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N
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© 2000 American College of Neuropsychopharmacology
Published by Elsevier Science Inc. All rights reserved 0893-133X/00/$–see front matter
655 Avenue of the Americas, New York, NY 10010 PII S0893-133X(99)00158-X
Alpha
-2 Adrenergic Receptor Agonists
Block Stress-Induced Reinstatement of
Cocaine Seeking
Suzanne Erb, Ph.D., Paul K. Hitchcott, D. Phil., Heshmat Rajabi, B.Sc., Devin Mueller, M.A.,
Yavin Shaham, Ph.D., and Jane Stewart, Ph.D.
The
alpha
-2 adrenergic receptor agonists, clonidine,
lofexidine and guanabenz, blocked stress- but not cocaine-
induced reinstatement of cocaine seeking at doses that
suppressed footshock-induced release of noradrenaline in
prefrontal cortex and amygdala. Rats were trained to self-
administer cocaine (0.5 mg/kg/infusion, i.v; 10–12 days) and,
after a drug-free period (7–13 days), were returned to the self-
administration chambers for daily extinction and
reinstatement test sessions. Both intermittent footshock (15
min, 0.6 mA) and cocaine priming (20 mg/kg, i.p.) reinstated
extinguished drug seeking. Pretreatment with either
clonidine (20, or 40
g/kg, i.p.) or lofexidine (50, 100, 150, or
200
g/kg, i.p.) attenuated footshock- but not cocaine-
induced reinstatement of cocaine seeking. Guanabenz (640
g/kg, i.p.), an
alpha
-2 agonist with low affinity for
imidazoline type-1 receptors, also attenuated footshock- but
not cocaine-induced reinstatement of cocaine seeking. The
results point to an important role for NE systems in the
effects of footshock on relapse to cocaine seeking.
[Neuropsychopharmacology 23:138–150, 2000]
© 2000 American College of Neuropsychopharmacology.
Published by Elsevier Science Inc. All rights reserved
KEY
WORDS
:
Clonidine; Lofexidine; Cocaine Self-
administration; Relapse; Stress; Microdialysis
“Priming” injections of an abused drug can induce
craving in abstaining addicts (e.g. Jaffe 1985) and rein-
state drug-seeking in monkeys and rats with a history
of drug self-administration (e.g., Stretch and Gerber
1973; de Wit and Stewart 1981). Another factor thought
to be important to relapse in humans is stress (Kreek
and Koob 1998). In a recent laboratory study, Sinha and
colleagues (1999) found that induction of psychological
stress provoked craving for drug in cocaine addicts. Us-
ing an animal model of stress-induced relapse, we and
others have found in rats that exposure to brief inter-
mittent footshock stress reliably reinstates heroin seek-
ing (Shaham and Stewart 1995), cocaine seeking (Erb et
al. 1996; Ahmed and Koob 1997; Mantsch and Goeders
1999), nicotine seeking (Buczek et al. 1999), and alcohol
seeking (Lê et al. 1998).
In a search for neurotransmitter systems involved in
stress-induced relapse, and for pharmacological manip-
ulations that might prevent it, we explored in the
present set of experiments the role of the noradrenergic
(NE) system, comprised of a dorsal stream of neurons
originating in the locus-coeruleus and a ventral stream
of neurons originating in the lateral tegmental nuclei
(Aston-Jones et al. 1995; Moore and Bloom 1979). Al-
though the stress-related functions of the ventral pro-
jections have been only partially characterized (Cole
and Robbins 1987; Hansen et al. 1980), NE projections
from the LC have been directly implicated in a number
From the Center for Studies in Behavioral Neurobiology (SE,
PKH, HR, DM, JS), Department of Psychology, Concordia Univer-
sity, Montreal, Quebec, Canada; Behavioral Neuroscience Depart-
ment(YS), IRP/NIDA, Baltimore, MD.
Address correspondence to: Dr. Jane Stewart, Center for Studies in
Behavioral Neurobiology, Department of Psychology, 1455 de Mai-
sonneuve Blvd. W., Montreal, Quebec, Canada H3G 1M8 Tel: 514-
848-2193, Fax: 514-848-2817, E-mail: Stewart@CSBN.concordia.ca.
Received September 20, 1999; revised December 21, 1999;
accepted December 28, 1999.
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alpha
-2 Agonists and Relapse to Cocaine Seeking
139
of stress-related responses. For example, electrical stim-
ulation of the LC has been found to induce anxiety and
to result in activation of the autonomic nervous system,
whereas local injections of the alpha-2 adrenergic recep-
tor agonist clonidine, which acts to inhibit NE cell firing
(Aghajanian and VanderMaelen 1982) and release
(Carter 1997), has been found to decrease physiological
and behavioral responses to stressors (Bremner et al.
1996; Redmond and Huang 1979).
We have recently reported that systemic or intracere-
broventricular (i.c.v.) injections of clonidine block foot-
shock-induced reinstatement of heroin seeking (Sha-
ham et al. 2000), a finding that suggests an important
role for brain NE in the mediation of stress-induced re-
lapse. In previous studies, we have shown that another
neurotransmitter system, corticotropin-releasing factor
(CRF), also plays an important role in mediating the
footshock-induced reinstatement of both cocaine (Erb et
al. 1998; Erb and Stewart 1999) and heroin seeking (Sha-
ham et al. 1997) via its actions at extrahypothalamic brain
sites. Interestingly, the CRF and NE systems interact in
the brain in the modulation of stress responses (e.g., Li et
al. 1998; Shimizu et al. 1994; Smagin et al. 1997; Valentino
et al. 1991; Valentino and Wehby 1988), raising the possi-
bility that an interaction between these systems underlies
stress-induced relapse to drug seeking.
