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Corticotropin-Releasing Factor, But Not Corticosterone, Is Involved
in Stress-Induced Relapse to Heroin-Seeking in Rats
Yavin Shaham,
2
Douglas Funk,
1
Suzanne Erb,
1
Theodore J. Brown,
3
Claire-Dominique Walker,
4
and
Jane Stewart
1
1
Department of Psychology, Center for Studies in Behavioral Neurobiology, Concordia University, Montre´ al, Que´ bec,
Canada H3G 1M8,
2
Biobehavioral Research Department, Addiction Research Foundation, Toronto, Ontario, Canada
M5S 2S1,
3
Department of Zoology, University of Toronto, Toronto, Ontario, Canada M6A 2EI, and
4
Department of
Psychiatry, McGill University, Douglas Hospital Research Center, Montre´ al, Que´ bec, Canada H4H 1R3
We showed previously that brief footshock stress and priming
injections of heroin reinstate heroin-seeking after prolonged
drug-free periods. Here, we examined whether the adrenal
hormone, corticosterone, and brain corticotropin-releasing fac-
tor (CRF) were involved in such reinstatement. We tested the
effects of adrenalectomy, chronic exposure to the corticoste-
rone synthesis inhibitor metyrapone (100 mg/kg, s.c., twice
daily), acute exposure to metyrapone, acute intracerebroven-
tricular injections of CRF (0.3 and 1.0
m
g), and intracerebroven-
tricular injections of the CRF antagonist
a
-helical CRF (3 and 10
m
g). Rats were trained to self-administer heroin (100
m
g/kg/
infusion, i.v.) for 12–14 d. Extinction sessions were given for
4–8 d (saline substituted for heroin). Tests for reinstatement
were given after priming injections of saline and of heroin (0.25
mg/kg, s.c.), and after intermittent footshock (15 or 30 min, 0.5
mA). Adrenalectomy (performed after training) did not affect
reinstatement by heroin but appeared to potentiate the rein-
statement by footshock. Chronic exposure to metyrapone (from
the beginning of extinction) or an acute injection of metyrapone
(3 hr before testing) did not alter the reinstatement of heroin-
seeking induced by footshock or heroin. Acute exposure to
metyrapone alone potently reinstated heroin-seeking. In addi-
tion, acute exposure to CRF reinstated heroin-seeking, and the
CRF antagonist
a
-helical CRF attenuated stress-induced re-
lapse. The effect of the CRF antagonist on reinstatement by
heroin was less consistent. These results suggest that CRF, a
major brain peptide involved in stress, contributes to relapse to
heroin-seeking induced by stressors.
Key words: adrenalectomy; corticosterone; CRF; metyra-
pone; opioid self-administration; reinstatement; relapse; stress
High rates of relapse to drug use after long periods of abstinence
characterize the behavior of experienced users of drugs of abuse
(Jaffe, 1990). In both humans and nonhumans, acute reexposure
to the self-administered drug is a potent event for provoking
relapse to drug-seeking (Stewart and de Wit, 1987; de Wit, 1996).
In addition, exposure to stress, an event long thought to be
important to relapse in humans (Shiffman and Wills, 1985), can
induce relapse to heroin- and cocaine-seeking in the rat (Shaham
and Stewart, 1995; Erb et al., 1996).
The mechanisms involved in the effect of stress on reinstate-
ment of heroin-seeking are not understood, but it appears that the
neurochemical events underlying reinstatement by footshock and
heroin are not identical. Reinstatement by heroin is blocked by
the opioid receptor antagonist naltrexone, the D
2
-like receptor
antagonist raclopride, a high dose of the D
1
-like antagonist SCH
23390, and the mixed dopamine (DA) receptor antagonist flu-
penthixol; only flupenthixol attenuates reinstatement of heroin-
seeking induced by footshock (Shaham and Stewart, 1996). Fur-
thermore, a “maintenance” dose of heroin (delivered
continuously via an Alzet osmotic minipump during extinction
and tests) attenuates reinstatement induced by heroin, but not by
footshock stress (Shaham et al., 1996).
Here, we determined whether the adrenal hormone corticoste-
rone and brain corticotropin-releasing factor (CRF) are involved
in the effects of stress and heroin-priming on relapse to heroin-
seeking. Corticosterone is involved in a variety of behavioral and
neurochemical effects of exposure to stress (Selye, 1956; Johnson
et al., 1992). There are reports that exposure to corticosterone
facilitates the initiation of self-administration of low doses of
psychostimulants (Piazza and Le Moal, 1996) and reinstates
cocaine-seeking (Deroche et al., 1996), whereas adrenalectomy
and chronic exposure to the corticosterone synthesis inhibitor
metyrapone (Jenkins et al., 1958; Haynes, 1990) decrease cocaine
self-administration during the maintenance phase (Piazza et al.,
1994; Goeders and Guerin, 1996).
CRF also mediates many of the behavioral and physiological
symptoms of the stress response via its effects on the hypothalam-
ic–pituitary–adrenal axis as well as via its extrahypothalamic ef-
fects (Dunn and Berridge, 1990; Johnson et al., 1992; Menzaghi et
al., 1993; de Souza, 1995). CRF plays a role in the anxiogenic and
aversive effects of withdrawal from drugs of abuse, including
opioid drugs (Menzaghi et al., 1994b; Heinrichs et al., 1995; Merlo
Pich et al., 1995; Sarnyai et al., 1995). These observations, and
those indicating a central role of CRF in the coordination of the
stress response, raise the possibility that CRF contributes to the
reinstatement effects of footshock stress.
In the present studies, we assessed the effects of adrenalectomy
Received Dec. 2, 1996; accepted Jan. 21, 1997.
This work was supported by grants from the National Institute of Drug Abuse, the
Medical Research Council of Canada, and Fonds pour la Formation de Chercheurs
et l’Aide a´ la Recherche (FCAR, Que´bec). Y.S. was supported by a Postdoctoral
Fellowship from the Medical Research Council of Canada. We thank Shirley Leung,
Demetra Rodaros, and Kathy Coen for their expert technical assistance.
