAc compared with placebo (P) and

morphine-dependant (M) rats, but ®ve times lower than the induction

observed in the striatum of the morphine withdrawn group (MW;

Figs 4 and 5D).

Phenotypic characterization and quanti®cation of c-fos-positiveneurons in the caudate putamen and in the nucleusaccumbens after chronic morphine treatment and precipitatedwithdrawal

In the striatum, phenotypic characterization of the neurons expressing

c-fos mRNA has been performed in placebo (P) and morphine-

dependent rats (M), in morphine-withdrawn rats (MW) injected with

naltrexone 20 mg/kg, and in placebo rats injected with naltrexone

FIG. 5. Localization and phenotypiccharacterization of striatal neurons expressingc-fos mRNA. (A±D) c-fos expression in theNAc after placebo (P), chronic morphine (M),naltrexone-precipitated morphine withdrawal(MW) and naltrexone (PW). Stars indicate theanterior commissure. (E±F) Phenotypiccharacterization of NAc neurons expressingc-fos mRNA following naltrexone-inducedmorphine withdrawal. Double in situhybridization detects preproenkephalin A(PPA) or D1 receptor mRNA withdigoxigenin-labelled riboprobe (stained cells),together with a 35S-labelled riboprobe for c-fosmRNA (silver grains). Black arrowheadspointing to c-fos-positive neurons correspondto striatopallidal PPA-positive neurons (in Eand F), and black arrows pointing to c-fos-positive neurons correspond to striatonigralD1-positive neurons (in E and F). Scale bars,0.3 mm (A±D) and 10 mm (D and E).



(PW). In the striatum, D1 and D2 dopamine receptor mRNAs are

segregated into distinct neuronal populations corresponding to D1/

substance P striatonigral neurons and to D2/enkephalin striatopallidal

neurons (Gerfen & Young, 1988; Gerfen et al., 1990; Le Moine &

Bloch, 1995). Based on these data and for methodological reasons,

we have used D1 mRNA and PPA mRNA to characterize the

phenotype of striatonigral neurons and striatopallidal neurons,

respectively. Figure 5E shows that after precipitated withdrawal, c-

fos mRNA was expressed both in PPA mRNA-positive and -negative

neurons. These data were corroborated by the expression of c-fos in

the same group both in D1 mRNA-positive and -negative neurons

(Fig. 5F). For all the groups, the neurons expressing c-fos have been

quanti®ed on the PPA/c-fos double labellings, in the entire NAc and

in an area representing the two-thirds of the caudate-putamen (level

+1.2 mm from bregma). Neurons have been classi®ed according to

two groups: neurons expressing both c-fos and PPA mRNAs

(corresponding to striatopallidal neurons) and neurons expressing

only c-fos mRNA (corresponding to striatonigral neurons). Figure 6

shows the results expressed in density of c-fos-positive neurons per

square mm. Placebo animals (P) exhibited a very low density of c-fos-

positive neurons (for the caudate-putamen, 4.1 6 1.6/mm2, and for

the Nac, 5.1 6 1.3/mm2) and the proportion in the two populations is

similar. The density of striatal c-fos-positive neurons was similar in

morphine-dependent (M) and in placebo groups, and the proportion

of c-fos/PPA-positive neurons also represented 50% of all c-fos-

positive neurons (Fig. 6).

Naltrexone-precipitated withdrawal (MW) produced a large

increase in the density of c-fos-positive neurons in the CPu and in

the NAc (P < 0.001 vs. M). In fact, in withdrawn rats the density of

c-fos-positive neurons was 117.6 6 15.1/mm2 in the NAc (compared

to 12.2 6 1.7/mm2 at M) and 130 6 14.3/mm2 in the CPu (compared

to 12.0 6 1.6/mm2 at M). Interestingly, the percentage of c-fos-

positive neurons in each population differs in the two areas (Fig. 6).

Indeed, 65% of c-fos-positive neurons in the CPu also expressed PPA

mRNA, while 35% expressed only c-fos mRNA. The NAc showed

the opposite distribution with 35% of c-fos/PPA-positive neurons and

65% of neurons which expressed only c-fos mRNA (Fig. 6).