The present set of experiments had four main objec-
tives. First, we assessed whether the effect of clonidine
on stress-induced reinstatement previously observed in
heroin-trained rats would generalize to rats with a his-
tory of cocaine self-administration. Second, we tested
whether another
alpha
-2 adrenergic receptor agonist,
lofexidine, would be equally effective. Third, because
clonidine is known to bind at high affinity to the imidazo-
line type-1 (I
1
) receptor, we studied the effect of an
alpha
-2
adrenergic receptor agonist with low affinity for the I
1
re-
ceptor, guanabenz (Piletz et al. 1994), on stress-induced
reinstatement. Finally, we studied the effect of clonidine,
lofexidine, and guanabenz on reinstatement of cocaine
seeking induced by a priming injection of cocaine.
Before conducting the behavioral experiments, we
assessed the ability of the three alpha-2 adrenergic re-
ceptor agonists to suppress footshock-induced release
of NE in two terminal regions, the prefrontal cortex
(PFC) and amygdala (AMG). The PFC is a major termi-
nal region of the dorsal NE system arising from the lo-
cus coeruleus, whereas the AMG receives input from
both the dorsal and ventral systems.
MATERIALS AND METHODS
Experiment 1: Effects of Clonidine, Lofexidine and
Guanabenz on Footshock-Induced NE Release
Subjects.
The subjects were male Long-Evans rats
(Charles River, Quebec) weighing 300–350 g at the be-
ginning of the experiment. Throughout the experiment,
animals were housed in a humidity- and temperature-
controlled colony room on a reversed light-dark schedule
(lights on 1730–0530 hours) and were given free access
to standard laboratory rat chow and water. The experi-
mental procedures followed the CCAC guidelines and
were approved by the Animal Care Committee, Con-
cordia University.
Surgery.
Before surgery, rats were anesthetized with
sodium pentobarbital (65 mg/kg, i.p.) and were in-
jected with atropine sulfate (0.6 mg/ml; 0.2 ml/rat, SC)
and antibiotic (Penlong, Rogar/STB Inc.; 0.1 ml/rat,
i.m.). Guide cannulae (20 gauge; Plastics One) were im-
planted for subsequent insertion of dialysis probes; one
was placed in PFC and one in AMG in opposite hemi-
spheres. The animals used to study the effects of guana-
benz received a single guide cannula aimed at PFC. The
hemisphere into which the respective guide cannulae
were implanted was counterbalanced across treat-
ments. The stereotaxic coordinates used (relative to
bregma and skull surface) were as follows: AMG, AP
⫺
3.0 mm, ML
⫾
4.5 mm, DV
⫺
6.7 mm; PFC, AP
⫹
3.2
mm, ML
⫾
0.5 mm, DV
⫺
1.5 mm (Paxinos and Watson
1997). Upon insertion, the shaft of the dialysis probe ex-
tended 1 mm from the guide cannulae. Implanted
guide cannulae were secured to the skull with stainless
steel screws and dental cement. Guide cannulae were
closed with screw-in obdurators. All animals were re-
turned to the colony room for a recovery period of no
less than 1 week prior to testing.
Microdialysis.
Microdialysis was conducted in four
hexagonal testing chambers (42
⫻
39
⫻
33.5 cm) built
from Plexiglas with wooden ceilings and stainless steel
rod floors. Dark curtains were drawn around each
chamber and lighting was provided on a reversed cycle
by overhead light bulbs (15 W). The dialysis probe con-
sisted of a 1.8 mm (AMG) or 3.5 mm (PFC) length of
semipermeable dialysis membrane (Spectra/Por; 240
m o.d., 13000 M.W. cutoff), closed at one end and at-
tached at the other to a 19 mm length of 26 gauge stain-
less-steel tubing. A 40- to 50-cm length of PE-20 tubing
connected the other end of the stainless steel shaft to an
infusion swivel stationed above the testing chamber that
was in turn connected via PE-20 tubing to a variable-
speed infusion pump. A small diameter, fused-silica
tube extended internally through the probe, with one
end resting 0.5 mm from the tip of the probe and the
other end exiting the PE tubing 35 cm below the infusion
swivel. The external length of PE-20 tubing was pro-
tected from damage by steel spring casings. The probes
were designed so that the entire length of semi-perme-
able membrane extended below the guide cannula tip.
Separate squads of animals were used to examine the
effects of each of the drugs tested. Within each squad,
each animal was randomly assigned to the treatment
140
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condition. Animals that received vehicle injections were
run in each squad, but were combined into a single
group for statistical analysis. Final group sizes (PFC/
AMG) were as follows: vehicle (n
⫽
15/17), clonidine
20
g/kg (n
⫽
7/6), clonidine 40
g/kg (n
⫽
9/5),
lofexidine 75
g/kg (n
⫽
6/6), lofexidine 150
g/kg (n
⫽
5/6), guanabenz 640
g/kg (n
⫽
4, PFC). With the ex-
ception of the animals receiving guanabenz, all subjects
were dialyzed twice, once in the PFC and once in the
AMG with an interval of 3–4 weeks between tests.
