Cocaine Self-Administration Induces Dopamine-Independent Self-Administration of Sigma
Agonists
Supplementary Material
Takato Hiranita, PhD1, Maddalena Mereu, PhD 1, Paul L. Soto, PhD 2, Gianluigi Tanda, PhD 1
and Jonathan L. Katz, PhD 1
1
Psychobiology Section, Molecular Targets and Medications Discovery Branch, Intramural
Research Program, Department of Health and Human Services, National Institute on Drug
Abuse, National Institutes of Health, Baltimore, MD 21224
2
Behavioral Biology Research Center, 5510 Nathan Shock Drive, Suite 3000, Johns Hopkins
University Medical School, Baltimore, MD 21224-6823
Detailed Methods
Subjects. Forty-two male Sprague-Dawley rats (Taconic Farms, Germantown, New
York) weighing approximately 300 g at the start of the study, served as subjects. Subjects were
acclimated to a temperature- and humidity-controlled vivarium for at least one week with food
(Scored Bacon Lover Treats, BIOSERV, Frenchtown, NJ) and tap water unrestrictedly available
in their home cages under a 12:12-h light:dark cycle with lights on at 07:00 hours. After
acclimation, weights of rats were maintained at approximately 320 g by adjusting their daily
food ration. Subjects were surgically implanted in the right external jugular vein with a chronic
indwelling catheter that exited at the mid-scapular region of the animal’s back. Catheter patency
was maintained over the course of the behavioral procedures, however, the subjects used of
microdialysis studies of dopamine concentrations in the shell of the nucleus accumbens were
surgically re-implanted with left external jugular catheters. Catheter implantation was performed
under anesthesia (ketamine 60.0 mg/kg, i.p. and xylazine 12.0 mg/kg, i.p.). Catheters were
infused daily with 0.1 ml of a sterile saline solution containing heparin (30.0 IU/ml), penicillin G
potassium (250,000 IU/ml) to minimize the likelihood of infection, and the formation of clots or
fibroids. All animals were allowed to recover from surgery for approximately seven days before
drug self-administration studies were initiated. Details of allocation of all the subjects to
individual experiments in the present study are supplied in Supplementary Table 1.
Care of the subjects was in accordance with the guidelines of the National Institutes of
Health and the National Institute on Drug Abuse Intramural Research Program Animal Care and
Use Program, which is fully accredited by AAALAC International.
Self-Administration Procedures. Experimental sessions were conducted with animals
placed in operant-conditioning chambers (modified ENV-203, Med Associates, St. Albans, VT)
that measured 25.5 x 32.1 x 25.0 cm, and were enclosed within sound-attenuating cubicles
equipped with a fan for ventilation and white noise to mask extraneous sounds. On the front wall
of each chamber were two response levers, 5.0 cm from the midline and 4.0 cm above the grid
floor. A downward displacement of a lever with a force approximating 20 g defined a response,
which always activated a relay mounted behind the front wall of the chamber producing an
audible “feedback” click. Three light-emitting diodes (LEDs) were located in a row above each
lever. A receptacle for the delivery of 45-mg food pellets via a pellet dispenser (Med Associates,
Model ENV-203-20), was mounted on the midline of the front wall between the two levers and
2.0 cm above the floor. A syringe infusion pump (Model 22, Harvard Apparatus, Holliston, MA)
placed above each chamber delivered injections of specified volumes from a 10 ml syringe. The
syringe was connected by Tygon tubing to a single-channel fluid swivel (375 Series Single
Channel Swivels, Plymouth Meeting, PA) which was mounted on a balance arm above the
chamber. Tygon tubing from the swivel to the subject’s catheter was protected by a surrounding
metal spring and completed the connection to the subject.
Experimental sessions were conducted daily with subjects placed in chambers for
experimental sessions that lasted for 120 min. After sessions subjects were returned to their
cages in the vivarium. Sessions started with the illumination of the LEDs above each lever.
Each downward deflection of the right lever turned off the LEDs and activated the infusion
pump for 10 sec (fixed-ratio or FR 1 schedule) followed by a 20-sec time-out period during
which LEDs were off and responding had no scheduled consequences. Drug injections were
cocaine (0.32 mg/kg/injection, n=6), PRE-084 (0.32 mg/kg/injection, n=6) or (+)-pentazocine
(0.32 mg/kg/injection, n=6). After the time out, the LEDs were illuminated and responding
again had the scheduled consequences. Responses on the left lever were recorded but had no
scheduled consequences. This condition remained in effect for 28 experimental sessions.
Acquisition of drug self-administration behavior was defined as less than 20% variation in
response rates across three consecutive sessions.