Correspondence should be addressed to Dr. Jane Stewart, Center for Studies in
Behavioral Neurobiology, Department of Psychology, Concordia University, 1455 de
Maisonneuve Boulevard, Montre´al, Que´bec, Canada H3G 1M8.
Copyright q 1997 Society for Neuroscience 0270-6474/97/172605-10$05.00/0
The Journal of Neuroscience, April 1, 1997, 17(7):2605–2614
and metyrapone pretreatment on reinstatement of heroin-seeking
in drug-free rats induced by intermittent footshock and by heroin.
We then tested the effects of intracerebroventricular (ICV) injec-
tions of CRF on reinstatement of heroin-seeking and the effects of
pretreatment with the CRF antagonist
a
-helical CRF on rein-
statement induced by footshock and by priming injections of
heroin.
MATERIALS AND METHODS
Experiment 1: adrenalectomy and metyrapone
Subjects. The subjects were 63 male Long–Evans rats (Charles River,
Raleigh, NC; 300–400 gm). Thirty-seven rats were studied for self-
administration of heroin. Four of these animals developed blocked cath-
eters, and their data are not included in the tests for reinstatement. They
were, however, used for hormonal measurements at the end of the
experiment. The animals were transferred from the animal housing
facility to operant chambers 1 week after surgery. The animals lived in the
operant chambers for 24 hr per day and were maintained on a reversed
light/dark cycle (lights on 10:00 P.M. to 10:00 A.M.) throughout the
experiment. Food and water were available ad libitum except during the
3 hr tests for reinstatement (see below). Twenty-six drug-naive rats were
used for hormonal measurements after exposure to metyrapone and
footshock. These rats were maintained on a reverse light/dark cycle in the
animal facility with food and water available ad libitum. The drug-naive
rats were brought to the testing apparatus 24 hr before the start of the
experiment.
Surgery. The animals were surgically implanted with intravenous SI-
LASTIC catheters (Dow Corning, Midland, MI) in the right jugular vein
under either sodium pentobarbital anesthesia (MTC Pharmaceutical,
Cambridge, ON; 65 mg/kg, i.p.) or a mixture of xylazine (Haver, Etobi-
coke, Ontario; 10 mg/kg, i.p.) and ketamine HCl (Vetrepharm, London,
Ontario; 100 mg/kg, i.m.). Atropine sulfate (MTC Pharmaceutical; 0.6
mg/ml; 0.3 ml/animal) and penicillin B (Wyeth-Ayerst, Montreal, Que´-
bec; 300,000 IU; 0.2 ml/animal) were given at the time of surgery. The
catheter was secured to the vein with a silk suture and passed subcuta-
neously to the top of the skull, where it exited into a connector (a
modified 22 gauge cannula, Plastic One, Roanoke, VA) mounted to the
skull with jeweler’s screws and dental cement. The catheters were flushed
daily with 0.1 ml of a saline–heparin solution (30 U/ml heparin, ICN
Biochemical, Cleveland, OH).
Apparatus. The operant chambers used had two levers located 9 cm
above the floor, but only one lever (an “active,” retractable lever; Med
Associates, Lafayette, IN) activated the infusion pump (Razel Scientific
Instruments, Stamford, CT). Presses on the other lever (“dummy,” sta-
tionary lever) were recorded but did not activate the infusion pump. A
given drug dose was infused at a volume of 0.13 ml during a 20 sec period.
During the infusion, a light located above the active lever was lit for 20
sec. Bar presses during those 20 sec were counted but did not lead to
additional infusions. Throughout the experiment, each session began by
the introduction of the retractable lever into the cage and the illumina-
tion of the white light above the lever for 30 sec. A red house light was
turned on for the entire session. The grid floors of the chambers were
connected to electric shock generators (Grason-Stadler, West Concord,
MA, or Med Associates, Lafayette, IN).
Drugs. Diacetylmorphine HCl (heroin) was obtained from Health and
Welfare, Canada, and dissolved in physiological saline. Metyrapone
[2-methyl-1,2-di(3-pyridyl)-2-propanone] was obtained from Sigma (St.
Louis, MO) and dissolved in distilled water containing 2% of Tween 80.
The latter solution was used for vehicle injections. Metyrapone was
injected subcutaneously at a dose of 100 mg/kg. This dose was chosen
based on previous studies (Piazza et al., 1994; Rouge-Pont et al., 1995).
Procedures. The animals were divided into four groups for the rein-
statement study [metyrapone-acute (MET-ACUTE, n 5 9), metyrapone-
chronic (MET-CHRONIC, n 5 6), adrenalectomy (ADX, n 5 9), and
sham-operated (SHAM, n 5 9; see below)]. The experiment was run in
four phases: initiation of heroin self-administration, maintenance, extinc-
tion, and reinstatement testing. During the initiation phase, all rats were
trained to self-administer heroin (100
m
g/kg per infusion) over 5–7 d
during which each lever press was reinforced except those made during
the infusion. Each day was divided into four 3 hr sessions (two during the
dark and two during the light), separated by 3 hr. The first session of each
day started at the beginning of the dark period, 10:00 A.M. During the
maintenance phase, rats were allowed to self-administer heroin for an
additional 7–8 d in one 6 hr session per day (10:00 A.M.–4:00 P.M.). On
the last day of the maintenance phase, the rats self-administered a lower
dose of heroin (50
m
g/kg per infusion). The animals showed the charac-
teristic increase in rate of responding when the dose of heroin was
decreased (mean 6 SEM numbers of infusions for 100 and 50
m
g/kg unit
doses per 6 hr were 25.9 6 1.8 and 41.0 6 3.0, respectively). The
extinction phase consisted of 4 d (one 6 hr session/day; 10:00 A.M.–4:00
P.M.) during which presses on the “active” lever resulted in saline
infusions. Before the first day of extinction, the rats were divided into the
four experimental groups.