The probes were inserted the day before the begin-
ning of microdialysis testing. To prevent occlusion, arti-
ficial CSF (145 mM Na
⫹
, 2.7 mM K
⫹
, 1.22 mM Ca2
⫹
,
1.0 mM Mg2
⫹
, 150 mM Cl-, 0.2 mM ascorbate, 2 mM
Na2HPO4, pH 7.4
⫾
0.1) was perfused overnight at a
rate of 0.06
l/min. Dialysate sampling and activity
monitoring began the next morning. The dialysate flow
rate was increased to 0.6
l/ min, and baseline dialy-
sate samples (
ⵑ
12
l/ sample) were collected every 20
min. Samples were collected until a stable baseline, de-
fined as a minimum of three consecutive samples in
which dialysate NE levels varied by
⫾
10%, was
achieved. Following the establishment of stable baseline
NE levels, all animals received an i.p. injection (1 ml/
kg) of the appropriate drug or vehicle which was given
40 min before a 10 min period of footshock. Shocks were
delivered on a variable time schedule at a mean interval
of 40 seconds (10–70 seconds range); each shock (0.6 mA)
was 0.5 seconds in duration. Samples were collected for
a further 120 minutes (6 samples). Samples were imme-
diately analyzed using one of two similar high-perfor-
mance liquid chromatography systems with electro-
chemical detection (HPLC-EC). The samples (10
l)
were loaded onto reverse-phase columns (15
⫻
0.46 cm;
HAISIL C18, 5
m; Sci. Products & Equipment, Concord,
Ontario) through manual injection ports (Reodyne
7125; 20
l loop); reduction and oxidation currents for
NE, dihydroxyphenylacetic acid (DOPAC), 5-hydoxy-
indoleacetic acid (5-HIAA) and homovanillic acid
(HVA) were measured with dual-channel ESA coulom-
etric detectors (Coulochem 5100, with a model 5021
conditioning cell and a model 5011 analytical cell, Sci.
Products & Equipment). The currents for NE were mea-
sured independent of those for DOPAC and HVA using
separate channels of the Coulochem detectors. The mo-
bile phases (sodium acetate 36 mM, SOS 3.1 mM, EDTA
100
M, 5% acetonitrile, adjusted to pH 3.7 using gla-
cial acetic acid) were circulated through each closed
system at a flow rate of 1.4 ml/min by Waters 510
HPLC pumps. The peaks obtained for NE, DOPAC and
HVA were integrated and quantified by EZChrom
Chromatography Data System (Sci. Products & Equip-
ment). Dialysate samples from individual rats always
were analyzed with the same HPLC-EC system, and the
assignment of animals to each system was counterbal-
anced across all treatment groups. Food was removed
from the chambers before sampling, but a water drink-
ing tube was available. At the completion of testing,
confirmation of correct probe placement was deter-
mined by examination of 30
m sections cut through
the sites of probe placement stained with Cresyl violet.
Experiment 2: Effects of Clonidine on Footshock-
and Cocaine-Induced Reinstatement
Subjects.
The subjects were 25 male Long-Evans rats
(Charles River, Quebec) weighing 350–425 g at the start
of the experiment. The animals were maintained as de-
scribed in Experiment 1.
Surgery.
Animals were prepared for surgery as de-
scribed in Experiment 1. An intravenous catheter (Dow
Corning) was implanted in the right jugular vein. A 3-cm
length of silastic tubing was inserted into the vein (in-
ner diameter 0.30 mm, outer diameter 0.64 mm) and
was connected with heat shrink tubing to a 9-cm length
of silastic tubing (inner diameter 0.51 mm, outer diame-
ter 0.94 mm) that was passed subcutaneously to the top
of the skull. The catheter was secured to the vein with
silk sutures and exited into a connector (a modified 22
gauge cannula; Plastics One, Roanoke, VA) that was
mounted to the skull with jeweler’s screws and dental
cement; a plastic cap was placed over the opening of the
cannula. Animals were allowed 1–2 weeks to recover
from surgery.
Apparatus.
The self-administration chambers used in
the experiments were equipped with a retractable lever
(Med Associates, St Albans, VT) and a non-retractable
“dummy” lever. Both levers were located 9 cm above
the floor. An infusion pump (Razel Scientific Instru-
ments, Stamford, CT) was activated by responses on the
retractable, or “active,” lever. Responses on the dummy
lever were recorded but did not result in activation of
the pump. Drug solution was delivered over a 10-s pe-
riod in a volume of 65
l. Throughout the infusion pe-
riod, a white stimulus light just above the active lever
was illuminated and additional responses during this
time were recorded, but did not result in reactivation of
the pump. Each self-administration chamber was fitted
to deliver constant-current, intermittent, inescapable,
electric footshock through a scrambler to the grid floor
(Grason-Stadler Generator #E1064GS or Med Associ-
ates). The footshock was delivered for 15 min according
to a variable time schedule at a mean interval of 40 sec-
onds (10–70 seconds range). Each shock (0.6 mA) was
0.5 seconds in duration. This intensity of footshocks has
been found using our apparatus to be the minimal re-
quired to induce reliable reinstatement.
Drugs.
Cocaine HCl was obtained from BDH Chemi-
cals (Toronto, Canada) and was dissolved in physiolog-
ical saline. Clonidine HCl was purchased from Sigma
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alpha
-2 Agonists and Relapse to Cocaine Seeking
141
(St Louis, MO) and was dissolved in physiological sa-
line and injected i.p. (0, 20, and 40
g/kg).
Procedure.
Phase 1: Training.
Rats were trained to self-
administer cocaine HCl (0.5 mg/kg/infusion, i.v.) on a
fixed-ratio-1 schedule of reinforcement during one
daily 3-h self-administration session. Each day, half of
the animals were brought to the operant chambers for
their self-administration session in the morning, ap-
proximately three hours after lights off, and half of the
animals were brought to the chambers in the afternoon,
approximately seven hours after lights off. The group of
animals assigned to the morning and afternoon sessions
alternated daily so that by the end of training all ani-
mals had received an equal number of self-administra-
tion sessions at both times. It was important that all ani-
mals had similar experience self-administering early
and late in the day since during Phase 2 all animals re-
ceived extinction sessions in the morning followed by a
test session that typically occurred in the afternoon. At
the beginning of each session, a red house light was il-
luminated for 10 seconds before introduction of the re-
tractable lever into the cage. The light just above the ac-
tive lever was lit for the initial 30 seconds following
presentation of the lever. The house light remained illu-
minated throughout the session. As indicated previ-
ously, responses on the active lever resulted in activa-
tion of the infusion pump and illumination of the light
above the lever for the 10 seconds of drug delivery. Ad-
ditional lever presses during the drug-delivery period
were recorded but did not result in reactivation of the
pump. Training conditions were in place for 10 to 12
days. At the end of training, animals were left undis-
turbed in the colony room for 7 to 13 days. Subse-
quently, extinction and testing took place. In this exper-
iment and in Experiment 3B, groups of animals were
assigned to different doses of the test drugs. All animals
in each group were then subjected to three test condi-
tions; saline, cocaine and footshock.