For the cocaine self-administration group, responses on the left rather than right lever
produced injections for the next seven sessions, with all other conditions as in the first 28
sessions. During the subsequent nine sessions, injections and accompanying stimulus changes
were discontinued (extinction) with other aspects of the sessions unchanged. Finally, responses
on the left lever again produced cocaine injections for five sessions under the FR 1 schedule as
described above (reacquisition).
During the initial 28 sessions with PRE-084 or (+)-pentazocine, responding was not
maintained by either drug. Subsequently the PRE-084 group was studied with 5 different doses
(0.03-1.0 mg/kg/inj) for 14 sessions each, after which they were allowed to self-administer
cocaine (0.32 mg/kg/inj) for 14 sessions under the FR 1 schedule as described above. The (+)pentazocine group was immediately changed to cocaine self-administration under the FR 1
schedule. After cocaine self-administration, all of the subjects were returned to the FR 1
schedule of PRE-084 or (+)-pentazocine self-administration, and the subsequent series of
sessions (change in active lever, extinction, reacquisition) for both groups was as described for
the cocaine group.
A separate group of subjects (n=6) were trained with food reinforcement (45 mg food
pellets, Bioserv, Rodent Purified Diet, Dustless Precision Pellets, Frenchtown, NJ) to press the
right lever under an FR 1-response schedule of reinforcement. All aspects of the schedule for
food delivery were similar to those for drug self-administration except that food pellets rather
than injections maintained responding. Similar to the study of drug self-administration, this
study of food reinforcement had the periods of acquisition of lever-pressing, alternating the lever
on which responses produced food, extinction, and reacquisition. After the reacquisition phase,
the subjects were catheterized, allowed to respond again for five sessions with food
reinforcement, and subsequently allowed to self-administer PRE-084 (0.32 mg/kg/inj) for 28
sessions.
The self-administration procedure was slightly modified to pharmacologically assess
mechanisms of the reinforcing effects of PRE-084 and to compare them to those of cocaine.
Subjects from the above described cocaine self-administration experiments and several
experimentally naïve subjects (N=18) were trained until their cocaine (0.32 mg/kg/inj) selfadministration was consistent from one session to the next under the FR 1 schedule described
above. Subsequently, the FR value was increased to five and the session was divided into five
20-min components, each preceded by a 2-min time-out period. This arrangement (Hiranita et
al, 2009) allowed the assessment of a range of self-administered doses in a single session. By
adjusting infusion volumes and durations, the drug dose per injection was incremented in the five
sequential components in an ascending dose-order as follows: no injection (also referred to as
extinction, or EXT, because responses had no scheduled consequences), 0.03, 0.10, 0.32, and 1.0
mg/kg/inj for cocaine. Infusion volumes and durations were respectively 0, 5.6, 18.0, 56.0, 180
μl and 0, 0.32, 1.0, 3.2 10.0 sec, based on a body weight of 0.32 kg. A sample injection of the
drug at the corresponding dose occurred independently of responding at the end of the time-out
period that preceded each component except the first.
Training continued until responding was maintained with less than 20% variation in
response rates across three consecutive sessions. With these stable performances the effects on
cocaine self-administration (0.03, 0.10, 0.32, and 1.0 mg/kg/inj) of presession i.p. injections of
the DA receptor antagonists (SCH 39166, L-741,626 or haloperidol) or the preferential σ1R
antagonist (BD1063) were assessed. These drugs were also examined in the same rats with PRE084 (0.03, 0.10, 0.32, and 1.0 mg/kg/inj) substituted for cocaine under otherwise identical
conditions. The effects of pre-session treatments on cocaine self-administration were separated
by a minimum of 72 hours. The antagonists were studied with a mixed order of drugs and doses.
In-vivo Microdialysis Procedures. Subjects were male Sprague–Dawley rats from selfadministration experiments shown in figure 3a and 3c (open triangles up). Rats were implanted
with microdialysis probes under a mixture of ketamine and xylazine (60.0 and 12.0 mg/kg i.p.,
respectively) anesthesia. Rats were placed in a stereotaxic apparatus, the skull was exposed, and
a small hole was drilled to expose the dura. Rats were then implanted with a concentric dialysis
probe (see below) aimed at the NAc shell either in the right or in the left brain side, as described
previously (Tanda et al 2005; Tanda et al 2008). Coordinates were from the rat brain atlas of
Paxinos and Watson (1987) [uncorrected coordinates: Anterior = + 2.0 mm from bregma, Lateral
= ± 1.0 mm from bregma, Vertical = -7.9 mm from dura]. After the surgery, rats were allowed to
recover overnight in hemispherical CMA-120 cages (CMA/Microdialysis AB, Solna, Sweden).
