Supplemental Material
General methods
Selective breeding paradigm
A number of measures were taken to maximize initial genetic variation and to reduce inbreeding
in the selectively-bred rat lines (for details see Stead et al., 2006). Briefly, the founding
population for the selectively bred lines was composed of 60 male and 60 female SpragueDawley rats purchased from Charles River Laboratories and obtained from three different
locations to increase genetic diversity (Kingston, NY, USA; Portage, MI, USA; and SaintConstant, QC, Canada). Animals from each of the three colonies contributed equally to the first
generation (S1) of selectively-bred rats. Behavioral testing for locomotor response to a novel
environment (see below) was performed on adult rats between postnatal days 55 and 65. Males
and females with the highest and lowest scores from locomotor testing were bred together to
generate the bred high-responder (bHR) and bred low-responder (bLR) lines, respectively.
Following completion of locomotor testing, rats were bred for the subsequent generation at 8090 days of age.
A. Locomotor response to novelty
As previously described (Stead et al., 2006; Clinton et al., 2007), each generation of selectivelybred rats were screened for locomotor activity in test chambers made of clear acrylic (43 x 21.5
x 25.5 cm (high) and equipped with infrared photocell emitters mounted 2.3 and 6.5 cm above
the grid floor. The animals were exposed to the test chamber for the first time on the day of
testing. Horizontal and rearing activity was monitored in 5-min intervals over a period of 60 min
via a computer. All testing was performed between 0800 and 1130. Total locomotion scores for
each rat were calculated by adding the total number of horizontal and rearing movements. bHR
rats whose scores fell one standard deviation below the bHR group average and bLR rats
whose scores fell one standard deviation above the bLR group average were not used for the
present studies.
B. Pavlovian conditional approach
B1. Pavlovian conditioning chambers (food, US)
Med Associates (St. Albans, VT) test chambers (21.6 cm x 17.8 cm floor area, 12.7 cm high)
located inside a sound-attenuating enclosure were used. Each chamber was equipped with a
food receptacle, which was located in the center of the 21.6-cm-wide-wall, 3 cm above the
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stainless steel grid floor. An illuminated retractable lever (Med Associates) was located
approximately 2.5 cm to the left or right of the food receptacle, 3 cm above the floor. The side of
the lever with respect to the food receptacle was counter-balanced to eliminate any side bias.
The lever required only a 10-gram force to operate, such that most contacts with the lever were
detected and recorded. Operation of the pellet dispenser (Med Associates) delivered one 45-mg
banana flavored food pellet (Bio-Serv®, Frenchtown, NJ) into the food receptacle. Head entries
into the food receptacle were recorded each time the rat broke a photobeam located inside the
receptacle.
B1. Pavlovian conditioning procedures (food US)
All training sessions were conducted between 1300 and 1800 hr. Banana-flavored food pellets
were placed into the rats’ home cages for 2 days prior to training to familiarize the animals with
this food (the US). Two pre-training sessions were conducted which consisted of the delivery of
50 food pellets that were randomly delivered on a variable 30-s schedule (25-min session) and it
was determined whether the rats were reliably retrieving the food pellets. Following pre-training,
Pavlovian training sessions were conducted as described in the main text.
B2. Jugular catheterization surgery for Pavlovian conditioning (cocaine, US)
Briefly, one end of silicone catheter was inserted into the external jugular vein and the other was
passed subcutaneously to exit the back of the animal, where it was connected to a pedestal
constructed from a 22 gauge cannula and connected to a piece of polyethylene mesh using
dental cement. For 2 weeks following surgery, catheters were flushed daily with 0.1 ml of sterile
saline containing gentamicin (10 mg/ml) to prevent occlusions and microbial buildup in the
catheter. After the first 2 weeks post-surgery, rats received a lower concentration of gentamicin
(0.1 mg/ml delivered in 0.1 ml sterile saline) on a daily basis. Before and after Pavlovian training
began catheters were checked for patency by injecting 0.1 ml of the short-acting barbiturate
sodium thiopental (i.v.; 20 mg/ml in sterile water). Rats that became ataxic within 5 s were
considered to have patent catheters.