Moreover, when the shell and core regions of the NAc were

delineated (following Paxinos & Watson, 1982), the proportions of

c-fos-positive neurons in these two regions for each population were

similar (Table 2). In terms of density of c-fos-positive identi®ed

neurons, no signi®cant variation was observed in placebo animals

FIG. 6. Quanti®cation of identi®ed striatal neurons expressing c-fos after withdrawal. Histograms showing the density of striatal neurons expressing c-fosmRNA in the NAc and the CPu following placebo treatment (P), chronic morphine (M), naltrexone-induced morphine withdrawal (MW), naltrexone (PW),and saline (MS). Phenotypic characterization of striatal neurons expressing c-fos mRNA was performed by double in situ hybridization. Neurons whichexpressed only c-fos mRNA are represented by white bars and c-fos/preproenkephalin A (PPA) -positive neurons by black bars. No signi®cant change of c-fos-positive neurons is observed after chronic morphine (M) and the proportion of c-fos-positive neurons only and c-fos/PPA-positive neurons remainunchanged as compared with placebo (1 : 1 ratio). MW shows a strong increase of the number of c-fos-positive neurons in the NAc as well as in the CPu (inthe Nac, 65% of c-fos only positive neurons vs. 35% of c-fos/PPA-positive neurons, and in the Cpu, 35% of c-fos only positive neurons vs. 65% of c-fos/PPA-positive neurons). An ANOVA followed by Newman±Keuls test for pairwise comparisons was performed for each region the primary values, i.e. thedensities of neurons per mm2. sP < 0.01 vs. M. dP < 0.01 between c-fos/PPA-positive and c-fos-positive neurons.

TABLE 2. Density of c-fos/PPA-positive neurons or neurons which expressed only c-fos mRNA in the shell and core parts of the nucleus accumbens following

placebo treatment (P), chronic morphine (M), naltrexone-precipitated morphine withdrawal (MW) and naltrexone (PW)

Treatmentgroup n

Core (neurons per mm2) Shell (neurons per mm2)

c-fos+/PPA+ c-fos+/PPA± c-fos+/PPA+ c-fos+/PPA±

P 6 2.0 6 1.0 4.5 6 1.3 2.0 6 1.0 1.0 6 0.6M 6 8.5 6 1.8 9.7 6 6.9 7.27 6 2.81 4.2 6 1.5MW 6 44.0 6 8.0* 72.7 6 11.7*,² 38.89 6 4.85* 76.3 6 7.6*,²PW 6 14.1 6 4.9 18.2 6 6.9 13.6 6 4.6 11.1 6 3.5

Rats were implanted with placebo or morphine pellets for a 6-day period. Withdrawal was induced by injection of naltrexone (20 mg/kg). c-fos mRNA wasdetected with double in situ hybridization. Values represent the mean 6 SEM of the number of c-fos mRNA-containing neurons per square mm. An ANOVA

followed by Newman±Keuls test for pairwise comparisons was performed for each region. *P < 0.01 vs. M. ²P < 0.01 between c-fos only positive neurons, andc-fos/PPA-positive neurons.



killed 1 h after naltrexone injection (PW) as compared with P and M

groups (Fig. 6). These control animals showed the same proportion of

c-fos/PPA-positive neurons or neurons which expressed only c-fos

mRNA. Interestingly, it is important to note that, despite an overall

lower level of c-fos expression, the proportions of c-fos-positive

neurons for the two striatal populations were similar in both regions

(CPu and NAc) for the other doses of naltrexone tested, 30 mg/kg and

1 mg/kg (Table 3).