Concentric dialysis probes were prepared with AN69 hollow fibers (Hospal Dasco, Bologna,
Italy). Briefly, two 4-cm pieces of silica fused capillary tubes (the inlet and outlet tubing of the
probes) were inserted into a 6-mm capillary (0.25 mm external diameter) dialyzing fiber (closed
by a drop of glue on the other side) with the inlet tubing set at about 0.1 mm from the closed end
of the fiber and the outlet set at about 1.8 mm from the inlet tip. The open end of the dialysis
membrane was then glued, and the protruding two silica fused capillary tubes were inserted and
glued into a 22-G stainless steel needle (2.4 mm length). The needle was attached to a CMA/10
clip (CMA/Microdialysis AB) and mounted in a stereotaxic holder. The exposed dialyzing
surface of the fibers, i.e., the portion not covered by glue, was limited to the lowest 1.8 mm of
the probes.
Experiments were performed on freely moving rats in the same hemispherical cages in
which they recovered from surgery. About 22 hours after implant, probes were connected to fluid
swivels (375/ D/22QM; Instech, Plymouth Meeting, PA, USA), and Ringer’s solution (147.0
mM NaCl, 2.2 mM CaCl2, and 4.0 mM KCl) was delivered by a 1.0 ml syringe operated by a
BAS Bee Syringe Pump Controller (BAS, West Lafayette, IN, USA) through the dialysis probes
at a constant flow rate of 1 μl/min. Collection of dialysate samples started after 30 min, and 10 μl
samples were taken every 10 min and immediately analyzed, as detailed below. After stable DA
values (less than 10% variability) were obtained for at least three consecutive samples (after
about 1 hour), rats were injected with increasing doses of PRE-084, 0.32, 1.0, 3.2, 10 mg/kg
i.v, spaced 1 hour apart. Dialysate samples were injected without purification into a HPLC
apparatus equipped with an MD 150 mm X 3.2 mm column, particle size 3.0 μm (ESA,
Chelmsford, MA, USA), and a coulometric detector (5200a Coulochem II, or Coulochem III,
ESA) to quantify DA. The oxidation and reduction electrodes of the analytical cell (5014B;
ESA) were set at +125 mV and -125 mV, respectively. The mobile phase, containing 100 mM
NaH2PO4, 0.1 mM Na2EDTA, 0.5 mM n-octyl sulfate, and 18% (v/v) methanol (pH adjusted to
5.5 with Na2HPO4), was pumped by an ESA 582 (ESA, Chelmsford, MA, USA) solvent
delivery module at 0.50 ml/min. Assay sensitivity for DA was 2 fmoles/sample.
At the end of each experiment, rats were sacrificed by pentobarbital overdose. Brains
were removed and left to fix in 4% formaldehyde in saline solution. Brains were then cut on a
vibratome 1000 Plus (The Vibratome Company, St. Louis, MO, USA) in serial coronal slices
based on Paxinos and Watson (1987). All of the probes were located inside the NAc shell have
with placements shown diagrammatically in Supplementary Figure 1.
Drugs. The drugs used in the present study were as follows: (-)-cocaine hydrochloride
(Sigma-Aldrich, St. Louis, MO), PRE-084 (Tocris, Ballwin, MO), (+)-pentazocine succinate
(National Institute on Drug Abuse), BD1063 (Tocris), SCH 39166 (Tocris), L-741,626 (SigmaAldrich) and haloperidol (Sigma-Aldrich). 0.9% NaCl was used as vehicle for all compounds
except L-741,626 and haloperidol. L-741,626 and haloperidol were dissolved in ethanol and
diluted to ≤6% ethanol (v/v) and ≤25% polyethylene glycol (v/v) in sterile water. Drugs used
were administered intravenously (cocaine, PRE-084, and (+)-pentazocine) or intraperitoneally
(BD 163, SCH 39166, L-741,626 and haloperidol). BD1063 was administered at 5 min before
sessions. All dopamine receptor antagonists were administered at 30 min before sessions. The
chemical identities of drugs used are as follows: BD1063; 1-[2-(3,4-dichlorophenyl)ethyl]-4methylpiperazine dihydrochloride; L-741,626: (±)-3-[4-(4-chlorophenyl)-4-hydroxypiperidinyl]methylindole; (+)-pentazocine: 2-dimethylallyl-5,9-dimethyl-2'-hydroxybenzomorphan
succinate; PRE-084: 2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate hydrochloride; SCH
39166: (6aS-trans)-11-chloro-6,6a,7,8,9,13bhexahydro-7-methyl-5H-benzo[d]naphth[2,1b]azepin-12-ol hydrobromide.