Animals in the MET-ACUTE group were not injected during the
extinction phase (metyrapone was injected only during the testing phase;
see below). Animals in MET-CHRONIC were injected with metyrapone
(100 mg/kg, s.c.) twice a day (7:00 A.M. and 5:00 P.M.) from the first day
of extinction; the injections continued throughout the extinction and
testing phases described below. This injection regimen was chosen on the
basis of previous studies (Piazza et al., 1994; Rouge-Pont et al., 1995).
Animals in the ADX group were adrenalectomized rapidly under light
methoxyflurane (Metofane, Jannsen Pharmaceutical, Mississauga, ON)
anesthesia. Surgery was performed 1 hr after the start of the light cycle in
the evening after the last session of the maintenance phase. Rats were
allowed 36 hr to recover from surgery before the start of the extinction
phase. After surgery, the rats were given physiological saline instead of
water in their drinking bottles. Animals in the SHAM group were exposed
to similar experimental manipulations as the rats in the ADX group,
except that the adrenal glands were not removed and they were not given
physiological saline in their drinking bottles.
At the end of the tests for reinstatement, rats in the ADX and SHAM
groups were killed by decapitation at the beginning of the dark cycle to
check for the completeness of the adrenalectomies. Trunk blood was
collected in heparinized tubes and centrifuged for 20 min at 3000 rpm at
48C. The plasma was extracted and stored at 2208C. The assays for the
ADX and SHAM animals were performed in Toronto by T.J.B. using
an [
125
I]corticosterone RIA kit (ICN Biomedical, Cost Mesa, CA). All of
the ADX animals had values below the detection limits of the assay;
mean (6SEM) 1.5 6 0.5
m
g/dl. The mean for the SHAM animals was
21.1 6 2.7.
Tests for reinstatement of heroin-seeking followed the extinction
phase. The duration of the test sessions was 3 hr (10:00 A.M. to 1:00
P.M.), and tests were conducted in the absence of food. Tests for
reinstatement in response to a priming injections of saline continued for
up to 3 d until the criterion of less than 15 responses on the active lever
was reached. Animals in the MET-ACUTE group were then given two
additional tests for the priming effects of saline overa2dperiod in a
counterbalanced order. In these tests, animals were given either metyrap-
one (100 mg/kg, s.c.) or the vehicle 3 hr before the start of the test
sessions; a SC priming injection of saline was given within 10 min of the
start of the test session. These animals were given four additional daily
tests for reinstatement in a counterbalanced order; after a priming
injection of heroin 10 min before the session (0.25 mg/kg, s.c.; two tests
48 hr apart); and after 15 min of intermittent footshock terminating just
before the start of the session [0.5 mA, 0.5 sec on with a mean off period
of 40 sec (range, 10–70 sec); two tests 48 hr apart]. Three hours before
each of these tests, animals were treated with either metyrapone or the
vehicle.
Animals in the MET-CHRONIC group were tested overa4dperiod
after the baseline tests with priming injections of saline; after exposure to
intermittent footshock (two tests 48 hr apart) and after heroin-priming
injections (two tests 48 hr apart) in a counterbalanced order. As men-
tioned, these animals were given the twice-daily metyrapone injections
during the testing phase.
Animals in the ADX and SHAM groups were tested after the baseline
tests with priming injections of saline, after exposure to priming injections
of heroin (two tests 48 hr apart) and after 15 min of intermittent
footshock (two tests 48 hr apart) in a counterbalanced order as described
previously. All tests for reinstatement were conducted under extinction
conditions during which presses on the active lever resulted in saline
infusions. The priming dose of heroin and the parameters of the foot-
shock stress were chosen on the basis of previous studies (Shaham and
Stewart, 1996; Shaham et al., 1996).
At the end of the tests for reinstatement, MET-ACUTE animals (n 5
10) were exposed twice to the intermittent footshock for 15 min at the
start of the dark cycle. Three hours before these daily shock sessions,
animals were injected with either vehicle or metyrapone. Tail blood was
collected in heparinized tubes immediately before exposure to footshock
2606 J. Neurosci., April 1, 1997, 17(7):2605–2614 Shaham et al. • CRF and Relapse to Heroin-Seeking
(prestress) and immediately after exposure to footshock (poststress) for
measurements of plasma corticosterone. MET-CHRONIC animals (n 5
9) were exposed to footshock 3 hr after an injection of metyrapone; tail
blood was collected before and after exposure to footshock as described
for the MET-ACUTE group. On the last day of the experiment, eight
animals from group MET-CHRONIC were either exposed (n 5 4) or not
exposed (n 5 4) to footshock 3 hr after exposure to the last metyrapone
injection; they were immediately decapitated, and trunk blood was taken
in tubes containing 20 ml of a 60 mg/ml solution of EDTA for measure-
ments of plasma ACTH.
Drug-naive rats (n 5 26) were used for additional characterization of
plasma levels of ACTH and corticosterone after exposure to metyrapone
and footshock. These rats were divided into four groups (n 5 6–7) for
treatment in a 2 (vehicle vs metyrapone) 3 2 (no footshock vs footshock)
factorial design: vehicle–no footshock (control); vehicle–footshock; 100
mg/kg metyrapone–no footshock; and 100 mg/kg metyrapone–footshock.
Metyrapone or vehicle was injected 3 hr before the start of the dark cycle,
and 15 min of intermittent footshock was administered at the start of the
dark cycle. Rats were decapitated, and trunk blood was collected either
after exposure to footshock or 3 hr after exposure to metyrapone in the
no-footshock conditions. The blood was centrifuged for 20 min at 3000
rpm at 48C, and the plasma was extracted and stored at 2708C for
subsequent radioimmunoassays to determine corticosterone and ACTH
levels.
Radioimmunoassays
Corticosterone. For the experiments on the effects of metyrapone and
stress on corticosterone levels, corticosterone was extracted from plasma
with anhydrous ethanol, and corticosterone levels were assayed in dupli-
cates with an antibody to corticosterone obtained from Endocrine Sci-
ences (Tarzana, CA) and
3
H-labeled corticosterone obtained from Du-
pont (Boston, MA). In routine plasma corticosterone determinations
performed in the Concordia laboratory, the intra- and interassay coeffi-
cients of variation are 4.0 and 10.0%, respectively.
