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The Journal of Neuroscience, November 1995, 75(11):
7189-7195
Stress-Induced Sensitization and Glucocorticoids. II.
Sensitizationof the Increase in Extracellular Dopamine Induced by
CocaineDepends on Stress-Induced Corticosterone SecretionFranqoise
RougbPont, Michela Marine lli, Michel Le Moal, Herv6 Simon, and
Pier Vincenzo Piazzal&chobio logie des Comportements
Adaptatifs, INSERM U259, Universitk de Bordeaux II, Domaine de
Carreire,33077 Bordeaux Cedex, France
Secretion of glucocorticoids seems to control stress-in-duced
sensitization of the behavioral effects of drugs ofabuse by acting
on the mesencephalic dopaminergic trans-mission, the principal
neural substrate of sensitization. Inorder to investigate the
mechanisms of this interaction be-tween glucocort icoids and
dopamine, we studied the sen-sitization of the increase in
extracellular concentration ofdopamine induced by cocaine in male
rats in which cot-&costerone secretion was either intact or
blocked. Extracel-lular concentrations of dopamine were evaluated
in the nu-cleus accumbens of free ly moving animals by means
ofmicrodialysis. Metyrapone, an inhibitor of
corticosteronesynthesis, was used to block stress-induced
cotticoste-rone secretion. Food-restriction (90% of the initial
bodyweight) was the stressor used to induce sensitization. Itwas
found that metyrapone (100 mg/kg S.C. twice a day for8 d)
suppressed stress-induced sensitization of the in-crease in
accumbens dopamine induced by cocaine (10mg/kg, i.p.) and
sensitization of cocaine-induced locomo-tion. Metyrapone suppressed
both the development andthe expression of sensitization. Thus,
sensitization wasequally blocked when the metyrapone treatment
started ei-ther 1 d before the start of food-restriction or 8 d
later, thatis, when food-restriction-induced sensitization to
cocainewas already established. In conclusion, our results
suggestthat glucocorticoids modi fy sensitization of the
behavioraleffects of cocaine by acting on extracellular
concentrationsof dopamine. Since addictive properties of
psychostimu-lants seem mediated by the increase in extracellular
con-centrations of dopamine they induce, these findings mayhave
implications for the development of new therapeuticstrategies of
addiction.
[Key words: glucocorticoids, stress-induced sensitiza-tion,
cocaine, dopamine, nucleus accumbens, drug abuse]Stress-induced
sensitization of the motor and addictive eff ectsof psychostimulant
and opioid drugs (Kalivas and Stewart, 1991;Robinson and Berridge,
1993) seems mediated by stress-inducedReceived Apr. 6, 1995;
revised June 13, 1995; accepted June 21, 1995.
This work was supported by lnstitu t National de la SantC et de
la RechercheMBdicale (INSER M), UniversitC de Bordeaux II , Consei
Regional dAquitaine,PBle MCdicame nt dAquitaine, Ministtire de la
Recherche et de IEnseigne mentSupCrieur. We thank Martine Kharouby
for precious technical help. M.M . wassupported by GI gran t of the
Dottora to di Ricerca in Neuroscienze Universityof
Rome.Correspondence should be addressed to Dr. Pier Vincenzo
PiaLLa, INSERMU259, Rue Camille Saint-S&x, 33077 Bordeaux
Cedex, France.Copyright 0 1995 Society for Neuroscience
0270-6474/95/l 57 189-07$05.00/O
secretion of glucocorticoids. First, stress-induced
sensitization ofthe motor e ffe cts of psychostimulants and opioids
is suppressedby the removal of the adrenal glands (Deroche et al.,
1992a.1993a, 1994), the principal source of endogenous
glucocorti-coids. Second, sensitization is reinstated in stressed
adrenalec-tomized rats by the administration of corticosterone, the
princi-pal glucocorticoid in the rodent, at doses reproducing
stress lev-els of the hormone (Deroche et al., 1995). Third,
repeated cor-ticosterone injections, to unstressed rats, induce
sensitization ofthe locomotor (Deroche et al., 1992b) and
reinforcing effects ofamphetamine (Piazza et al., I99 I a).