Data analysis. Response rates were determined by dividing responses by elapsed time,
excluding time outs after drug injections or food presentations. The significance of effects on
response rates was assessed by ANOVA. A Bonferroni t-test was used to compare a series of
response rate on drug injections, or food reinforcement. For studies involving dose-effect curves
for drug self-administration, a post-hoc Bonferroni t-test was used for all pairwise comparisons.
A two-way repeated-measures ANOVA was used to assess the effects of successive response
rates during drug self-administration or food presentation (factors were session and lever: right
or left). A two-way, repeated-measures ANOVA was also used to assess the drug-dose effects
(factors were drug-dose and lever: right or left) or effects of pre-session treatment with
antagonists on self-administration (factors were drug-dose and component: no injection or doses
of cocaine or PRE-084). For all analyses, the criterion for significance was set at p<0.05.
The in-vivo microdialysis results were expressed as a percentage of basal DA values,
which were calculated as means of the three consecutive samples immediately preceding the test
drug or saline injection. All results are presented as group means (±SEM). Dose-effect curves
were analyzed using standard ANOVA and linear regression techniques, from which ED50 values
(doses producing 50% of maximal effect) were calculated from points on the linear part of the
curves (Snedecor and Cochran, 1967). To assess whether cocaine experience altered the effects
of PRE-084 data were analyzed by standard parallel-line bioassay techniques (Finney, 1964).
The relative-potency value obtained represents the dose of PRE-084 in subjects with cocaine
experience equal to 1 mg/kg PRE-084 in subjects without cocaine experience. A relative
potency value with 95% confidence limits that include 1.00 is not significant.
Results
A separate group of experimentally-naïve rats was trained to lever-press with food
reinforcement under an FR 1-response schedule (Fig. S2). Following that acquisition, the active
lever was switched from the right to the left which produced a corresponding change in the lever
on which responses were emitted. Pellet deliveries were subsequently discontinued (extinction)
with a resulting decrease in response rates to low levels. When food pellets were again delivered
after each response responding was re-acquired (Fig. S2). A two-way repeated-measures
ANOVA indicated significant effects on response rates (p values ≤ 0.006) of session number
(F49,245=12.8), right or left lever (F1,245=20.5), and their interaction (F49,245=28.3).
Supplementary Figure Legends
Supplementary Figure 1: Drawings of forebrain sections, based on those of Paxinos and
Watson (1998), showing the microdialysis probe tracks. All of the dialyzing portions of the
microdialysis probes were within the boundaries of the NAC shell.
Supplementary Figure 2: Reinforcing effects of food pellets in experimentally naïve rats. Each
point represents the mean ±SEM in 6 subjects. Food reinforcement readily increased rates of
responding (right lever) when each response produced a food pellet, without increasing rates of
responding on the alternate (left) lever which had no scheduled consequences. When food pellets
were available only for responses on the previously inactive (left) lever, responding switched to
that lever. When food pellets were no longer available responding on both levers decreased to
low levels. When food was again available for responses on the left lever, responding increased
on that lever. *p<0.05, **p<0.01, ***p<0.001, compared with responding on the inactive lever
(Bonferroni t-test). A two-way repeated-measures ANOVA indicated significant effects on
response rates (p values ≤ 0.006) of session number (F49,245=12.8), right or left lever
(F1,245=20.5), and their interaction (F49,245=28.3).
Supplementary Table 1: The experimental conditions for each group of subjects
Group
1
Initial Training
Cocaine
2
Cocaine
3
Cocaine
4
PRE-084
5
6
7
PRE-084
(+)-Pentazocine
Food
Experiment
1. Comparison of acquisition of cocaine and σagonist self-administration; cocaine group
2. Antagonist treatments on cocaine self
administration
Antagonist treatments on PRE-084 self
administration
Antagonist treatments on PRE-084 self
administration
1. Comparison of acquisition of cocaine and σagonist self-administration; PRE-084 group
2. Acquisition of cocaine self-administration
after exposure to PRE-084
3. PRE-084 self-administration after cocaine
self-administration
4. Measurement an extracellular dopamine
concentration
1. Dose-effect curve for PRE-084 selfadministration before cocaine exposure
2. Measurement an extracellular dopamine
concentration
1. Acquisition of (+)-pentazocine selfadministration
2. Acquisition of cocaine self-administration
after exposure to (+)-pentazocine
3. (+)-Pentazocine self-administration after
cocaine self-administration
1. Acquisition of responding maintained by
food presentation under FR 1
2. PRE-084 substitution for food under FR 1
Results shown
Figure 1a
Figures 4a-e
Figures 4f and 4j
Figures 4g-i
Figures 1b and 2c
Figures 2a and 3c
Figures 3a, 3c and
2c
Figure 5
Figure 3c
Figure 5
Figure 1c
Figure 2b
Figure 3b
Supplementary
Figure 1
Figures 3d and 3e