B2. Habituation sessions prior to Pavlovian conditioning (cocaine, US)
Rats were habituated to lever presentation prior to Pavlovian training in order to obtain a stable
measure of baseline responding. Habituation sessions were initiated by activation of the house
light and white noise generator, both of which remained on throughout the sessions. The first
two habituation sessions consisted of 24 trials in which the illuminated lever was extended for 8
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s and the infusion pump activated for 2.8 s (as during Pavlovian training). The ITI varied
randomly with a mean interval of 120 s. Following the first two habituation sessions, rats
underwent jugular catheterization surgery (described above), and after recovery from surgery
rats were exposed to 8 additional habituation sessions. The habituation session was altered
such that it now consisted of 14 trials with a mean ITI of 60 s. The last habituation session was
conducted using the same paradigm described below (but with saline instead of cocaine) in
order to obtain measures of baseline behavior comparable to that during the training session.
B2. Pavlovian conditioning procedures (cocaine US)
Pavlovian training sessions were conducted between 0800 and 1300. Rats were brought to the
chambers and connected to infusion lines via their backport. Sessions began with the activation
of the red house light and white noise generator. One session consisted of 6 trials (CS-US
pairings) and each trial consisted of the 8 s presentation of the illuminated lever (CS) paired with
the non-contingent intravenous injection of 0.5 mg/kg (weight of the salt, dissolved in 0.9%
sterile saline) of cocaine (US). The infusion pump was activated upon insertion of the lever
because of the delay involved with an injection, and the injection itself took 2.8 s.
The number of approaches and the latency to approach the lever was recorded with a plastic
insert that was equipped with photocells aligned 1 cm in front of the extended lever. This
modification was made to the test chambers because it had previously been found that when
paired with the intravenous delivery of cocaine, animals approach and investigate the lever, but
seldom come into contact with it, and therefore lever presses do not provide a good measure of
approach behavior when cocaine is used as the US (Uslaner et al., 2006).
C. Impulsive behavior
C1. Delay-discounting procedures
Behavioral testing occurred between the hours of 1300 and 1700. On the first day of training,
rats were brought to the chambers and received 50 banana pellets delivered on a random
interval schedule with a mean ITI of 30 s to familiarize the rats with retrieving pellets from the
food cup. This session lasted ≈25 min. Rats were then trained on a fixed ratio (FR) 1 schedule
of reinforcement to lever press for the banana pellets. These sessions lasted 30 min, or until the
rat reached a criterion of 50 presses/session. Alternating levers (i.e. left or right) were presented
during each training session such that rats were exposed to 3 FR1 sessions with the left lever
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and 3 FR1 sessions with the right lever. By the end of this training phase all rats had reached a
criterion of 50 presses in 30 min for both levers.
Rats were then trained on a simplified version of the delay-discounting task. These sessions
consisted of 90 trials, each lasting 40 s. Each trial began with activation of the house light and
the large cue light placed above the food cup. The rat was required to poke its nose into the
food cup within 10 s, which resulted in the cue light being turned off and the presentation of the
illuminated lever (either the right or the left, randomly selected). Initiation of a trial as a result of
a head entry into the food cup ensured that the rat was centrally located between the two levers
prior to each trial. If the rat failed to initiate the trial by placing its nose into the food cup within
10 s of activation of the cue light, the trial was aborted and the houselight and cue light turned
off. Likewise, if the rat failed to press the lever within 10 s of presentation, the trial was aborted,
the illuminated lever retracted, and the houselight turned off. If the rat successfully pressed the
lever within the 10 s period, the lever was retracted, a single banana pellet was delivered into
the food cup, and the large cue light above the food cup was activated. Once the rat retrieved
the food pellet (or after 10 s had elapsed), the cue light was turned off and the house light was
turned off 6 s later. The forced-choice training procedures were prolonged for this task to ensure
that there were no group differences in successfully completing this simplified version of the
task prior to moving to the test phase. Rats that did not complete at least 60 successful trials for
3 consecutive days were exposed to additional sessions for a maximum of 24 days. Once a rat
reached stable levels of responding, they were then exposed to the training procedures just
once every 5 days to ensure that their performance did not change. All of the rats underwent
training for the last 5 sessions (days 20-24) at which time all were successfully meeting the
criterion of at least 60 successfully completed trials.
Each test session of the delay-discounting task consisted of 60 trials (at 100-s intervals) which
began with the houselight turned off and the levers retracted. Similar to the forced-choice
training phase described above, at the onset of each trial the houselight and cue light above the
food cup were activated and the rat was required to poke its nose into the food cup within 10 s
to prevent abortion of the trial. Following successful head entry into the food cup, the cue light
was turned off and either one (during forced-choice) or both levers (during free-choice) were
extended. Forced choice trials were included to encourage the rat to sample from both levers.