Glucocorticoids seem to control stress-induced sensitizationby
acting on the mesencephalic dopaminergic transmission, theprincipal
neural substrate of sensitization (Robinson and Becker,1986;
Kalivas and Stewart, 1991; Robinson and Berridge, 1993).Thus,
sensitization of the locomotor responses to either am-phetamine or
morphine, injected, respectively , into the nucleusaccumbens or the
ventral tegmental area (VTA), are abolishedby the suppression of
stress-induced corticosterone secretion(Deroche et al., 1995). The
locomotor activation induced by theinjection of psychostimulants
(Kelly and Iversen, 1976; Delfs etal., 1990) and opioids (Joyce and
Iversen, 1979; Kalivas et al.,1983; Vezina and Stewart, 1984) in
these cerebral area dependson the mesencephalic dopaminergic
transmission.
The mechanisms by which glucocorticoids control the
sensi-tization of dopamine-dependent effects of drugs is still
largelyunknown. Modulation by glucocorticoids of drug-induced
in-creases in the extracellular concentration of dopamine is a
pos-sibility: (I) an increase in extracellular dopamine mediates
motorand reinforcing eff ects of psychostimulants and at least in
partthose of opioids (Fibiger and Phillips, 1988; Koob and
Bloom,1988; Wise and Rompre, 1989; Le Moal and Simon, 1991); (2)the
expression of behavioral sensitization is associated with
anenhancement of drug-induced increases in extracellular
concen-tration o f dopamine (Kalivas and Stewart, 1991); (3)
mesence-phalic dopaminergic neurons have glucocorticoid
receptors(HCirfstrand et al., 1986) and glucocorticoids can modify
dopa-mine metabolism (Ho-Van-Hap et al., 1967; Versteeg et
al.,1983; Rothschild et al., 1985) and extracellular
concentrationsof dopamine (Imperato et al., 1989; Mittleman et al.,
1992).
The possible modulation by glucocorticoids of
psychostimu-lant-induced increase in extracellular concentrations
of dopa-mine was studied in this report. In particular, the
developmentand the expression of stress-induced sensitization of
the dopa-minergic response to cocaine was compared in rats in
which
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7190 Rouge-Pont et al. . Sensitization of Accumben s Dopamine
and Glucocotticoids
corticosterone secretion was either intact or blocked.
Sensitiza-tion of cocaine-induced locomotion was also studied.
Extracellular concentrations of dopamine were measured inthe
nucleus accumbens of free ly moving animals by means
ofmicrodialysis. Stress-induced corticosterone secretion wasblocked
by metyrapone [2-methyl-I,2-di(3-pyridy1)-2-propa-none] an
inhibitor of the enzyme I I-P-hydroxylase (Jenkins etal., 19.58;
Chart and Sheppard, 1959). Food-restriction was usedas a stressor,
since the behavioral sensitization it induces (Camp-bell and
Fibiger, I97 I ; Carroll et al., 1979; Carroll and Meisch,198 I ;
Papasava and Singer, 1985; De Vry et al., 1989) dependson both
glucocorticoids and dopamine (Deroche et al., 1995).Materials and
MethodsSubjectsMale Sprague-Dawley rats (I ffa Credo, Lyon, France)
weighing 280-300 gm at the beginning of the experiments were used.
Animals wereindividually housed with ad libitum access to food and
water. A constantlight-dark cycle (on 12 P.M., of f 12 A.M.) was
maintained in the animalhouse, in which temperature (22C) and
humidity (60%) were con-trolled.Gene& methodsDrugs and dnq
udministrufion. Metyrapone [2-methyl-l
&di&pyri-dyl)-2-propanone] (Sigma) was freshly dissolved in
a 0.9% NaCl salinesolution containing 3% of Tween 80, and was
injected subcutaneouslyat a dose of 100 mg/kgR ml twice a day (at
IO A.M. and 6 PM.) for8 d. Controls received. with an identical
schedule, injections of vehiclesolution (0.9% NaCl + 3% Tween 80).