Phase 2: Extinction and testing.
During extinction and
testing, which occurred over four consecutive days, ani-
mals were housed 24 hours per day in the self-adminis-
tration chambers. Food and water were freely available
to animals, except during daily extinction and test ses-
sions. Animals were brought to the chambers in the
evening preceding the first day of extinction and test-
ing. Since two separate groups of rats were run each
day during the training phase, one group of animals
was tested in the first four days of Phase 2 and the other
group of animals was tested in the next four days. Dur-
ing extinction and testing, all of the conditions that
were present during training were maintained except
that lever presses did not result in cocaine infusions.
In this and all subsequent reinstatement experi-
ments, animals were given several daily 1-h extinction
sessions separated by intervening periods in which the
lever was withdrawn. On day 1, animals were given
four extinction sessions; on days 2–4, animals were
given two to three extinction sessions, sufficient to es-
tablish a baseline level of responding of 15 or fewer re-
sponses in one hour, followed by a 180 min test session.
The duration of the intervening periods was kept con-
stant for each experiment and corresponded to the de-
lay required for drug absorption between pretreatment
injections and the start of the test. In the present experi-
ment, the duration of intervening periods was 40 min.
At test, separate groups of animals were pretreated
with either 0, 20, or 40
g/kg, i.p., clonidine before each
of three tests for reinstatement (saline, cocaine, and
footshock), given on consecutive days and in a counter-
balanced order. Clonidine was injected 40 min before
the lever was inserted. For the saline and cocaine prim-
ing tests, animals were given a non-contingent, i.p., in-
jection of saline or cocaine (20 mg/kg) 5 min before le-
ver insertion. For the footshock tests, animals were
exposed to the 15-min brief intermittent footshock stress
immediately before insertion of the lever. Test sessions
were 3-h in duration. The dose of cocaine and footshock
parameters were chosen on the basis of previous work
from this laboratory (Erb et al. 1996; Erb et al. 1998; Sha-
ham et al. 1998). The doses of clonidine were chosen on
the basis of reinstatement experiments we have con-
ducted with heroin-trained rats (Shaham et al. 2000).
Experiment 3A: Effects of Lofexidine on High Rates
of Responding for Sucrose
Because the pharmacological profile for lofexidine has
not been as well characterized as that for clonidine, an
experiment was conducted to establish whether, and at
what doses, lofexidine induces performance deficits.
The doses in the tests for reinstatement were chosen on
the basis of results obtained in this experiment.
Subjects.
The subjects were 8 male Long-Evans rats
(Charles River, Quebec) weighing 400–550 g at the be-
ginning of the experiment. Rats were previously used
in an experiment aimed at studying footshock-induced
reinstatement of sucrose seeking (Buczek et al. 1999).
Animals were maintained under similar conditions to
those described in Experiment 1.
Apparatus.
Each self-administration chamber was
equipped with two stationary levers, symmetrically
centered on one side panel, 5 cm above the floor. Re-
sponses on one lever, the “active” lever, activated a
pump (Razel Sci. Stamford, CT). Responses on the other
lever, the “dummy” lever, were recorded but were
without consequence. Activation of the pump resulted
in a 20-second delivery of 0.18 ml sucrose solution to a
liquid drop receptacle located between the two levers.
Throughout the sucrose delivery period, a white stimu-
lus light just above the active lever was illuminated and
142
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additional responses during this time were recorded
but did not result in reactivation of the pump.
Drugs.
Lofexidine HCl was generously supplied by
Keith Davies, Britannia Pharmaceuticals Ltd., UK. The
drug was dissolved in physiological saline and injected
i.p. (0, 80, 120, 160 or 200
g/kg).
Procedure.
Animals that had previously learned to
self-administer sucrose in a different study (Buczek et
al. 1999) were subsequently given five sessions over
five consecutive days in which they self-administered a
30% sucrose solution on an FR-1 schedule. In subse-
quent daily sessions, animals were injected with either
vehicle (0
g/kg) or lofexidine (80, 120, 160 or 200
g/
kg, i.p.) 60 min before the start of the self-administra-
tion session. All animals received all doses of lofexidine
in a counterbalanced order; the highest dose of lofexi-
dine was tested twice. Each session in which animals
were treated with lofexidine was followed by a saline
pretreatment session to minimize the possibility of
carry-over effects of the drug.
Experiment 3B: Effects of Lofexidine on Footshock-
and Cocaine-Induced Reinstatement
Subjects.
The subjects were 49 male Long-Evans rats
(34 from Charles River, Quebec; 15 from Harlan Sprague
Dawley, USA) weighing 350–400 g at the beginning of
the experiment. The animals were maintained as de-
scribed in Experiment 1. There were no obvious differ-
ences in responding between animals from the two sup-
pliers in any phase of the experiment.
Procedure.
Phase 1: Training.
Animals were trained to
self-administer cocaine under the conditions described
in Experiment 2.
Phase 2: Extinction and testing.
In this experiment the
intervening periods, as described above, were 60 min in
duration. At test, separate groups of animals were pre-
treated with either 0, 50, 100, 150, or 200
g/kg, i.p.,
lofexidine before each of three tests for reinstatement
(saline, cocaine, and footshock stress), given on consec-
utive days and in a counterbalanced order (see Experi-
ment 2). Lofexidine was injected 60 min before the in-
sertion of the lever. Test sessions were 3-h in duration.