The ‘immediate’ and the ‘delay’ lever were right-left counterbalanced across animals. If the rat
failed to press the lever within 10 s, the trial was aborted, houselight turned off and levers
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retracted. If the rat pressed the immediate lever, the lever was retracted, the cue light turned on
and one food pellet was immediately delivered into the food cup. If the rat pressed the delay
lever, the lever was retracted, the cue light activated, and four food pellets were delivered at the
specified delay (see main text). As described above, after the rat retrieved the food pellet(s) or
after 10 s had elapsed the cue light was turned off and the houselight was turned off 6 s later.
C2. Probabilistic choice procedures
Behavioral testing occurred between the hours of 1300 and 1700. The training phase for the
probabilistic-choice task was the same as that described for the delay-discounting task (i.e.
retrieval of food pellets from food cup followed by FR1 training for each lever, and forced-choice
training). The forced-choice training procedures were prolonged for this task to ensure that there
were no group differences in successfully completing this simplified version of the task prior to
moving to the test phase. Rats that did not exhibit stable or peak levels of responding for 3
consecutive days were exposed to the forced-choice trials for a maximum of 28 days. Once a
rat reached stable levels of responding, they were then exposed to the training procedures just
once every 5 days to ensure that their performance did not change. All of the rats underwent
training for the last 5 sessions (days 23-28) at which time all were successfully completing
approximately 90% of the trials. Though it is noteworthy that bHR rats acquired this behavior at
a faster rate than bLR rats, it is unlikely that these differences during training affected
performance during the test sessions—especially since the data that were analyzed and
reported were collected after weeks of exposure to the probabilistic-choice task (days 20-24).
Probabilistic-choice test sessions consisted of 80 trials (at 40-s intervals) which began with the
houselight turned off and the levers retracted. Similar to the delay-discounting task, at the onset
of each trial the houselight and cue light above the food cup were activated and the rat was
required to poke its nose into the food cup within 10 s to prevent abortion of the trial. Following
successful head entry into the food cup, the cue light was turned off and either one (during
forced-choice trials) or both levers (during free-choice trials) were extended. One lever was
designated the large/uncertain lever and the other the small/certain lever (right-left
counterbalanced across animals). If the rat failed to press the lever within 10 s the trial was
aborted, houselight turned off, and levers retracted. If the animal pressed the small/certain lever,
the lever was retracted, the cue light turned on, and one food pellet was delivered into the food
cup. If the rat pressed the large/uncertain lever, the lever was retracted, the cue light activated,
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and four food pellets were delivered into the food cup if a given probability was met (see main
text).
C3. DRL training
The training phase for the DRL task began a few days after the delay-discounting task. Even
though the rats had been trained previously for the delay-discounting procedure, they briefly
underwent retraining prior to DRL testing to ensure that there were no bHR/bLR differences in
operant behavior that might affect interpretation of the results. Rats were trained for FR1
responding for banana pellets for two 45-in sessions prior to the DRL task.
D. Response to quinpirole
Separate groups of rats received either drug or vehicle injections. The study was designed in
this manner in order to meet the needs of another experiment that was run in parallel and to
minimize the number of animals that were used. Due to the large number of rats being tested
and the limited number of activity chambers, rats received one injection per week. Thus, the 3
doses of the drug (or vehicle) were administered in counterbalanced order with 1 week elapsing
between injections. Prior to each injection, rats were allowed to habituate to the testing
chambers for 2 hrs. Rats then remained in the activity chamber for 1 hr following each injection
and behavior was videotaped during this time.
Activity chambers and video recording devices
Chambers were constructed from expanded PVC (33.02 cm x 68.58 cm x 60.96 cm tall) with a
stainless woven wire cloth grid floor (30.48 cm x 60.96 cm, 7.62 cm x 7.62 cm), complete with a
catch tray. Directly above each activity chamber was a camera (CCTV Specialty Bullet
Cameras, Lake Worth, FL) used to record behavior. A Pelco (Clovis, CA) DX9100 digital video
recorder (DVR) was used to transfer the videos to a computer for automated analysis of
behavior (Clever Sys, Inc. Drug Scan software).
Automated behavioral analysis
Clever Sys, Inc. (Reston, VA, USA) Drug Scan software was used to analyze video-recorded
behavior as previously described (Flagel and Robinson, 2007; Flagel et al., 2008). For the
purpose of this study, we analyzed locomotor activity and repetitive head movements as two
indices of psychomotor activation. Locomotor activity was measured using the distance traveled
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and the velocity of individual bouts of locomotion. The frequency of head movements was
determined by dividing the total number of lateral head movements by the time spent in place.
