SUPPLEMENTARY MATERIALS AND METHODS
Self-administration Contexts
Rats were randomly assigned to receive self-administration training in operant-conditioning
chambers equipped with two distinctly different sets of multi-modal contextual stimuli. Context
1 contained a continuous red house light (0.4 fc brightness), intermittent pure tone (80 dB, 1
kHz; 2 s on, 2 s off), pine-scented air freshener strip, and wire mesh floor (26 cm × 27 cm).
Context 2 contained an intermittent white stimulus light above the inactive lever (1.2 fc
brightness; 2 s on, 2 s off), continuous pure tone (75 dB, 2.5 kHz), vanilla-scented air freshener
strip, and a slanted ceramic tile wall that bisected the bar floor (19 cm × 27 cm). Rats had no
exposure to these contextual stimuli prior to cocaine self-administration training. These stimuli
were presented throughout each session independent of responding, as in our previous studies
(Fuchs et al, 2007; Fuchs et al, 2005; Fuchs et al, 2008; Xie et al, 2010).
Reinstatement Testing
After extinction training, the rats in experiments 1, 2, and 4 received two 1-h test sessions in the
previously cocaine-paired context and two 1-h test sessions in the extinction context, as
described previously (Xie et al, 2010). In experiment 3, rats received a single 1-h test session in
the previously cocaine-paired context or exposure to the home cage, followed by euthanasia.
Microinfusions were administered over 2 min immediately before each test session or before
home cage exposure in Experiment 3. The injectors were left in the guide cannulae for 1 min
before and after the infusion. Testing order in the two contexts (cocaine-paired context first,
extinction context first), treatment order (antagonist/inhibitor first, vehicle first), and treatment
dose of AP5, PP2, or Ro25-6981 were counterbalanced based on cocaine intake, when
appropriate. Between test sessions, rats received additional extinction training sessions until they
re-obtained the extinction criterion (≤25 active lever responses/session on at least 2 consecutive
days). During each test session, responses on the active and inactive levers were recorded, but
had no programmed consequences.
In experiment 1, rats (N=7/dose) received bilateral microinfusions of one dose of AP5
(Tocris Bioscience, Ellisville, MO; 0.25 or 2.5 µg/0.5 µl/hemisphere) or vehicle (phosphatebuffered saline; 0.5 µl/hemisphere) into the DH. These doses of AP5 in the nucleus accumbens
core have been shown to disrupt explicit cue-induced cocaine-seeking behavior (Bäckström and
Hyytiä, 2007).
In experiment 2, rats (N=8/dose) received bilateral microinfusions of one dose of PP2
(Tocris Bioscience, Ellisville, MO; 6.25 or 62.5 ng/0.5 µl/hemisphere) or vehicle (0.1% DMSO;
0.5 µl /hemisphere) into the DH. The 6.25ng dose of PP2 in the DH has been shown to disrupt
extinction of contextual fear conditioning (Isosaka et al, 2009), and the 62.5ng dose was selected
based on our pilot experiments.
In experiment 3, rats (N=6-9/dose) received bilateral microinfusions of PP2 (62.5 ng/0.5
µl/hemisphere) or vehicle (0.1% DMSO; 0.5 µl/hemisphere) into the DH. The behaviorally
effective dose of PP2 was selected based on the results of experiment 2.
In experiment 4, rats (N=8/dose) received bilateral microinfusions of one dose of Ro25-6981
(Tocris Bioscience, Ellisville, MO; 0.2 or 2 µg/0.5 µl/hemisphere) or vehicle (phosphatebuffered saline; 0.5 µl/hemisphere) into the DH. Administration of the same dose of Ro25-6981
into the anterior cingulate cortex inhibits acquisition of contextual fear conditioning (Zhao et al,
2005).
Locomotor Activity
In experiments 1, 2, and 4, two locomotor activity test sessions were conducted in a novel
context, using a partial within-subjects design, at least 72 h after the last reinstatement test
session. Before testing, a subset of rats (N=7-8/group) received bilateral microinfusions of the
behaviorally effective dose of AP5, PP2, Ro 25-6981 or vehicle into the DH using the infusion
procedures described above. Assignment to treatment groups and treatment order were
randomized. Horizontal locomotor activity was measured in novel Plexiglas chambers (42 × 20 ×
20 cm high), as described previously (3, 4). The total number of photobeam breaks was recorded
by a computerized activity system (San Diego Instruments, San Diego, CA, USA) during each
1h test session.