The doses of lofexidine were chosen on the basis of the
results obtained in Experiment 3A.
Experiment 4: Effects of Guanabenz on Footshock-
and Cocaine-Induced Reinstatement
Subjects.
The subjects were 18 male Long-Evans rats
(Charles River, Quebec) weighing 350–400 g at the be-
ginning of the experiment. The animals were main-
tained as described in Experiment 1.
Drug.
Guanabenz was purchased from Sigma (St
Louis, MO) and was dissolved in physiological saline
and injected i.p. (0, and 640
g/kg).
Procedure.
Phase 1: Training.
Animals were trained to
self-administer cocaine under the conditions described
in Experiment 2.
Phase 2: Extinction and testing.
In this experiment the
intervening periods, as described above, were 60 min in
duration. At test, animals were pretreated with 0 or 640
g/kg guanabenz, i.p., before each of two tests for rein-
statement (no footshock, footshock or saline, cocaine),
given on consecutive days (see Experiment 2). Guana-
benz was injected 60 min before the insertion of the le-
ver. Test sessions were 3-h in duration. The dose of gua-
nabenz was chosen on the basis of its substitutability
for 40
g/kg clonidine in a drug discrimination proce-
dure (Bennett and Lal 1982).
Statistical Analyses
Microdialysis data were analyzed using a mixed-factor
ANOVA for concentration of extracellular NE in 20 min
dialysate samples; the between-subjects factor was dose
of clonidine (0, 20, 40
g/kg), lofexidine (0, 75, or 150
g/kg), or guanabenz (0 or 640 g/kg) and the re-
peated measure was time. Significant time by dose in-
teractions were subjected to one-way ANOVAs for the
factor of dose at specific time points. When appropriate,
post hoc tests (Fisher’s LSD, p ⬍ .05) were conducted to
compare vehicle (0 g/kg) to drug conditions.
The two dependent measures in the tests for rein-
statement were number of responses on the active lever
(infusions ⫹ time out responses) and number of re-
sponses on the inactive lever in three hours. All behav-
ioral data are presented as means ⫾ SEM. Because of
considerable variability in the number of responses
made in the different tests for reinstatement, the non-
parametric statistics for related (Friedman and Wil-
coxon) and unrelated (Kruskal-Wallis and Mann-
Whiney) samples were used where appropriate.
For the sucrose study (Experiment 3A) the depen-
dent measures were the number of responses on the ac-
tive (reinforced ⫹ timeout responses) and inactive le-
vers and the number of reinforcements obtained.
Repeated measures ANOVAs were conducted with
dose as the within-subject factor. When appropriate,
post hoc tests (Fisher’s LSD, p ⬍ .05) were conducted to
compare vehicle (0 g/kg) to drug conditions.
RESULTS
Experiment 1: Effects of Clonidine, Lofexidine and
Guanabenz on Footshock-Induced NE Release
Figure 1 shows basal and footshock-induced levels of
NE in PFC (top row) and/or AMG (bottom row) after
injection at Time 0 with clonidine (left panel), lofexidine
(center panel), or guanabenz (right panel). Mixed-factor
ANOVAs revealed significant Time by Dose interac-
NEUROPSYCHOPHARMACOLOGY 2000–VOL. 23, NO. 2alpha-2 Agonists and Relapse to Cocaine Seeking 143
tions in both brain regions and for each of the three
drugs. The statistics for each of these interactions are in-
dicated in the figure legend. One-way ANOVAs con-
ducted at specific time points were, when statistically
significant, followed by post-hoc comparisons (Fisher’s
LSD). Significant differences are indicated in Figure 1.
See Figure 2 for a diagram showing the areas of the PFC
and AMG where the active regions of the dialysis
probes were located.
Experiment 2: Effects of Clonidine on Footshock-
and Cocaine-Induced Reinstatement
Training and Extinction. The mean (⫾SEM) number
of infusions of 0.5 mg/kg cocaine made in the 3-h ses-
sion on the last two days of training was 32.24 (⫾2.77)
and 25.76 (⫾2.96), respectively. The mean (⫾SEM)
number of responses (infusions ⫹ time out responses)
made during the first three 1-h extinction sessions on
day 1 of Phase 2, was 28.92 (⫾2.81), 13.16 (⫾2.03), and
9.68 (⫾2.04). There was no difference in the rate of ex-
tinction between animals tested in the first four days of
Phase 2 and those tested four days later.
Tests for reinstatement. The number of responses
made on the active and inactive levers during each 3-h
test for reinstatement following pretreatment with
clonidine is shown in Figure 3A. Figure 3B shows the
number of responses on the active lever during each
hour of testing. It can be seen that pretreatment with 20
or 40 g/kg clonidine blocked footshock-induced re-
Figure 1. Microdialysis – Prefrontal cortex and amygdala: Mean (⫾SEM) NE concentrations in 20 min dialysate samples in
prefrontal cortex and amygdala. Animals were injected with either clonidine (0, 20, or 40 g/kg, i.p.), lofexidine (0, 75, or
150 g/kg, i.p.), or guanabenz (0 or 640 g/kg, i.p.) at Time 0, and 40 min later were exposed to 10 min of intermittent foot-
shock stress (0.6 mA). Significant Time by Dose interactions were obtained for the prefrontal cortex and amygdala after
clonidine (F(16,192) ⫽ 4.40, p ⬍ .01 and F(16,168) ⫽ 2.03, p ⬍ .03, respectively), lofexidine (F(16,168) ⫽ 5.09 p ⬍ .01 and
F(16,192) ⫽ 2.09, p ⬍ .03, respectively), and guanabenz (F(8,120) ⫽ 6.22, p ⬍ .001). * Different from high dose only, p ⬍ .05.; ⫹
different from both other doses, p ⬍ .05.