ACTH. Plasma ACTH concentrations were measured by a sensitive
radioimmunoassay as described previously (Walker, 1995). Briefly, 50
m
l
of plasma was incubated with a specific ACTH antiserum generously
supplied by Dr. W. Engeland (University of Minnesota). The [
125
I]ACTH
was purchased from Incstar (Stillwater, MN). The limit of detection was
0.78 pg/tube, and interassay and intra-assay coefficients of variation were
26% (n 5 12) and 8% (n 5 16), respectively.
Statistical analyses. The data used for the statistical analyses in the
reinstatement study were those from the last test for the priming effect of
saline and, when appropriate, the mean values from each of the two tests
for reinstatement with the heroin prime and footshock. Data were col-
lected for each hour of testing. Three separate analyses were carried out.
The first was performed on the data from the MET-ACUTE group and
examined the effect of acute metyrapone pretreatment on reinstatement
after priming injections of saline or heroin or exposure to footshock. Data
were analyzed by repeated-measures ANOVA using the factors of Test
Condition (saline, heroin, and footshock); Pretreatment Condition (me-
tyrapone or vehicle); and Hour (each hour of test). The second analysis
examined the data from the MET-CHRONIC group and determined the
effect of chronic metyrapone injections on footshock- and heroin-induced
reinstatement. The data were analyzed by repeated-measures ANOVA
for Test Condition (saline, heroin, and footshock) and Hour. A third
analysis was performed on the data from the ADX and the SHAM groups
and examined the effect of adrenalectomy on reinstatement. Repeated-
measures ANOVA used the between-subject factor of Adrenalectomy
(adrenalectomy vs sham) and the within-subject factors of Test Condition
(saline, heroin, and footshock) and Hour.
The data from the hormonal assays were analyzed by ANOVAs. In the
case of the heroin-trained animals in group MET-ACUTE, there was one
between factor (vehicle vs metyrapone) and one within factor (Pre- and
Poststress). For the drug-naive animals, there were two between factors
(vehicle vs metyrapone and no stress vs stress). In all cases, post hoc
analyses were performed using the Fisher least significant difference test
with a significance level of p 5 0.05 (two-tailed).
Experiment 2: CRF and
a
-helical CRF
Subjects and surgery. The subjects were 56 male Long–Evans rats. Twenty
rats were excluded because of catheter failure during training, loss of
head caps, sickness, or improper cannula placement. Rats were also
excluded if they did not meet the extinction criterion after four ICV
vehicle injections (see below). The rats were maintained under the
conditions described in experiment 1. During the surgery for intravenous
catheterization, each rat was also implanted with a 22 gauge guide
cannula (Plastic One) above one of the lateral ventricles. The stereotaxic
coordinates used were 20.9 mm from bregma, 1.4 mm lateral from the
midline, and 23.0 mm from the skull surface. The incisor bar was set
at 23.5 mm.
Drugs and injection procedures
Human/rat CRF and
a
-helical CRF [9–41] were obtained from Sigma.
CRF (0.3 and 1
m
g/rat) was dissolved in physiological saline and
a
-helical
CRF (3 and 10
m
g/rat) was dissolved in distilled water with the pH
adjusted to 6.7. The drugs were injected ICV at a volume of 5
m
l, and the
intracranial injections were given over a 30–45 sec period. The 28 gauge
injector was extended 1 mm below the tip of the guide cannula. The
injectors were retained in position for an additional 30–45 sec after the
injection. At the end of the experiment, rats were overdosed and perfused
transcardially with 0.9% saline followed by 10% formalin. The brains
were removed and sliced in 40 mm frozen sections for verification of the
placement of the cannulae. ICV injections were given within 15 min
before the start of the test sessions. The doses of CRF and
a
-helical CRF
are based on previous reports (Cador et al., 1993; Heinrichs et al., 1994;
Menzaghi et al., 1994a).
Procedures
The training and extinction procedures for heroin self-administration
differed to some degree from those used in experiment 1. Animals were
trained to self-administer heroin (100
m
g/kg per infusion) as described
above over an 11–14 d period. Each day was divided into three 3 hr
sessions separated 3 hr apart. The first session of each day started at 10:00
A.M. (the start of the dark period). On the last day of training, the rats
self-administered a lower dose of heroin (50
m
g/kg per infusion). The
animals showed the characteristic increase in rate of responding when the
dose of heroin was decreased (mean 6 SEM numbers of infusions for 100
and 50
m
g/kg/U doses per 3 hr were 10.1 6 0.9 and 16.3 6 1.4, respec-
tively). The extinction phase consisted of 4–5 d. The first day consisted of
three 3 hr sessions, and subsequent days consisted of one 3 hr session
(10:00 A.M.–1:00 P.M.) during which presses on the “active” lever re-
sulted in saline infusions.
For tests of reinstatement, the rats were divided to three groups. In one
group (n 5 10), the effect of ICV injections of CRF on reinstatement of
heroin-seeking was examined. For comparison purposes, the effects of
footshock and heroin-priming on reinstatement were also determined.
All animals were initially exposed to an ICV injection of the vehicle once
a day for one to four sessions until they met the extinction criterion of less
than 15 responses per 3 hr. Subsequently, they were tested after ICV
injections of two doses of CRF (0.3 and 1
m
g/rat), two durations of
footshock (15 and 30 min), and a priming injection of heroin (0.25 mg/kg,
s.c.) in a counterbalanced order.
The other two groups were tested for the effect of acute pretreatment
with the CRF antagonist
a
-helical CRF on reinstatement induced by
priming injections of saline and heroin (0.25 mg/kg, s.c.) and exposure to
footshock (15 min). Each group of rats was tested with only one ICV dose
of
a
-helical CRF (3 or 10
m
g; n 5 14 and 12, respectively), and lever press
scores were compared with those obtained after ICV vehicle injections.