Food-reinforced Instrumental Behavior
After the last reinstatement test session, a second subset of rats (N=7-8/group) were trained to
lever press for food pellets (45 mg, Purina) during daily 2-h sessions in a neutral context (operant
conditioning chamber without the presence of contextual stimuli). As in our previous study (2),
the rats received free access to an additional 100 food pellets in their home cages 1h before each
session in order to elicit similar lever response rates – thus similar sensitivity to the rate-altering
effects of the pharmacological manipulations – as those seen in the cocaine reinstatement
experiments. During all training and test sessions, each active lever press resulted in the delivery
of a single food pellet (45 mg, Purina) under an FR 1 reinforcement schedule with a 20s timeout
period. Inactive lever presses had no programmed consequences. After active lever responding
stabilized (i.e., ≤ 20% variability across two consecutive sessions), two 1h test sessions were
conducted using a counterbalanced within-subject test design. Before testing, rats received
bilateral microinfusions of the behaviorally effective dose of AP5, PP2, Ro25-6981, or vehicle
into the DH using the infusion procedure described above. Between test sessions, rats received a
minimum of two food self-administration training sessions.
Data Analysis
In all experiments (i.e. Experiment 1-4), potential pre-existing differences between the treatment
groups in (a) lever responses and cocaine intake during the last three days of self-administration
training, (b) lever responses during the first seven days of extinction training, and (c) the number
of days needed to reach the extinction criterion were analyzed using mixed factorial ANOVAs.
In these ANOVAs, experimental group (Experiment 1, 2, 3, and 4) and treatment group
(Experiment 1 – AP5: 0.25 µg and 2.5 µg; Experiment 2 – PP2: 6.25 ng and 62.5 ng; Experiment
3 – PP2: vehicle and 62.5 ng; Experiment 4 – Ro25-6981: 0.2 µg and 2.0 µg) were included as
between-subjects factors, and subsequent treatment order (Experiment 1 – AP5 first, vehicle
first; Experiment 2 – PP2 first, vehicle first; Experiment 4 – Ro25-6981 first, vehicle first) and
time (day) was included as the within-subjects factor, where appropriate.
Mixed factorial ANOVAs were used to determine whether the vehicle data could be
collapsed for each experimental group (i.e. Experiment 1, 2, and 4). In Experiment 1, nonreinforced active and inactive lever presses on the vehicle test days were analyzed separately
using mixed factors ANOVAs with additional treatment (AP5 0.25 µg, AP5 2.5 µg) and test
order (extinction context first, cocaine-paired context first) as between-subjects factors and
testing context (extinction, cocaine-paired) as the within-subjects factor. In Experiment 2, nonreinforced active and inactive lever presses on the vehicle test days were analyzed separately
using mixed factorial ANOVAs with additional treatment (PP2 6.25 ng, PP2 62.5 ng) and test
order (extinction context first, cocaine-paired context first) as between-subjects factors and
testing context (extinction, cocaine-paired) as the within-subjects factor. In Experiment 4, nonreinforced active and inactive lever presses on the vehicle test days were analyzed separately
using mixed factors ANOVAs with additional treatment (Ro25-6981 0.2 µg, Ro25-6981 2.0 µg)
and test order (extinction context first, cocaine-paired context first) as between-subjects factors
and testing context (extinction, cocaine-paired) as the within-subjects factor.
To assess the effects of intra-DH AP5, PP2, and Ro25-6981 on the test days, active and
inactive lever responses were analyzed separately using mixed factorial ANOVAs with drug
treatment (vehicle, Experiment 1 – AP5: 0.25 µg, 2.5 µg; Experiment 2 – PP2: 6.25 ng, 62.5 ng;
Experiment 3 – PP2 62.5 ng; Experiment 4 – Ro25-6981: 0.2 µg, 2.0 µg) as the between-subjects
factor and testing context (extinction, cocaine-paired) as the within-subjects factor.
To assess the effects of intra-DH PP2 and vehicle infusions on active lever responding in
cocaine-paired context across time in Experiment 3, active lever responding were analyzed using
mixed factors ANOVAs with treatment (PP2 62.5 ng, vehicle) as the between-subjects factor and
time (20-min intervals) as the within-subjects factor.