144 S. Erb et al. NEUROPSYCHOPHARMACOLOGY 2000–VOL. 23, NO. 2
lapse to cocaine seeking, but had no effect on relapse in-
duced by a priming injection of cocaine. Kruskal-Wallis
tests conducted at each hour of testing revealed a signif-
icant effect of dose of clonidine in the footshock condi-
tion in Hour 1, when most of the responding occurred
Figure 2. Diagrams showing the regions (shaded areas) o
f
the prefrontal cortex (top panel; 46 placements) and
amygdala (bottom panel; 40 placements) in which the active
region of the dialysis probes were found to be placed after
histological analysis. The values of the right of each figure
indicated mm from bregma (Paxinos and Watson 1997).
Figure 3. Systemic clonidine – reinstatement: (A) Mean
(⫾SEM) number of responses (infusions ⫹ timeout
responses) on the inactive and previously active levers in the
three hours after an i.p. injection of saline, exposure to 15 min
of intermittent footshock stress (0.6 mA), or an i.p. priming
injection of cocaine (20 mg/kg). (B) Mean (⫾SEM) number of
responses on the previously active lever in each hour after a
saline injection, exposure to footshock stress, or a priming
injection of cocaine. Rats were pretreated with vehicle (0) (n ⫽
9) or 20 (n ⫽ 8) or 40 (n ⫽ 8) g/kg clonidine, i.p., 40 min
before the start of the self-administration session. Lever
presses were without consequence during the tests. * Differ-
ent from vehicle (0) condition, ⫹ different from other doses,
p ⬍ .05.
NEUROPSYCHOPHARMACOLOGY 2000–VOL. 23, NO. 2alpha-2 Agonists and Relapse to Cocaine Seeking 145
(X2 (2) ⫽ 6.0, p ⬍ .05, footshock); both doses of clonidine
were effective (p ⬍ .05). There were no significant ef-
fects of clonidine in either the saline or cocaine condi-
tions in Hour 1; no effects were seen in any of the test
conditions in Hours 2 or 3. Figure 3A shows that re-
sponding on the inactive lever was low under all pre-
treatment and test conditions. This was the case in all
subsequent reinstatement experiments, where there
were no significant effects of the number of responses
on the inactive lever.
Experiment 3A: Effects of Lofexidine on Responding
for Sucrose
Figure 4 shows the total number of responses (rein-
forced ⫹ time out responses) on the active and inactive
levers, and the number of sucrose reinforcements
made, following injections of vehicle (0 g/kg) or lofex-
idine. There was no effect of lofexidine on the number
of responses on the active lever, F(4,28) ⫽ 1.79, ns.
There were, however, dose effects on the number of re-
inforcements and number of responses on the inactive
lever (F(4,28) ⫽ 2.80, p ⬍ .05 and F(4,28) ⫽ 2.85, p ⬍ .05,
respectively); this reflects the fact that when compared
with the vehicle (0 g/kg) condition, animals obtained
fewer sucrose reinforcements and made fewer re-
sponses on the inactive lever following pretreatment
with the highest dose of lofexidine (200 g/kg).
Experiment 3B: Effects of Lofexidine on Footshock-
and Cocaine-Induced Reinstatement
Training and Extinction. The mean (⫾SEM) number
of cocaine infusions made in the 3-h session on the last
two days of training was 30.07 (⫾2.37) and 30.91
(⫾2.06), respectively. On day 1 of extinction, the mean
(⫾SEM) number of responses made in the first three 1-h
extinction sessions (infusions ⫹ time out responses)
was 31.32 (⫾3.22), 16.84 (⫾3.29), and 16.23 (⫾2.35).
There was no difference in the rate of extinction be-
tween animals tested in the first four days and those
tested in the subsequent four days of Phase 2.
Tests for Reinstatement. Figure 5A shows the num-
ber of responses made on the active and inactive levers
during each 3-h test for reinstatement following pre-
treatment with vehicle (0 g/kg) or lofexidine. Figure
5B shows the number of responses on the active lever
during each hour of testing. It can be seen that both
footshock stress and priming injections of cocaine in-
duced reinstatement of cocaine seeking behavior. Only
the effect of footshock stress was attenuated by lofexi-
dine. This is confirmed by the Kruskal-Wallis tests con-
ducted for the 3-h tests for reinstatement where the only
significant effect was found in the footshock condition
(X2 (4) ⫽ 10.50, p ⬍ .05). Subsequent Mann-Whitney
comparisons revealed a significant difference between
the 0 and 150 g/kg and 0 and 200 g/kg lofexidine
doses (ps ⬍ .05); furthermore, the response to footshock
after 150 and 200 g/kg doses did not differ from the
responses in the saline condition. Separate analyses
conducted at each hour of testing revealed a significant
effect, again, only in Hour 1 of testing (X2 (4) ⫽ 17.31, p ⬍
.01, footshock), when all doses of lofexidine blocked the
footshock effect (ps ⬍ .05).
Experiment 4: Effects of Guanabenz on Footshock-
and Cocaine-Induced Reinstatement
Training. The mean (⫾SEM) number of infusions of
cocaine made in the 3-h session on the last two days of
training was 36.63 (⫾3.56) and 40.12 (⫾3.65), respectively.
Tests for Reinstatement. Figure 6 shows the number
of responses made on the active lever in tests for rein-
statement after pretreatment with either 0 or 640 g/kg
guanabenz. It can be seen that guanabenz attenuated
the effect of footshock stress. A Friedman analysis on
the footshock and no footshock tests revealed a signifi-
cant effect of test condition (X2(3) ⫽ 8.36, p ⬍ .05) and
subsequent comparisons using the Wilcoxon test
showed that the footshock (0 g/kg) condition differed
from the others (ps ⬍ .05). Inspection of Figure 6 also re-
veals what appears to be a reduction in responding in
the cocaine test following pretreatment with guana-
benz. However, a comparison of the scores on the co-
caine test using the Mann-Whitney revealed no signifi-
Figure 4. Systemic lofexidine – responding for sucrose:
Mean (⫾SEM) number of responses on the inactive and
active levers 60 min after pretreatment with 0, 80, 120, 160
and 200 g/kg lofexidine, i.p. (n ⫽ 8). Responding on the
active lever was reinforced with 0.18 ml of a 30% sucrose
solution. * Different from vehicle (0) condition, p ⬍ .05.