The order of these conditions was counterbalanced. All animals were
initially tested for one to four daily sessions after ICV injections of vehicle
and SC injections of saline until they met the extinction criterion of less
than 15 responses per 3 hr.
Statistical analyses
The data from the CRF and the CRF antagonist groups were treated
separately. For the CRF agonist group, the effects of CRF, footshock, and
heroin-priming on reinstatement were examined in separate repeated-
measures ANOVAs. The baseline condition for these analyses was the
last vehicle pretreatment condition in which the criterion of extinction
was met. The statistical analyses for the effect of the CRF antagonist on
reinstatement were done in separate ANOVAs in which the heroin-
priming condition and the footshock condition were compared with the
saline-priming condition. As mentioned, each rat was exposed to the
vehicle and one dose of the CRF antagonist and tested for the effects of
saline-priming, heroin-priming, and footshock on reinstatement. The
factors in these analyses were the Pretreatment Condition (vehicle vs CRF
antagonist, within-subject factor); Antagonist Dose (3 vs 10
m
g, between-
subject factor); Test Condition (either saline-priming vs heroin-priming or
saline-priming vs footshock, within-subject factor); and Hour.
Shaham et al. • CRF and Relapse to Heroin-Seeking J. Neurosci., April 1, 1997, 17(7):2605–2614 2607
RESULTS
Experiment 1: adrenalectomy and metyrapone
Acute metyrapone
Figure 1 shows the mean number of lever presses on the active
lever made during the tests for reinstatement after acute pretreat-
ment with either metyrapone or vehicle. Surprisingly, pretreat-
ment with metyrapone, 3 hr before the priming injection of saline,
reinstated heroin-seeking. In subsequent tests, both heroin injec-
tions and footshock reinstated heroin-seeking, regardless of the
pretreatment condition. The ANOVA revealed a significant effect
of Pretreatment Condition (F
(1,8)
5 6.9, p , 0.05) and marginally
significant effect of Pretreatment Condition 3 Test Condition
(F
(2,16)
5 3.1, p 5 0.074). Analyses within the pretreatment
conditions revealed significant effects of Test Condition (F
(2,16)
5
5.6, p , 0.05) in the vehicle pretreatment condition, but not in the
metyrapone pretreatment condition (F
(2,16)
5 1.2, NS). This latter
finding was attributable to the fact that in the saline condition,
metyrapone injections brought about levels of responding as high
as those seen after heroin and footshock. Significant differences
between groups are indicated in Figure 1. No significant differ-
ences were observed for lever presses on the inactive lever (data
not shown).
Chronic metyrapone
Figure 2 shows the mean number of lever presses on the inactive
and active levers made during the 3 hr tests for reinstatement after
exposure to saline, heroin, and footshock in animals chronically
treated with metyrapone. It can be seen that the metyrapone
treatment regimen did not block reinstatement by either priming
injections of heroin or footshock. The statistical analysis revealed
significant effects of Test Condition (F
(2,10)
5 10.5, p , 0.05) and
Test Condition 3 Hour (F
(4,20)
5 9.3, p , 0.05). The interaction
effect reflects the fact that most of the responses after exposure to
footshock and heroin occurred in the first hour of testing. The
results of the post hoc tests are indicated in Figure 2. No signifi-
cant effects of Test Condition were obtained for lever presses on
the inactive lever. It should be noted that chronic metyrapone
injections had deleterious effects on the overall health of the rats,
including skin irritation around the injection areas and swollen
glands.
Adrenalectomy
Figure 3 shows the mean number of lever presses on the active
lever made during the 3 hr of tests for reinstatement after expo-
sure to saline, heroin, and footshock in animals from the ADX
and SHAM groups. Adrenalectomy did not block reinstatement in
response to priming injections of heroin or footshock. The statis-
tical analysis revealed significant effects of Test Condition (F
(2,32)
5 13.6, p , 0.05); Test Condition 3 Adrenalectomy (F
(2,32)
5 4.0,
p , 0.05); and Test Condition 3 Hour (F
(4,64)
5 7.4, p , 0.05). The
interaction reflects the fact that ADX animals made a greater
number of responses after footshock, but not after exposure to
saline- or heroin-priming, than animals in the SHAM group. The
results of the post hoc tests are indicated in Figure 3. No signifi-
cant effects were obtained for lever presses on the inactive lever.
Acute metyrapone and the first day of extinction
An unexpected observation was that in the MET-CHRONIC
group, the first injection of metyrapone given on the first day of
extinction increased lever-pressing to levels 2 to 3 times that seen
in animals in the ADX, SHAM, and MET-ACUTE groups not
given metyrapone (Group: F
(3,29)
5 4.4, p , 0.05 and Group 3
Hour: F
(5,145)
5 43.4, p , 0.05). Figure 4 shows the mean number
of lever presses during each hour of the first extinction session.
Significant differences between groups are indicated. No signifi-
cant differences between groups were observed on any of the
subsequent extinction sessions (data not shown).
Hormonal response to metyrapone and footshock
Table 1A shows the mean plasma levels of corticosterone in
heroin-trained animals from the reinstatement study taken before
and after exposure to 15 min of footshock. It can be seen that
acute injections of metyrapone (MET-ACUTE, n 5 10) did not
Figure 1. A, Mean (6 SEM) number of lever presses on the previously
active lever in the 3 hr after noncontingent priming SC injections of saline
and heroin (0.25 mg/kg, s.c.) and exposure to 15 min of intermittent
footshock stress in rats in the MET-ACUTE group (n 5 9). Rats were
pretreated with either metyrapone (100 mg/kg, s.c.) or vehicle 3 hr before
the start of the test of reinstatement. Lever presses result in saline
infusions during the tests. B, Time course of lever-pressing. *Different
from the vehicle condition, p , 0.05.