This dose and schedule of treat-ment was chosen because we have
previously shown that, in identicalconditions, this metyrapone
treatment selectively blocks stress-inducedcorticostero ne secretio
n (Piazza et al., 1994). Coca ine hydrochloridewas dissolved in
0.9% NaCl solution and was injected intraperitoneallyat a dose of
IO mg/kg/ml.
Loconroror ucti\~ify. Animals were tested for locomotor activity
in acircula r corridor ( IO cm wide and 70 cm in diameter). Four
photoele c-tric cells placed at the perpendicular axes of this
apparatus automaticallyrecorded locomotion. Since it has been
previously shown that locomotorresponse to novelty is correlated to
the dopam inergic activity in thenucleus accumbens (Hooks et al.,
1991 b; Piazza et al., 1991 b; RougC-Pont et al., 1993) and to the
sensitiv ity to the psychomo tor effects ofdrugs (Piazza et al.,
1989; Hooks et al., 199la; Deroche et al., l993b).we ensured a
homogenous distribution of this factor throughout thedifferent
experimental groups. For this purpose, after a period of I weekof
habituation to the housing conditions, and before any other
manip-ulation , anim als w ere tested for their locomo tor response
to novelty andevenly distribute d in the different experimental
groups according totheir activity score cumulate d over the 2 hr of
testing .Food-restrktion. Animals were weighed daily and the ration
of foodwas progressively reduced in order to bring, over 4 d, the
body weightto 90% of its initial value. Food-restricted animals
were then maintainedat this weight throughout the entire
experiment. 90% of food-restrictionhas been choosen in the present
report because in preliminary experi-ments this level of
restriction has been used to test the specificity ofmetyrapone
effects (Piazza et al., 1994; M. Marinelli, M. Le Moal, andP V.
Piazza unpublished observation).Microdiulysis. Rats were
chronically implanted with a guide cannula(CMA/I l-Carnegie Medicin
Sweden) in the nucleus accumbens undersodium pentobarbital
anesthesia (SO mg/kg i.p.). The guide cannula waslowered to 2 mm
above the locati on of the probe tip. The stereotaxiccoordin ates
relative to bregma were: A/P = +3.6, L = +2.0, V =~6.5 from the
surface of the skull, with the incis or bar set at +S.O mm
with a lateral angle of 6 according to the stereotaxic atlas of
Pellegrinoet al. ( 1979). A recovery period of IO d after surgery
was given priorthe start of all other manipu lations . The dial
ysis probe (CMA/I 1, Car-negie Medicin, 2 mm membrane length) was
inserted through the guidecann ula, 48 hr before starting the
perfusion (Osborne et al., 199 1 a), andduring this period the rat
was returned to its home cage. The day of theexperiment animals
were transferred to the dialysis cage (32 X 32 X22 cm), the probe
connected via a dual channel swivel (Instech) to aHarvard syringe
micro liter pump 22 and the perfusion started imme -diately at a
flow rate of 2 kl/min. The perfusion fluid was a modified
artificial cerebrospinal fluid (145 m M NaCl I .2 mM CaCl?, 2.7
mM K CI,I mM MgC$, and 0.2 mM NaZHPO,/NaH,PO, buffered at pH
7.4).
These condltlons were used because they have been described to
efti-ciently reflec t DA synaptic release and to minimize the
influence of anyedema induced by the implantation of the probe
(Moghaddam and Bun-ney, 1989 ; Osborne et al., 1991 b).Dopamine
ussa~. Brain dialysis was performed with a fully automatedon-line
system. Dlalysate was collected in a 40 pl sample loop. Every21 min
the dialysate was automatically injected into the HPLC
system(Rheodyne 712.5) in combination with a sample/event
controller (Tou-zart et Matignon, France). The HPLC system cons
isted of a ShimadzuLC-9A pump and an analytical column (Hypersil
BDS-C IX, 3 km, IO0X 4.6 mm; Shandon, France). The mobile phase was
a sodium phos-phate buffer (75 mmol) containing 20 pmol of EDTA,
I.5 mmol ofsodium dodecyl sulfate, 100 pl triethylamine, 15%
methanol, and 13%acetonitrile, pH 5.6 delivered at a constant flow
of 0.9 ml/min. A Cou-lometric detector (Coulochem II, ESA, USA)
with a SO14 High Perfor-mance Analytical Cell was used. A model
5020 guard cell was posi-tioned before the column to oxidize at
+3SO mV. The first electrodereduced at - 175 mV and the second
electrode oxidized at I75 mV toquantify only dopamine. Signals were
recorded with a D2000 Megaintegrator (Merck). The retention time of
dopam ine was of 6.S min andthe detection limit of this compound
during the assay was of 0.5 pg.Experimentul gmups. Five identical
groups of animals were used forthe two experiments of this report.