146 S. Erb et al. NEUROPSYCHOPHARMACOLOGY 2000–VOL. 23, NO. 2
cant difference between the two groups (0 and 640g/
kg; z ⫽ 0.73, p ⫽ .46).
DISCUSSION
Two major findings emerge from the present series of
experiments. First, systemic injections of clonidine and
lofexidine attenuate the footshock-induced reinstate-
ment of cocaine seeking, but have no effect on reinstate-
ment induced by priming injections of cocaine. Second,
the alpha-2 adrenergic receptor agonist, guanabenz,
which unlike clonidine and lofexidine has a low affinity
for I1 receptors (Piletz et al. 1994), also attenuates foot-
shock-induced reinstatement. These effects were ob-
tained by drugs that at similar doses and under similar
stress parameters to those used in the tests for reinstate-
ment suppressed the footshock-induced increase in NE
release in AMG and/or PFC in naïve animals. These
findings suggest that reduction of brain NE activity
may be an effective way to prevent stress-induced re-
lapse to cocaine seeking.
The finding that clonidine, lofexidine, and guana-
benz attenuate footshock-induced but not cocaine-
Figure 5. Systemic lofexidine – reinstatement: (A) Mean
(⫾SEM) number of responses (infusions ⫹ timeout
responses) on the inactive and previously active levers in the
three hours after an i.p. injection of saline, exposure to 15
min of intermittent footshock stress (0.6 mA), or an i.p.
priming injection of cocaine (20 mg/kg). (B) Mean (⫾SEM)
number of responses (infusions ⫹ timeout responses) on the
previously active lever in each hour after saline injection,
exposure to footshock stress, or a priming injection o
f
cocaine. Rats were pretreated with vehicle (0) (n ⫽ 9) or 50 (n
⫽ 11), 100 (n ⫽ 11), 150 (n ⫽ 9), or 200 (n ⫽ 9) g/kg lofexi-
dine, i.p., 60 min before the start of the self-administration
session. Lever presses were without consequence during the
tests. * Different from vehicle (0) condition, ⫹ different from
other doses, p ⬍ .05.
Figure 6. Systemic guanabenz – reinstatement: Mean
(⫾SEM) number of responses (infusions ⫹ time out
responses) on the previously active lever during 3-hour test
sessions (no footshock and footshock; saline and cocaine)
after pretreatment with 0 or 640 g/kg guanabenz. * Differ-
ent from all other conditions, p ⬍ .05. # different from saline
test condition, p ⬍ .05.
NEUROPSYCHOPHARMACOLOGY 2000–VOL. 23, NO. 2alpha-2 Agonists and Relapse to Cocaine Seeking 147
induced reinstatement was not unexpected in view of
our previous findings that stress- and drug-induced re-
lapse can be dissociated pharmacologically. As men-
tioned, CRF receptor antagonists attenuate footshock-
induced, but not drug-induced relapse (Erb et al. 1998;
Shaham et al. 1997). From these data it seems logical to
consider that footshock stress may have its effects on re-
instatement through an interaction between the CRF
and NE systems. Although the present data do not bear
directly on where in the brain this interaction might
take place, we have recent data that do (Erb and Stew-
art 1999; Shaham et al. 2000). We found first that
6-OHDA lesions of the ventral NE pathway attenuate
footshock-induced reinstatement of heroin seeking,
whereas injections of clonidine or ST-91 (a less diffus-
ible charged analogue of clonidine) directly into the lo-
cus coeruleus were not effective (Shaham et al. 2000).
On the basis of this work and additional studies on the
site of action of CRF and its receptor antagonist, D-Phe
CRF12-41, we have proposed that the effects of footshock
on relapse may be mediated by the interaction between
NE and CRF in the bed nucleus of the stria terminalis
(BNST) and/or the central nucleus of the AMG. We have
found that local injections of CRF and its receptor antag-
onist, D-Phe-CRF12-41, in the BNST act to induce and
block stress-induced reinstatement, respectively (Erb
and Stewart 1999). The ventral NE bundle provides the
primary source of NE to both the BNST and central nu-
cleus of the AMG (Aston-Jones et al. 1995; Terenzi and In-
gram 1995) and both structures are known to contain CRF
cell bodies (Phelix and Paull 1990; Veinante et al. 1997);
furthermore, the CRF cell bodies of the central nucleus of
the AMG project to the BNST (Sakanaka et al. 1986).
Whereas footshock-induced reinstatement may be
mediated by the NE and CRF systems, the effects of
priming injections of psychostimulant and opioid drugs
on relapse to drug seeking have been shown to be me-
diated by the midbrain dopaminergic system (Self and
Nestler 1998; Stewart 1984; Stewart and Vezina 1988). It
is interesting, however, that clonidine is known to have
effects on the functioning of midbrain dopaminergic
neurons (Lategan et al. 1990). For example, it has been
shown that systemic injections of alpha-2 adrenergic re-
ceptor agonists decrease dopamine release in the nu-
cleus accumbens (Murai et al. 1998) and the PFC
(Gobert et al. 1998). These effects may be due to the ef-
fect of alpha-2 adrenergic receptor agonists on dopa-
mine cell firing. Grenhoff and Svensson (1989) have
shown that clonidine regularizes midbrain dopamine
cell firing in anesthetized rats, an effect that should re-
duce dopamine release from terminals (Roth et al.