In Experiment 3, the effect of intra-DH PP2 on NR2B activation was analyzed using a twoway ANOVA with treatment (PP2 62.5 ng, vehicle) and context (home cage, cocaine-paired) as
between-subjects factors. In addition, to evaluate the relationship between active lever
responding during the first 20-min interval of reinstatement testing and NR2B activation,
separate Pearson’s correlation coefficients were calculated for the vehicle-treated and PP2treated groups. To evaluate the relationship between cocaine intake and NR2B activation, the
Pearson’s correlation coefficient was calculated for the vehicle-treated rats.
The number of photobeam breaks during the locomotor activity tests was assessed using a
repeated-measures ANOVA with drug treatment (vehicle, Experiment 1 – AP5 2.5 µg;
Experiment 2 – PP2 62.5 ng; Experiment 4 – Ro25-6981 2.0 µg) and time (20-min intervals) as
within-subjects factors. Food-reinforced instrumental responding was assessed using a repeated
measures ANOVA with drug treatment (vehicle, Experiment 1 – AP5 2.5 µg; Experiment 2 –
PP2 62.5 ng; Experiment 4 – Ro25-6981 2.0 µg) and lever (active, inactive) as within-subjects
factors.
SUPPLEMENTARY RESULTS
Cocaine Self-administration and Extinction History
The descriptive statistics for drug intake and behavioral training history are summarized in Table
S1. Generally, all experimental groups of rats in Experiment 1-4 exhibited stable responding on
the active lever during the last three self-administration training days with a within-subject
variability of < 10% in daily cocaine intake. Collapsed across groups, the mean numbers of
active lever responses was 68.52 ± 6.46, and the mean daily cocaine intake ± standard error of
mean (SEM) was 13.39 ± 0.59 mg/kg per session based on body weight (26.78 ± 1.19 infusions).
Separate analyses for each experiment revealed no pre-existing differences between the groups
that were subsequently assigned to receive different dose of AP5, PP2, or Ro25-6981 in active or
inactive lever responding during last three days of cocaine self-administration training (all
treatment main and interaction effects, F(1-2, 12-28) = 0.01-2.46, p = 0.13-0.99).
Upon the removal of cocaine reinforcement, active and inactive lever responding
gradually declined in each experimental groups (all time main effects, F(6, 366) = 20.09-37.99, p =
0.0001). There was no pre-existing difference between experimental groups in active or inactive
lever responding during the first seven days of extinction training (all experiment main and
interaction effects, F(3-18,
61-366)
= 0.22-1.18, p = 0.32-0.97) or in the mean number of daily
sessions (± SEM; 7.31 ± 0.05) needed to reach the extinction criterion (F(3, 63) = 2.36, p = 0.08).
Separate ANOVAs for each experiment indicated that active and inactive lever responding
gradually declined to criterion in the treatment groups that were assigned to subsequently receive
different dose of AP5, PP2, or Ro25-6981 (all time main effects, F(6, 72-102) = 7.90-31.84, p =
0.0001). Furthermore, with each experiment, there was no pre-existing difference between the
treatment groups in active or inactive lever responding during the first seven days of extinction
training (all treatment main and interaction effects, F(1-6, 12-102) = 0.01-2.09, p = 0.07-0.99) or in
the mean number of daily sessions needed to reach the extinction criterion (F(1, 12-17) = 0.01-1.53,
p = 0.23-1.00).