Four groups of rats were submittedto food-restriction, whereas the
fifth group was fed ad libitum andserved as control (ad libitu m
fed controls). Food-restricted anim als weretreated w ith either
metyrapone or vehicl e. Two of these groups, onetreated with
metyrapone, the other with vehicle, were used for the studyof the
developmen t of sensi tization . The other two groups were
utilizedfor the study of the expression of sens itization . For the
study on thedevelopment of sensi tization , the vehicle or
metyrapone treatment start-ed one day before the beginn ing of food
restrictio n. For the study onthe expression of sensitiz ation, the
treatment started after 8 d of food-restriction. Since the two
food-restricted groups treated with vehicle didnot differ for all
the parameters studied, they were cumulated in all ofthe figures
and defined as food-restricted controls. The metyraponegroups used
for the study of the developme nt and expression of sens i-tization
were respectively named metyrapone treated before food-re-strictio
n and metyrapone treated after food-restriction.Hisrology. At the
end of the experiments, the animals were anesthe-tized with sodium
pentobarbital and perfused transcardially with SO mlof 0.9% NaCl
saline solution and then with SO ml of 10% formalinsolution. The
brains were removed and stored in 10% formalin solutionuntil
verification of cannula placement. For this purpose, brains werecut
on a freezing microtome (Kryostat System, Dittes-Dispu va.
Ger-many), and the precise location of the probe determined in
coronal serialsections usin g thionin staining. Only the animals
with correctly placedimpla ntation s (probes membrane in the
medial-an terior part of the nu-cleus accumbens) were included in
the statistical analysis.ProceduresExperimenr 1: ef fec t of
stress-induced corticosterone secretiorr CM thesensitization of the
dopaminergic response to cocaine it) the r7wleu.sclccunzhens.
Animals were divided in the five experimental groups de-scribed
above. On the day of the dialysis test, animals were placed inthe
dialysis cage at 1 I A.M. After 2 hr of habituation to this
apparatus,the animals received an injection of cocaine (IO mg/kg
i.p.). Thus, ratswere tested 3 hr after the last inje ction of
metyrapone or vehicle . San-ples of dialysate were obtained from
the nucleus accumbens over 20min intervals for 2 hr after the coca
ine injec tion. For this experimentnine food-restricted controls
(respectively II = 5 and II = 4 for eachtime conditio n), eight
food-restricted rats treated with metyrapone (n =4 for each time
condition), and seven ad libitum fed controls were used.A supplemen
tary set of anim als was tested in order to verify the effectsof
food-restriction and metyrapone treatments on the dopam inergic
re-sponse to the injection of 0.9% NaCl saline solution. For this
purposesix food-restricted contro ls (n = 3 for each time cond
ition) and sixfood-restricted rats treated with metyrapone (II = 3
for each time con-dition) were compared to four ad libitu m fed
controls .Experiment 2: +ct qfstress-induced c~orric~osterorle
secretion 012 hesensitization of the locomotor response to cocaine.