2608 J. Neurosci., April 1, 1997, 17(7):2605–2614 Shaham et al. • CRF and Relapse to Heroin-Seeking
alter baseline levels of plasma corticosterone, but blocked
footshock-induced corticosterone release in these animals. This is
evident from the significant interaction of Metyrapone 3 Foot-
shock (F
(1,9)
5 6.0, p , 0.05). Similarly, there was no effect of
footshock stress on corticosterone levels in animals from group
MET-CHRONIC, n 5 9, (F
(1,8)
5 2.3, NS). At the end of the
experiment, trunk blood was taken from animals in group MET-
CHRONIC to measure ACTH levels before and after footshock
stress. The levels of ACTH were elevated in these animals regard-
less of exposure to footshock stress (No Stress, 2028.7 6 321.0
pg/ml; Stress, 2220.7 6 639.5 pg/ml). For comparison, see the
levels in the drug-naive animals exposed to acute injections of
metyrapone (Table 1, Drug naive). Table 1 shows the mean
plasma levels of corticosterone and ACTH in drug-naive animals
treated with an acute injection of metyrapone or vehicle and
subjected or not subjected to footshock stress. It can be seen again
that the acute injection of metyrapone blocked the corticosterone
response to stress seen after injections of the vehicle alone. The
ANOVA revealed a significant effect of Metyrapone (F
(1,22)
5
10.0, p , 0.05) and Footshock (F
(1,22)
5 9.7, p , 0.05). ACTH
levels rose in vehicle-treated animals subjected to stress, but the
Figure 2. A, Mean (6 SEM) number of lever presses on the previously
active and inactive levers in the 3 hr after noncontingent SC priming
injections of saline and heroin (0.25 mg/kg, s.c.) and exposure to 15 min
of intermittent footshock stress in rats in the MET-CHRONIC group (n 5
6). Rats were pretreated with metyrapone (100 mg/kg, s.c., twice per day)
starting on first day of extinction. B, Time course of lever-pressing on the
active lever. *Different from the priming injections of saline, p , 0.05.
Figure 3. A, Mean (6 SEM) number of lever presses on the previously
active lever in the 3 hr after noncontingent SC priming injections of saline
and heroin (0.25 mg/kg, s.c.) and exposure to 15 min of intermittent
footshock stress in rats in the ADX and SHAM groups (n 5 9 per group).
Adrenalectomy was performed at the end of the maintenance phase. B,
Time course of lever-pressing. #Different from the SHAM group, p , 0.1.
Shaham et al. • CRF and Relapse to Heroin-Seeking J. Neurosci., April 1, 1997, 17(7):2605–2614 2609
levels were equally high in metyrapone-treated animals, whether
or not they were subjected to stress. Neither the effect of Metyrap-
one (F
(1, 21)
5 3.4, p 5 0.08) nor of Footshock (F
(1,21)
5 2.9, p 5
0.10) reached statistical significance. Significant differences be-
tween groups are indicated in Table 1.
Experiment 2: CRF and
a
-helical CRF
CRF
Figure 5 shows the mean number of lever presses on the active
lever made during the 3 hr tests for reinstatement after exposure
to ICV saline and CRF, heroin-priming, and footshock. As ex-
pected, both heroin and footshock reinstated heroin-seeking.
More importantly, both doses of CRF reinstated heroin-seeking.
The ANOVA for the vehicle condition and the two CRF doses
revealed significant effects of Pretreatment Condition (F
(2,18)
5 7.6,
p , 0.01) and Pretreatment 3 Hour (F
(4,36)
5 3.0, p , 0.05),
indicating that most of the responses after exposure to CRF were
made in the first hour of testing. There were no significant
differences between the two doses of CRF. The ANOVA for the
vehicle condition and the two stress conditions revealed signifi-
cant effects of Stress (F
(2,18)
5 7.0, p , 0.01) and Stress 3 Hour
(F
(4,36)
5 6.6, p , 0.01), indicating that most of the responses
after exposure to the stressor were done in the first hour of
testing. The ANOVA for the vehicle condition and the heroin-
priming condition revealed significant effects of Heroin (F
(1,9)
5
10.3, p , 0.01) and Heroin 3 Hour (F
(2,18)
5 4.5, p , 0.05),
indicating that most of the responses after exposure to heroin
were done in the first 2 hr of testing. Post hoc group differences
are indicated in Figure 5.
a
-Helical CRF
Figure 6 shows the mean number of lever presses on the active
lever made during the 3 hr tests for reinstatement after pretreat-
ment with ICV vehicle or
a
-helical CRF. Both doses of the CRF
antagonist attenuated reinstatement induced by footshock. Rein-
statement by heroin-priming was slightly attenuated by the low
dose of the CRF antagonist, but not with the high dose. It is
interesting to note that the high dose of
a
-helical CRF, given
alone, partially reinstated heroin-seeking. The statistical analysis,
comparing the footshock condition with the saline-priming con-
dition, revealed significant effects of Pretreatment Condition
(F
(1,24)
5 7.9, p , 0.01); Test Condition (F
(1,24)
5 34.5, p , 0.01);
Pretreatment Condition 3 Test Condition (F
(2,48)
5 16.2, p , 0.01);
and Pretreatment Condition 3 Test Condition 3 Hour (F
(2,48)
5
15.6, p , 0.01). These analyses indicate that pretreatment with the
CRF antagonist attenuated stress-induced reinstatement, an ef-
fect that was most pronounced in the first hour of testing. There
was no significant effect of Antagonist Dose (F
(1,24)
5 0.7, NS).
The statistical analysis, comparing the heroin-priming condition
with the saline-priming condition, revealed significant effects of
Antagonist Dose (F
(1,24)
5 8.2, p , 0.01); Test Condition (F
(1,24)
5
18.4, p , 0.01); and Pretreatment Condition 3 Test Condition
(F
(2,48)
5 7.7, p , 0.01), indicating that pretreatment with the
CRF antagonist attenuated heroin-induced reinstatement at the
low dose, but not at the high dose. The CRF antagonist did not
affect presses on the inactive lever. Post hoc group differences are
indicated in Figure 6.