SUPPLEMENTARY TABLE
Table S1. Descriptive statistics of behavioral training history
Cocaine Self-administration
Extinction (Lever Presses)
Exp/Group
Lever
Intake
First Day
Last Day
Presses
VEH/0.25µg
69.09 ± 12.48 69.29 ± 13.50
3.43 ± 1.17
27.90
±
2.14
AP5
(2.62 ± 2.50)
(9.43 ± 5.58)
(1.57 ± 0.81)
1
VEH/2.5µg
66.14 ± 12.92
67.43 ± 6.45
6.14 ± 1.30
26.71 ± 1.90
AP5
(3.43 ± 2.17)
(10.00 ± 4.07) (3.86 ± 3.54)
VEH/6.25ng
75.71 ± 9.51
74.87 ± 14.23
3.00 ± 0.84
25.96 ± 2.88
PP2
(1.71 ± 3.92)
(11.63 ± 6.03) (2.50 ± 1.22)
2 VEH/62.5ng
67.58 ± 6.14
68.38 ± 8.18
7.63 ± 2.74
21.67 ± 3.18
PP2
(4.37 ± 2.05)
(14.36 ± 4.82) (1.75 ± 1.37)
VEH
51.44 ± 9.94
59.17 ± 11.68
6.00 ± 1.24
27.91 ± 2.14
(HC)
(4.06 ± 3.83)
(15.50 ± 4.75) (3.83 ± 1.64)
62.5ng PP2
57.29 ± 9.20
68.14 ± 19.50
2.86 ± 4.13
26.71 ± 3.70
(HC)
(3.81 ± 1.28)
(17.00 ± 4.18) (2.57 ± 4.19)
3
VEH
52.59 ± 5.23
71.00 ± 16.68
1.89 ± 0.54
22.59 ± 4.27
(COC-paired)
(6.70 ± 8.17)
(17.22 ± 3.58) (4.00 ± 2.11)
62.5ng PP2
63.52 ± 11.99 56.67 ± 17.93
1.00 ± 2.80
26.15 ± 4.70
(COC-paired)
(4.59 ± 2.28)
(6.44 ± 3.26)
(1.89 ± 0.99)
VEH/0.2µg
67.96 ± 18.46 74.25 ± 21.15
8.00 ± 2.46
25.71 ± 4.71
Ro25-6981
(8.87 ± 7.41)
(20.50 ± 5.96) (2.75 ± 1.82)
4
VEH/2.0µg
75.04 ± 18.46 78.63 ± 16.50
7.75 ± 2.45
30.96 ± 7.07
Ro25-6981
(2.21 ± 7.13)
(14.75 ± 7.22) (5.12 ± 2.59)
Days to
EXT
7.43 ± 0.20
7.29 ± 0.18
7.38 ± 0.18
7.38 ± 0.18
7.33 ± 0.26
7.14 ± 0.13
7.18 ± 0.15
7.56 ± 0.18
7.13 ± 0.12
7.38 ± 0.18
Table S1: Cocaine intake (mean ± SEM infusions/session for the last 3 training sessions), active
and inactive (in parentheses) lever responses during self-administration (SA; mean ± SEM for
the last 3 training sessions) and extinction training sessions, and the number of days required to
meet the extinction criterion (Days to EXT). VEH = vehicle; HC = home cage; COC-paired =
cocaine-paired context.
20
Veh
0.25 g AP5
2.5 g AP5
10
0
EXT
COC-paired
Context
C
B
20
Veh
6.25 ng PP2
62.5 ng PP2
*
10
0
EXT
COC-paired
Context
Inactive lever presses/1h
A
Inactive lever presses/1h
Inactive lever presses/1h
SUPPLEMENTARY FIGURES
20
Veh
0.2 g Ro 25-6981
2 g Ro 25-6981
*
10
0
EXT
COC-paired
Context
Fig. S1. Effects of bilateral AP5 (A: N = 7-14/dose; mean/1h + SEM), PP2 (B: N = 8-16/dose;
mean/1h + SEM), or Ro25-6981 (C: N = 7-14/dose; mean/1h + SEM) pretreatment in the DH on
non-reinforced inactive lever responses during testing in the extinction (EXT) and previously
cocaine-paired contexts (COC). Asterisks represent significant difference relative to responding
in the extinction context (ANOVA context main effect, p < 0.05).
60
Veh
62.5 ng PP2
*
40
†
20
0
EXT
COC-paired
Context
Inactive lever presses/1h
Active lever presses/1h
A
B
60
Veh
62.5 ng PP2
40
20
0
*
EXT
COC-paired
Context
Fig. S2. Effects of bilateral PP2 (N = 9/group; mean/1h + SEM) pretreatment in the DH on nonreinforced active (A) and inactive (B) lever responses in the extinction (EXT, last extinction
session) and previously cocaine-paired contexts (COC). Asterisks represent significant difference
relative to responding in the extinction context (Panel A: ANOVA context simple main effect,
Tukey test, p < 0.05; Panel B: ANOVA context main effect, p < 0.05). Dagger represents
significant difference relative to vehicle treatment (ANOVA treatment simple main effect, Tukey
test, p < 0.05).
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