Similar results were obtained in a c-fos/D1 double labelling

experiment (see Fig. 5F, Table 4). In the same groups, quantitative

data also showed the expression of c-fos both in D1 mRNA-positive

and -negative neurons (Table 4). These data con®rmed that

naltrexone-precipitated withdrawal (MW) produced a large increase

in the density of c-fos-positive neurons in the CPu and in the NAc

(P < 0.001 vs. M), and that the proportion of activated neurons were

different in the CPu (65% of neurons which expressed only c-fos

mRNA, P < 0.001) and in the NAc (60% of c-fos/D1-positive

neurons, P = 0.08) (Table 4).

Discussion

Our study shows that c-fos mRNA expression is induced in several

brain regions during morphine withdrawal. One hour after injection

of naltrexone to morphine-dependent rats, c-fos mRNA was induced

in the projecting areas of the dopaminergic neurons (NAc, CPu,

prefrontal cortex, olfactory tubercle), of noradrenergic neurons

(BNST, amygdala nuclei, septal areas), and in several brain regions

expressing opiate receptors (LC, thalamus, hypothalamus, cortex).

The dose of 20 mg/kg of naltrexone was chosen to precipitate

withdrawal because it produces sustained maximal decrease in

dopamine levels in the NAc accompanied by typical withdrawal

symptoms (Pothos et al., 1991). However, doses of 30 mg/kg and

1 mg/kg of naltrexone also induced c-fos expression in similar areas,

especially the NAc and CPu, but at lower levels.

After chronic morphine, only basal level of c-fos mRNA is

observed in the areas mentioned above. This absence of c-fos

induction is likely to be speci®c to our model of dependence and

related to the constant and low rate of drug release induced by

subcutaneous morphine implantation which lead to a continuous

steady-state from day 1 (Yoburn et al., 1985). As reported by Nye &

Nestler (1996) in a similar model of morphine-dependant rats, the

level of Fos was not increased in the striatum. Interestingly the

authors showed the induction of novel Fos-like proteins termed

chronic Fos-related antigens (FRAs), which could mediate some of

the long-term effects that morphine exerts in speci®c brain regions

after chronic exposure. These data show the differences in the

intracellular mechanisms involved in gene regulation in other models

of dependence induced by repeated and intermittent injections of

morphine which are ef®cient to induce c-fos (Erdtmann-Vourliotis

et al., 1998). After naltrexone injection in placebo rats, marked c-fos

mRNA inductions were observed in some brain areas, such as the

central nucleus of the amygdala (CEA) and the piriform cortex, and a

lower induction c-fos induction was also observed in the NAc.

Recently, the functional signi®cance of naltrexone-induced Fos

immunoreactivity has been studied especially in the CEA and in

the NAc (Carr et al., 1999). These authors suggest that under basal

conditions endogenous opiates produce an inhibitory control on cells

within the NAc via the m receptors and on cells within the CEA via

the m and kappa (k) receptors.

It has been shown that enhanced c-fos mRNA expression occurs

within neural circuits known to be involved in the symptomatology of

morphine withdrawal (Koob, 1992b). According to a previous

TABLE 4. Density of c-fos/D1-positive neurons or neurons which expressed only c-fos mRNA in the nucleus accumbens and caudate putamen following

placebo treatment (P), chronic morphine (M), naltrexone-precipitated morphine withdrawal (MW) and naltrexone (PW)

Treatmentgroup n

Nucleus accumbens (neurons per mm2) Caudate putamen (neurons per mm2)

c-fos+/D1+ c-fos+/D1± c-fos+/D1+ c-fos+/D1±

P 2 27.1 6 6.2 27.1 6 8.7 24.1 6 3.3 24.1 6 4.1M 3 20.5 6 1.5 17.6 6 1.2 11.7 6 2.1 12.9 6 1.1MW 5 103.3 6 12.9* 67.5 6 5.5* 45.4 6 5.5* 87.4 6 6.1*,²PW 5 34.3 6 6.7 34.8 6 6.3 28.2 6 6.2 31.2 6 6.2

Rats were implanted with placebo or morphine pellets for a 6-day period. Withdrawal was induced by injection of naltrexone (20 mg/kg). c-fos mRNA wasdetected with double in situ hybridization. Values represent the mean 6 SEM of the number of c-fos mRNA-containing neurons per square mm. An anovafollowed by Newman±Keuls test for pairwise comparisons was performed for each region. *P < 0.01 vs. M, ²P < 0.01 between c-fos only positive neurons, and c-fos/D1-positive neurons.