DISCUSSION
A major finding in the present report is the involvement of CRF
in the reinstatement effect of footshock stress. Acute ICV injec-
tions of CRF reinstated heroin-seeking, whereas pretreatment
with the CRF antagonist
a
-helical CRF attenuated stress-induced
reinstatement. In contrast, a role for CRF in reinstatement by
heroin is less clear, because although the low dose of
a
-helical
CRF somewhat attenuated the priming effect of heroin, the higher
dose was without an effect. This latter finding may be related to
the partial agonist effects of the high dose of
a
-helical CRF, as
manifested by its ability to partially reinstate heroin-seeking (Fig.
6B). The CRF antagonist at this dose range has also been shown
to produce CRF-like effects in other behavioral procedures (Hein-
richs et al., 1994; Menzaghi et al., 1994a). Thus, it is possible that
at a low dose,
a
-helical CRF slightly attenuated the heroin-
priming effect by inhibiting brain systems directly involved in the
reinstatement effect of heroin, whereas at a high dose, the CRF-
Figure 4. A, Mean (6 SEM) number of lever presses on the previously
active and inactive levers in the 6 hr of the first extinction day for the four
experimental groups. Metyrapone-treated rats (MET-CHRONIC group)
were injected for the first time with 100 mg/kg, s.c., 3 hr before the start
of the extinction session. Animals in the No Injection condition were those
in the MET-ACUTE group before any treatments. The extinction session
for the SHAM and ADX group began 36 hr after surgery. B, Time course
of lever-pressing. *Group differences, p , 0.05.
2610 J. Neurosci., April 1, 1997, 17(7):2605–2614 Shaham et al. • CRF and Relapse to Heroin-Seeking
like effects of the drug counteracted this effect. A possible ana-
tomical site for this interaction is the mesolimbic DA system that
mediates reinstatement by heroin (Stewart, 1984; Stewart and
Vezina, 1988). Previous studies have shown that CRF induces
sensitization to the locomotor activating effects of d-amphetamine
(Cador et al., 1993) and increases DA utilization in the prefrontal
cortex and the nucleus accumbens (Dunn and Berridge, 1987;
Lavicky and Dunn, 1993; but see Kalivas et al., 1987).
It is possible that the observed effects of CRF on drug-seeking
are related to the extra hypothalamic effects of the neuropeptide.
CRF has been shown to have actions in many areas of the brain
(Potter et al., 1994), including those involved in emotional re-
sponses to stress such as the amygdala and locus coeruleus (Gray,
1993; Valentino et al., 1993). A recent study using in vivo micro-
dialysis further indicates that restraint stress induces the release of
CRF in the amygdala (Merlo Pich et al., 1995). It cannot be ruled
out, however, that the observed effects of CRF on reinstatement
are attributable to the activation of anterior pituitary neuropep-
tides such as ACTH. The CRF doses used in our study increase
ACTH release (Cador et al., 1992); ACTH is self-administered by
laboratory rats (Jouhaneau-Bowers and Le Magnen, 1979), and it
has been shown that acute injections of ACTH increase rates of
lever-pressing during extinction of food-reinforced behavior (Gar-
rud et al., 1973; De Weid and Jolles, 1982).
It is important to note that our data also indicate that neuro-
transmitters in addition to CRF are involved in the reinstatement
effect of stressors. That is, even in the presence of a high dose of
a
-helical CRF, footshock retained its ability to reinstate drug-
seeking to levels that were similar to those observed after priming
heroin injections (Fig. 6). Furthermore, doses of CRF that have
behavioral and neurochemical actions similar to those observed
after exposure to stressors (Dunn and Berridge, 1990; Johnson et
al., 1992; de Souza, 1995) reinstated heroin-seeking to a lesser
degree than footshock itself (Fig. 5).
A second major finding of the present study is that corticoste-
rone appears not to be involved in reinstatement of heroin-
seeking. Neither adrenalectomy nor chronic exposure to a synthe-
sis inhibitor of corticosterone metyrapone or acute exposure to
metyrapone interfered with the ability of priming injections of
heroin or footshock stress to reinstate heroin-seeking. Further-
more, adrenalectomy even appeared to potentiate the reinstate-
ment effect of footshock (Fig. 3). This latter effect may be related
to the lack of inhibitory control by corticosterone on stress-
induced increases in CRF utilization in the ADX rats compared
with the sham-operated rats (Imaki et al., 1995). It should be
noted, however, that the role of corticosterone in stress-induced
reinstatement was only determined for one form of stress, namely,
uncontrollable, intermittent footshock, a highly effective reinstat-
ing stimulus. Thus, the possibility that corticosterone is involved in
reinstatement of drug-seeking after exposure to other stressors
with a more moderate effect on reinstatement cannot be ruled out.
An unexpected finding in the present study was that acute
injections of metyrapone at a dose that blocked footshock-
induced corticosterone release and increased plasma ACTH lev-
els potently reinstated extinguished responding (Fig. 1). Further-
more, these same injections significantly increased the number of
lever presses made on the first day of extinction (Fig. 4). These
effects of metyrapone may be understood in the context of the
regulatory role of corticosterone in the hypothalamic–pituitary–
adrenal axis. Corticosterone provides inhibitory control over pi-
tuitary ACTH and CRF as well as other hypothalamic and pitu-
itary neuropeptides (Keller-Wood and Dallman, 1984; Tilders et
al., 1993). Injections of metyrapone, by interfering with the syn-
thesis of corticosterone, increase the release and synthesis of
ACTH, CRF, and other pituitary and hypothalamic neuropep-
tides (Plotsky and Sawchenko, 1987; Conte-Devolx et al., 1992). It
appears, however, that the behavioral actions of acute injections
of metyrapone cannot be satisfactorily explained only in terms of
the regulatory role of corticosterone on CRF release. Based on
the potent effect of metyrapone on heroin-seeking (Fig. 1) com-
pared with the effect of CRF (Fig. 5A), other factors would appear
to be involved. Metyrapone may serve as a nonspecific pharma-
cological stressor that activates other neurotransmitter systems
that are mobilized by exposure to stressors (e.g., norepinephrine,
excitatory amino acids). As mentioned in Results, metyrapone
affected the health of the rats and thus could serve as a nonspecific
pharmacological stressor.