1987). These changes, however, may not have been suf-
ficient to prevent increases in extracellular dopamine in
the nucleus accumbens, in the face of cocaine-induced
blockade of dopamine reuptake. It is also possible that
previous exposure to cocaine during the self-adminis-
tration phase of the study served to increase dopamine
availability in the nucleus accumbens, thereby increas-
ing sensitivity to a priming injection of the drug (Kali-
vas and Duffy 1993).
The effects of clonidine and lofexidine observed in
this study could have been mediated through their ac-
tions at I1 rather than alpha-2 receptors. The nM affinity
of clonidine for the alpha-2 receptor is 28 ⫾ 2.6 and for
the I1 receptor is 0.99 ⫾ 0.43 (Ernsberger et al. 1990); the
affinity of lofexidine for these receptors is almost identi-
cal (Buccafusco et al. 1995). Alpha-2 and I1 receptors
have been shown, however, to be differentially in-
volved in biochemical and behavioral effects of alpha-2
adrenergic receptor agonists (for a review see Piletz et
al. 1994). For example, in drug discrimination studies,
alpha-2 receptors appear to be responsible for a cloni-
dine-induced cue, for which both lofexidine and guana-
benz (a compound with nM affinity for alpha-2 receptor
of 7.2 ⫾ 0.6 and for the I1 receptor of ⬎ 1,000,000) substi-
tute dose-dependently (Bennett and Lal 1982; Jordan et
al. 1993; Lal and Yaden 1985). On the other hand, I1 re-
ceptors of the medulla are thought to mediate the anti-
hypertensive actions of these drugs (Buccafusco et al.
1995; but see Guyenet 1997). Although not definitive in
view of the fact that guanabenz was not tested indepen-
dently for rate-reducing effects, it appears unlikely that
the attenuation of the footshock-induced reinstatement
of cocaine seeking was I1-receptor mediated; guana-
benz, a drug with a low affinity for the I1 receptor, at-
tenuated footshock-induced reinstatement of cocaine-
seeking at a dose that has been shown to substitute for
40 g/kg clonidine in a drug discrimination task (Ben-
nett and Lal 1982).
Three issues might be seen to complicate the inter-
pretation of the present findings. First, clonidine, lofexi-
dine and guanabenz might affect motor performance
(see Monti 1982; Stanford 1995); second, these drugs
might act at peripheral rather than central receptors
(Buccafusco et al. 1995); and finally, it is possible that
the analgesic actions of these drugs (see Codd et al.
1995) mediate their effect on footshock-induced rein-
statement. With respect to the first, it is unlikely that the
effects were due to a performance deficit. In the saline
and cocaine priming conditions, neither clonidine,
lofexidine nor guanabenz reduced responding on the
active lever. Furthermore, the doses of clonidine and
lofexidine used in the tests for reinstatement were cho-
sen because they had little or no effect on high rates of
responding for sucrose. After the highest dose of cloni-
dine tested (40 g/kg) in sucrose-trained animals (Sha-
ham et al. 2000), animals responded nearly 100 times in
20 min; likewise, in Experiment 3A, animals injected
with the highest dose of lofexidine (200 g/kg) ob-
tained close to 40 reinforcements in 30 min and demon-
strated no significant reduction in response rate.
The possibility that the effects observed in the
148 S. Erb et al. NEUROPSYCHOPHARMACOLOGY 2000–VOL. 23, NO. 2
present study were due to the actions of the alpha-2 ago-
nists at peripheral rather than central receptors also
seems unlikely. First, we found in a study with heroin-
trained rats that i.c.v. injections of clonidine were as ef-
fective as systemic injections in blocking the footshock-
induced reinstatement (Shaham et al. 2000). Additionally,
we have given systemic injections of 40 g/kg ST-91
(the charged analogue of clonidine which does not ef-
fectively cross the blood brain barrier) to cocaine-
trained animals and have found that ST-91-treated
animals show a comparable footshock-induced rein-
statement of responding (90.75 ⫾ 26.37 in three hours)
to vehicle-treated animals (115.63 ⫾ 24.49 in three
hours; unpublished data). It would appear, therefore,
that the effects of systemic injections of clonidine on
footshock-induced reinstatement of both heroin and co-
caine seeking are mediated centrally.
Finally, it seems unlikely that footshock-induced re-
instatement of drug seeking is mediated by analgesic
effects of these drugs at the doses used. Observations
made during the footshock sessions revealed that both
vehicle- and drug-pretreated animals reacted similarly
to the footshocks throughout the shock period. These
observations are consistent with the finding that cloni-
dine, within a similar dose range to the one used in the
present study, does not alter threshold sensitivity to
footshock (Soderpalm and Engel 1988).
In conclusion, the present findings may have clinical
implications. First, the results presented here may pro-
vide a rationale for the use of alpha-2 adrenergic recep-
tor agonists in treatment programs for the prevention of
relapse to drug use. Currently, these drugs are being
used with some success in the short-term treatment of
opioid withdrawal (Lin et al. 1997; Warner et al. 1997).
The present findings suggest that they may be effective
in the treatment of cocaine users (McDougle et al. 1994)
if given for a more prolonged period (see Herman and
O’Brien 1997). Second, the present experiments demon-
strate a similar efficacy of clonidine and lofexidine in
preventing footshock-induced reinstatement of cocaine
seeking. This finding is of potential clinical significance
in that lofexidine has been reported in humans to be as-
sociated with fewer adverse side effects than clonidine,
in particular fewer hypotensive effects (Carnwath and
Hardman 1998; Kahn et al. 1997; Lin et al. 1997).
ACKNOWLEDGMENTS
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 la Recherche (Que-
bec). S.E. was supported by graduate fellowships from the
Natural Science and Engineering Research Council of Canada
and Concordia University. We thank Dr. Yvona Buczek and
Angela Wang for their expert technical assistance in carrying
out the experiment with sucrose-trained rats.
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