TABLE 3. Density of c-fos/PPA-positive neurons or neurons which expressed only c-fos mRNA in the nucleus accumbens and caudate putamen following

chronic morphine treatment (MS), naltrexone-precipitated morphine withdrawal (MW)

Treatmentgroup n

Nucleus accumbens (neurons per mm2) Caudate putamen (neurons per mm2)

c-fos+/PPA+ c-fos+/PPA± c-fos+/PPA+ c-fos+/PPA±

MS 2 4.4 6 1.7 5.6 6 2.9 6.6 6 1.2 6.5 6 3.5MW (30 mg/kg) 2 9.6 6 2.5 20.2 6 4.5 33.4 6 13.4 26.2 6 14.6MW (1 mg/kg) 2 13.2 6 6.8 30.5 6 17.5 59.4 6 25.1 28.5 6 8.1MW (20 mg/kg) 6 41.0 6 5.4 76.6 6 9.7 83.8 6 7.9 46.2 6 6.6

Rats were implanted with morphine pellets for a 6-day period. Withdrawal was induced by injection of naltrexone (30 mg/kg; 1 mg/kg; 20 mg/kg). c-fos mRNAwas detected with double in situ hybridization. Values represent the mean 6 SEM of the number of c-fos mRNA-containing neurons per square mm.



Northern blotting study (Hayward et al., 1990), we show here by in

situ hybridization that the NAc and the VTA, which may mediate the

rewarding and motivational aspects of drugs of abuse (Mucha &

Herz, 1985; Koob, 1992b), exhibited an increased c-fos mRNA

expression after precipitated withdrawal. Particularly, a strong c-fos

induction was observed in the two major populations of the NAc (i.e.

striatonigral substance P/D1-positive and striatopallidal PPA/D2-

positive neurons). The VTA exhibited a generally moderate increase

that appeared particularly intense in a few neurons (whose topog-

raphy may correspond to GABA interneurons). In addition to the

dopaminergic structures, some noradrenergic structures also showed a

clear activation during withdrawal. Indeed, substantial evidence

indicates that central noradrenergic systems are strongly involved in

morphine withdrawal (Aghajanian, 1978; Gold et al., 1980;

Maldonado et al., 1992; Maldonado & Koob, 1993; CailleÂ et al.,

1999; Delfs et al., 2000). For example, the BNST and its

noradrenergic afferences, such as the nucleus tractus solitarius

(NTS) and the A1 area (Moore, 1978; Swanson & Hartman, 1975;

Phelix et al., 1992), exhibited intense c-fos mRNA expression during

withdrawal. Our results suggest that neurons in these regions are

activated during withdrawal, and give additional evidences for the

implication of the noradrenergic system during morphine withdrawal.

Our data showed that the pattern of c-fos mRNA expression after

precipitated withdrawal globally overlapped with opiate receptor

distribution (Mansour et al., 1988). Overlap occurred within cortical

areas, NAc, CPu, thalamic and hypothalamic nuclei and the LC. This

correlation between opiate receptor expression and c-fos expression

suggest that this IEG might be induced as a consequence of cessation

of opiate receptor stimulation during withdrawal. Indeed, opiate

receptors are negatively coupled to adenylate cyclase (for review see

Akil et al., 1997). Thus, the cessation of opiate receptor stimulation

that occurs during naltrexone-precipitated withdrawal may have an

activating effect on neurons. For example, this activating effect

would possibly explain the c-fos induction observed in the LC.