For this experiment,the five experimental groups were placed in the
circula r corridor at I IA.M. After a 2 hr period of habituation,
animals received an injectionof either saline or cocaine. The
locomotor response to the two injections
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The Journal of Neuroscience, November 1995, 15(11) 7191
VEHICLE COCAINE
Ad libifum Fed ControlsFood-restricted ControlsMetyrapone
Treated before Food-restrictionMetyrapone Treated after
Food-restriction i
-40 -20 0 20 40 60 -40 -20 0 20 40 60 80 100 120TIME (min) TIME
( min)
Figure 1. Extracellular concentrations of dopamine in the
nucleus accumbens in response to the intraperitoneal injection of
either 0.9% NaClsolution (SALINE) or cocaine (10 mg/kg). The
different experimental groups did not dif fer fo r their
dopaminergic response to saline. In contrast,food-restricted
animals pretreated with vehicle (Food-restricted Controls) showed a
higher increase in extracellular concentrations of dopamine thanad
libitum fed controls. This difference was significant during the
first hour after the injection of cocaine [F(2,12) = 8.04, P <
0.0061. Developmentand expression of food-restriction-induced
sensitization were suppressed by pretreatment with metyrapone.
Food-restricted rats in which the treatmentwith metyrapone (100
mg/kg s .c., twice a day for 8 d) started either I d before the
beginning of food-restriction (Metyrapone treated before
food-restriction) or 8 d later (Metyrapone treated after
food-restriction) had a lower response to cocaine than
food-restricted animals [F( I, 13) = 10.45,P < 0.0061 and did
not diff er from ad libitum fed controls.
was recorded, over 10 min intervals , for a period of 1 hr after
the salineinjection and of 2 hr after the cocaine injection. All
the food-restrictedgroups contained eight subjects, whereas the ad
libitum fed controlscontained seven rats.Sraristid unrr~~is. Basal
extracellular dopamine concentrations, anddopamine concentrations
after saline or cocaine injections (expressed aspercentage o f
baseline), and locomotor response to saline or cocainewere compared
by analysis of variance (ANOVA) for repeated mea-sures.
Newman-Keuls test was used for post hoc
comparisons.ResultsExperiment I: effe ct of stress-induced
corticosterone secretionon the sensitizution of the dopaminergic
response to cocainein the nucleus accumbensThe injection of cocaine
nduceda significant increasen nucleusaccumbens extracellular
concentrations of dopamine [F(5,90) =48. I I, P < O.OOl] that
was maximal 20-40 min after the injec-tion. This effect was
modified by food-restriction and metyra-pone treatments Treatment
effect, F(4,18) = 3.31 P < 0.051 ina time dependent manner
[Treatment X Time interaction,F(20,90) = 1.88, P < 0.051.
Effect s of,food-restriction on cocaine-induced increase in
do-putnine. Food-restriction ncreased he efflux of dopamine n
re-sponse o cocaine. Food-restricted groups treated with vehicleand
ad libitum fed controls significantly differed [F(2,12) =4.04, P
< 0.051 n a time dependentmanner Treatment ;ts Timeinteraction
F(10,60) = 1.99, P < 0.051. This difference waspresent during
the first hour after the injection of cocaine[F(2,12) = 8.04, P
< 0.006] but not during the second one[F(2,12) = 0.80, P =
0.4691. Post hoc comparisons evealed
that animalssubjected o either 7 or 16 d of food-restriction
hada higher increase n cocaine-induceddopamineefflux than adlibitum
fed controls(P < 0.05 andP < 0.01 respectively). How-ever the
two food-restricted groups did not differ (P = 0.44).For this
reason he two food-restricted groups treated with ve-hicle, in
Figure 1, are cumulated as food-restricted controls.Eff ects of
metyrapone on cocaine-induced increase in dopa-mine. Metyrapone
significantly reduced the enhancement f do-pamineoverflow observed
n food-restricted animals.This effectwas similar when the
metyrapone treatment was started eitherbefore or after the start of
food-restriction (Fig. I, right panel).Indeed, a bifactorial