Adrenalectomy also increases the levels and synthesis of
ACTH, CRF, and other pituitary and hypothalamic neuropep-
tides (Tilders et al., 1993; de Souza, 1995). However, unlike the
acute injection of metyrapone, adrenalectomy did not alter drug-
seeking during extinction and after priming injections of saline. A
Table 1. The effect of metyrapone pretreatment on plasma levels of corticosterone (
m
g/dl) and ACTH (pg/ml) in response to footshock stress in
heroin-trained and drug-naive animals
Heroin trained
Pretreatment
Corticosterone (
m
g/dl)
MET-ACUTE MET-CHRONIC
Prestress Poststress Prestress Poststress
Vehicle 15.2 6 4.6 27.3 6 4.1* NA NA
Metyrapone 14.2 6 1.7 14.9 6 1.5 14.2 6 2.0 16.6 6 1.8
Drug naive
Pretreatment
Corticosterone (
m
g/dl) ACTH (pg/ml)
No stress Stress No stress Stress
Vehicle 23.3 6 4.2 39.3 6 4.3
†
604.8 6 86.9 1013.0 6 135.0
Metyrapone 17.7 6 2.3 22.7 6 1.7 1004.7 6 138.6 1228.7 6 306.4
†
*Significantly different from the Prestress condition, p , 0.05.
†
Significantly different from the Vehicle No Stress condition, p , 0.05.
Shaham et al. • CRF and Relapse to Heroin-Seeking J. Neurosci., April 1, 1997, 17(7):2605–2614 2611
possible explanation for this difference is that the acute injection
of metyrapone caused a phasic elevation in CRF (or ACTH)
levels, whereas adrenalectomy performed several days before the
test resulted in a tonic increase in the levels and synthesis of CRF
(or ACTH). It seems more likely that the phasic changes, or the
acute disruptions in homeostasis, would be involved in drug-
seeking, rather than the gradual changes that occur in the levels
and synthesis of CRF, ACTH, and other pituitary and hypotha-
lamic neuropeptides after adrenalectomy. In addition, it can be
noted that after chronic exposure, metyrapone alone did not
induce reinstatement in the MET-CHRONIC group, suggesting
that after the initial injection of this drug, the state induced by
metyrapone lost its ability to induce drug-seeking by being asso-
ciated with the extinction conditions. Support for this possibility is
the observation after the first day of extinction, animals in group
MET-CHRONIC did not lever press at a higher rate than the
other groups throughout extinction.
The results of this study reinforce further the view that the
neural substrates involved in reinstatement induced by stress and
heroin-priming are not identical. As mentioned, in previous stud-
ies we found that the opioid antagonist naltrexone, a maintenance
dose of heroin, the selective D1-like and D2-like DA antagonists
SCH 23390 and raclopride, and chronic exposure to the mixed DA
antagonist flupenthixol decanoate all attenuated heroin-induced
reinstatement. In contrast, the only effective manipulation against
reinstatement by footshock was a chronic blockade of DA recep-
tors by the mixed antagonist. We also found that over a range of
heroin doses (0.125–0.5 mg/kg, s.c.) and different duration of
intermittent footshock (10–60 min), the stressor appears to be a
more effective stimulus for reinstatement, whereas the priming
Figure 5. A, Mean (6 SEM) number of lever
presses on the previously active lever in the 3 hr
after ICV injections of CRF or vehicle, priming
injections of heroin (0.25 mg/kg, s.c,), and expo-
sure to footshock stress (n 5 10). B, Time course
of lever pressing. *Different from the vehicle
condition, p , 0.05.
2612 J. Neurosci., April 1, 1997, 17(7):2605–2614 Shaham et al. • CRF and Relapse to Heroin-Seeking
injections of heroin are more effective in eliciting locomotor
activity and DA release in the nucleus accumbens (Shaham and
Stewart, 1996; Shaham et al., 1996; Shaham, 1997). Taken to-
gether, these data suggest that stress-induced reinstatement is to
a large degree opioid-independent and that both dopaminergic
and nondopaminergic mechanisms participate in reinstatement by
stress. Here, we find that a CRF antagonist attenuates stress-
induced reinstatement, whereas it only slightly affects the rein-
statement effect of heroin.
The findings from our recent studies may have implications for
the understanding of relapse to heroin use. Our previous data
(Shaham and Stewart, 1996) support the view that heroin-priming
elicits drug-seeking by activating an incentive motivation system
(Stewart et al., 1984; Robinson and Berridge, 1993). The anatom-
ical component of this system is most likely the mesolimbic DA
system with its afferent and efferent connections (Stewart, 1984;
Stewart and Vezina, 1988). We now tentatively suggest that stress
reinstates heroin-seeking by inhibiting a putative behavioral inhi-
bition system. Gray (1987, 1990) has suggested, on the basis of
studies on the behavioral effects of anxiolytic drugs and of septal
and hippocampal lesions, that a behavioral inhibition system exists
to stop ongoing activity in the presence of punishment or nonre-
ward (e.g., during extinction). This behavioral inhibition system
has anatomical connections that are to a large degree distinct
from the reward system (Gray, 1990). Many studies indicate that
stressors can disinhibit a variety of behaviors that are usually
under inhibitory control, including aggression, sexual behavior
(Gray, 1987), and extinguished operant responding (Brimer, 1970;
Bouton and Swartzentruber, 1991). Thus, it is possible that stress
induces relapse to heroin-seeking by disrupting behavioral inhibi-
tion. We can speculate further that this system may be particularly
vulnerable to disruption in animals with a history of drug use.
In conclusion, CRF, but not corticosterone, contributes to re-
lapse to heroin-seeking induced by stressors. We suggest that
brain systems and neurotransmitters involved in the inhibition of
ongoing behavior may be involved in the reinstatement of drug-
seeking by stressors.
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