Indeed, an increased expression of c-fos mRNA during morphine

withdrawal has been described in this structure which showed an

intense expression of m opiate receptors (Hayward et al., 1990;

Couceyro & Douglass, 1995; Chahl et al., 1996). Alternatively, it is

possible that other neurotransmitters, such as glutamate, mediate a

part of the c-fos mRNA induction in the LC, as previously shown in

electrophysiological and behavioural studies (Rasmussen, 1995;

Rasmussen et al., 1996; Vandergriff & Rasmussen, 1999). In

contrast, it has been shown that the total speci®c lesions of LC

noradrenergic neurons, do not modify either naloxone-induced place

aversion or somatic withdrawal syndrome (CailleÂ et al., 1999),

suggesting that the c-fos induction in the LC is not directly correlated

to the acute behavioural expression of withdrawal.

c-fos mRNA expression after a decreased stimulation of receptors

coupled to Gi proteins, has been shown in a model of acute lesion of

dopaminergic neurons. Indeed, the inhibition of dopamine release in

the striatum after an acute injection of 6-hydroxydopamine in the

median forebrain bundle causes an induction of c-fos mRNA in

dopamine D2 receptor-containing striatopallidal neurons

(Svenningsson et al., 1999). Similarly, it is possible that the blockade

of opiate receptors during withdrawal induced a direct expression of

c-fos mRNA in neurons expressing these receptors.

Also, despite the dif®culty in discriminating the in¯uence of stress

from the direct effects of opiate withdrawal itself on c-fos mRNA

expression, the possibility that stress participates in the neuronal

activation observed during withdrawal should be considered.

Previous studies have indeed indicated that corticotropin-releasing

factor contributed to the anxiogenic and aversive states associated

with morphine withdrawal (Heinrichs et al., 1995; Iredale et al.,

2000). Based on these ®ndings, it is possible that stress contributes to

withdrawal behaviour and regulation of c-fos mRNA expression in

speci®c brain areas.

Taken together, our data support the view that levels of c-fos

expression re¯ect acute changes in the physiological states of

neurons, and could be used to identify neuronal populations that

are activated during morphine withdrawal.

Interestingly, our study demonstrates that naltrexone-precipitated

withdrawal induces the expression of c-fos mRNA in the two

populations of striatal efferent neurons: the striatonigral neurons

(containing substance P/dynorphin and D1 receptors) and the

striatopallidal neurons (containing enkephalins and D2 receptors).

However, the proportion of neurons of these two populations

expressing c-fos mRNA differs between the NAc and the CPu.

Indeed the majority of c-fos-positive neurons in the NAc are

striatonigral/D1-containing neurons, while the majority in the CPu

are striatopallidal/D2-containing neurons.

Regulation of c-fos mRNA expression was analysed in previous

reports from our group and has provided a more complete

understanding of the mechanisms through which dopamine regulates

c-fos mRNA induction in striatal neurons (Le Moine et al., 1997;

Svenningsson et al., 1999). Indeed, the acute blockade of dopamine

release leads to a strong induction of c-fos mRNA in striatopallidal

neurons (Svenningsson et al., 1999). Moreover, microdialysis studies

have clearly shown an inhibition of dopamine release after an

injection of opiate receptor antagonist to morphine-dependent rats

(Pothos et al., 1991; Rossetti et al., 1992; Crippens & Robinson,

1994; Spanagel et al., 1994). This inhibition has also been

demonstrated in the NAc and in the CPu by biochemical procedures

(Honkanen et al., 1994; Ghosh et al., 1998). Therefore, the induction

of c-fos mRNA in striatopallidal D2-positive neurons of the NAc and

CPu after administration of naltrexone to morphine-dependent rats

probably re¯ects a direct in¯uence of the decrease in dopamine levels

on these D2 receptor-containing neurons. Accordingly, a recent study

has shown that the stimulation of the D2 receptor attenuates the

expression of FRAs in the NAc during opiate withdrawal (Walters

et al., 2000). However, it is dif®cult to correlate the induction of c-fos

mRNA in the population of striatonigral D1-positive neurons, with

the inhibition of dopamine release occurring during withdrawal.

Based on our previous results which established that m and k opioid

receptor mRNAs are speci®cally expressed in neurons expressing D1

receptors (Georges et al., 1999), we propose that c-fos induction in

this population could result from a disactivation of m and k opiate

receptors. This could also account for the largest proportion of c-fos

striatonigral positive neurons in the NAc, which shows a higher

density of k opiate receptors than the CPu (Mansour et al., 1988).