analysisshoweda significant effect of me-tyrapone [F( 1.13) =
10.45, P < 0.006] but did not reveal anyinteraction between this
effect and the scheduleof treatment[F(1,13) = 0.380, P = 0.5451.The
effect of metyraponevariedover time [metyrapone X Time interaction
F(5.65) = 3.28, P
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7192 Roug&Pont et al. - Sensitization of Accumbens Dopamine
and Glucocort icoids
VEHICLE COCAINE
g8
350 -tA5 300 -ri w20E 250 -
E 200 -5a 150-55s
loo-
Ad libifum Fed Controlsd libifum Fed ControlsFood-restricted
Controlsood-restricted ControlsMetyrapone Treated before
Food-restrictionetyrapone Treated before Food-restrictionMetyrapone
Treated after Food-restrictionetyrapone Treated after
Food-restriction
- 350 68
- 300 g250 $-
fj- 200 52
10 20 30 40 50 60 20 40 60 80 100 120TIME (min) TIME (min)
Figurr 2. Locomotor response to the intraperitoneal injection of
either 0.9% NaCl solution (SALINE) or cocaine (IO mg/kg). The
differentexperimental groups did not dif fer for their locomotor
response to saline. In contrast, food-restricted-animals treated
with vehicle (Food-restrictedConrr&), showed a higher locomotor
response than ad libitum fed controls. This difference was
significant during the firs t hour after the injectionof cocaine
[F( I,2 I ) = 7.13, P < 0.02]. Development and expression of
food-restriction-induced sensitization were suppressed by
metyrapone. Food-restricted rats in which the treatment with
metyrapone (100 mglkg s.c. , twice a day for 8 d) started either 1
d before the beginning of food-restriction(Mrtyrupone Treuted
before Food-restr iction) or 8 d later (Metyrupone Treuted uffer
Food-restricf ion) had a lower locomotor response to cocainethan
food-restricted animals [F( 1,28) = 20.46, P < 0.00021 and did
not differ from ad libitum fed controls.
saline [F(4,12) = 0.318, P = 0.801 (Fig. 1, lef t panel).
Thismanipulation per se did not modify extracellular
concentrationsof dopamine [F(2,22) = 0.18, P = 0.831. Furthermore,
therewere no group differences in baseline extracellular
concentra-tions of dopamine (pg/40 ~1) the hour preceding the
saline andcocaine injections [F(2,32) = 0.27, P = 0.701. Baseline
valueswere: ad libitum fed controls, 3.66 + 0.7; food-restricted
con-trols, 3.44 +- 0.8, and metyrapone-treated animals, 2.898 ?
0.6.Experiment 2: effe ct of stress-induced corticosterone
secretionon the sensitization of the locomotor response to
cocaineFood-restriction and metyrapone treatments significantly
modi-fied the locomotor response to cocaine [Treatment eff ect
,F(4,34) = 6.39, P < 0.0061 in a time dependent manner
[Treat-ment X Time interaction, F(44,374) = 2.80, P <
O.OOl].Effects qf food-restriction on cocaine-induced locomotion.
Inparallel to what was observed for dopamine, animals subjectedto 8
or 16 d of food-restriction did not dif fer [F(l,l4) = 0.53,P =
0.4801. For this reason the two food-restricted groups arecombined
in Figure 2 as food-restricted controls. Food-restrictedanimals
treated with vehicle showed a higher locomotor re-sponse to cocaine
than ad libitum fed controls [F( I,2 1) = 5.46,P < 0.031 and
this difference changed over time [F( 11,231) =3.5, P < O.OOl].
Thus, the effect of food-restriction was presentduring the first
[F( I,2 I) = 7.13, P < 0.021 but not during thesecond hour
[F(l,2l) = 0.36, P = 0.5541 after the injection ofcocaine.
Effect s of metyrapone on cocaine-induced locomotion.
Metyr-apone significantly reduced the increase in the locomotor eff
ects
of cocaine observed in food-restricted animals. The effects
ofmetyrapone were similar both when the treatment started before(1
d) and after (8 d) the beginning of food-restriction. Indeed,
abifactorial analysis showed a significant eff ect of metyrapone[F(
1,28) = 20.46, P < 0.0002] but did not reveal any
interactionbetween this ef fec t and the schedule of treatment [F(
l,28) =0.036, P = 0.8511. The eff ect of metyrapone varied over
time[metyrapone X Time interaction F(l 1,308) = 6.70, P <
O.OOl],but was significant both during the first [F( 1,28) = 19.96,
P