Another hypothesis to explain the induction of c-fos mRNA in the

striatonigral neurons is a direct opposite in¯uence of dopaminergic

and opioidergic systems at the single-cell level. We have previously

showed that m and k mRNAs are expressed in striatonigral neurons

together with D1 mRNA (Georges et al., 1999). Previous studies have

demonstrated by biochemical measures of cAMP levels, a direct

interaction between m and D1 receptors which resulted in an

inhibitory effect of morphine on D1-mediated dopaminergic activity

(Schoffelmeer et al., 1995). Thus, precipitated withdrawal by

naltrexone could shunt the inhibitory in¯uence of m opioid receptors

by morphine, and then allow a stimulatory effect of residual

dopamine via D1 receptors.

Alternatively, it is possible that other neurotransmitters, such as

glutamate, mediates a part of the c-fos mRNA induction especially in

the striatum. In fact, previous studies have described a large increase



of extracellular level of glutamate in the NAc following naltrexone-

precipitated withdrawal (Sepulveda et al., 1998). On the other hand, it

has been clearly demonstrated that some symptoms of opiate

withdrawal syndrome can be inhibited by MK801 (Marek et al.,

1991; Trujillo & Akil, 1991). Intracerebral injection of glutamate to

morphine-dependent rats is also able to induce a withdrawal

syndrome, and this glutamate-precipitated withdrawal is prevented

by pretreatment with MK801 (Tanganelli et al., 1991; Tokuyama

et al., 1996). Taken together, these data support the possibility that

during withdrawal, the increase of glutamate release may participate

in the induction of c-fos mRNA in the two populations of the

striatum. This induction may occur via the stimulation of NMDA

receptors which are expressed by both striatopallidal and striatonigral

neurons, and which modulate D1 and D2 receptor activity (Micheletti

et al., 1992; Lannes et al., 1995; Healy & Meador-Woodruff, 1996).

In conclusion, naltrexone-precipitated morphine withdrawal in-

creases c-fos mRNA levels in structures strongly associated with the

behavioural expression of withdrawal. In particular, morphine

withdrawal induces c-fos expression in the two efferent populations

of the striatum (i.e. striatonigral and striatopallidal neurons). The

proportion of activated neurons are different in the CPu (majority of

striatopallidal neurons) and in the NAc (majority of striatonigral

neurons). Our data strongly suggest that the induction of c-fos gene

expression in striatal populations during morphine withdrawal and

especially in striatonigral neurons, is not a direct consequence of the

accompanying changes in dopamine release, but probably involves

interactions between dopamine and opioid systems, as well as other

neurotransmitters.

Acknowledgements

The authors thank S. CailleÂ, M. Cador and B. Bioch for their major interest tothe present study and for helpful discussions. We also thank C. Vidauporte forexpert photographic artwork and M. Manse for excellent and skilful technicalassistance. This work was supported by the Centre National de la RechercheScienti®que (CNRS), the UniversiteÂ Victor Segalen Bordeaux 2, the ConseilReÂgional d'Aquitaine, and by special funds from the Mission InterministeÂriellede Lutte contre le Drogue et la Toxicomanie (number 96CO20).

Abbreviations

BNST, bed nuclei stria terminalis; CEA, central nucleus amygdala; CPu,caudate putamen; D1, D1-dopamine; D2, D2-dopamine; IEGs, immediateearly genes; LC, locus coeruleus; M, morphine; MS, morphine and salinetreatment; MW, morphine withdrawn; NAc, nucleus accumbens; P, placebotreatment; PPA, preproenkephalin A; PW, placebo withdrawn; SNc, substantianigra pars compacta; SNr, substantia nigra pars reticulata; SSC, sodiumchloride sodium citrate buffer; VTA, ventral tegmental area.

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Mapping of c-fos gene expression in the brain during morphine dependence and precipitated...

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