E. Dopamine regulation in bHR and bLR rats
E1. Dopamine D2 receptor mRNA expression (In situ hybridization)
Rats were sacrificed by rapid decapitation under basal conditions (without any prior
manipulation) and brains were immediately removed, frozen in isopentane (-30º to -40ºC), and
stored at -80ºC. Coronal brain sections (10 µm) were cut on a cryostat (at 100-µm intervals) and
thaw mounted onto Superfrost/Plus slides (Fisher Scientific, Pittsburgh, PA, USA). Slides were
stored at -80ºC until further processing. Regions of interest were identified with cresyl violet
staining of adjacent sections.
In situ hybridization was performed as previously described (see Kabbaj et al., 2000; Flagel et
al., 2007). Briefly, post-fixed sections were hybridized with a 35S-labeled cRNA probe produced
using standard in vitro transcription methodology. The D2 receptor probe was a 495-base-pair
fragment directed against the rat D2 mRNA. The probe was diluted in hybridization buffer, and
brain sections were coverslipped and incubated overnight at 55ºC. Following post-hybridization
rinses and dehydration, slides were apposed to Kodak Biomax MR film (Eastman Kodak,
Rochester, NY, USA). Sections were exposed to film for 6 days. The specificity of the
hybridization signal was confirmed by control experiments using sense probes.
Autoradiograms were captured and digitized using Microtek ScanMaker 1000XL (Fontana, CA,
USA) and the scanner was driven by Lasersoft Imaging (SilverFast) software (Sarasota, FL,
USA). The magnitude of the signal from the hybridized 35S-cRNA probe was determined using
National Institutes of Health Image software. A macro was used (Dr. Serge Campeau,
University of Colorado, Boulder, CO, USA) which enabled signal above background to be
automatically determined. The relative integrated optical density (IOD) of these signal pixels
was obtained by multiplying the size of the area quantified by the signal intensity. The person
quantifying was blind to group assignments. D2 mRNA was quantified in the nucleus
accumbens (NAcc; between bregma levels 1.7 and 1.0) and caudate putamen (CPu; between
bregma levels 1.7 and 0.7). Optical density measurements were taken from the left and right
sides of at least four brain sections per animal. A mean IOD value was then generated for each
region of interest to yield one data point per animal for statistical analysis.
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E2. D2 binding assay
Rats were sacrificed by rapid decapitation under basal conditions (without any prior
manipulation) and brains were immediately removed and the left and right dorsal striata
(caudate-putamen) were dissected and stored at -70ºC until further processing. The dorsal
striatum (rather than the ventral striatum) was used here because of the amount of tissue
required for the assays.
Dissected striata were homogenized in buffer (4 mg frozen tissue per ml buffer), using a Teflonglass homogenizer with the piston rotating at 500 rpm and 10 up and down strokes of the glass
container. The buffer contained 50 mM Tris-HCl (pH 7.4 at 20 ˚C), 1 mM EDTA, 5 mM KCl, 1.5
mM CaCl2, 4 mM MgCl2, and 120 mM NaCl. The homogenate was washed three times by
centrifugation at 10,000 rpm at 4˚C and by resuspending the pellet in 15 ml of buffer.
D2 receptor binding was measured by the competition of dopamine with [3H]domperidone.
[3H]Domperidone was custom synthesized as [phenyl-3H(N)]domperidone (41.4 Ci/mmol) by
Moravek Radiochemicals Inc. (Brea, CA) and used at a final concentration of 2 nM. Because the
Kd of [3H]domperidone was 0.47 nM at D2 for rat striatum, the final concentration of 2 nM
occupied 81% of the D2 receptors, using the equation f = C/(C + Kd), where f was the fraction of
receptors occupied by [3H]domperidone, and C was 2 nM (Seeman et al., 2003).
For the competition assay, each incubation tube (12 x 75 mm, glass) received, in the following
order, 0.5 ml buffer (containing dopamine at various concentrations, and with or without a final
concentration of 10 µM S-sulpiride to define nonspecific binding to the dopamine D2 receptors),
0.25 ml [3H]domperidone and 0.25 ml of tissue homogenate. The tubes, containing a total
volume of 1 ml, were incubated for 2 h at room temperature (20C), after which the incubates
were filtered using a 12-well cell harvester (Titertek, Skatron, Lier, Norway) and bufferpresoaked glass fiber filter mats (Whatman GF/C). After filtering the incubate, the filter mat was
rinsed with buffer for 15 s (7.5 ml buffer). The filters were pushed out and placed in scintillation
polystyrene minivials (7 ml, 16 x 54 mm; Valley Container Inc., Bridgeport, Conn.). The minivials
received 4 ml each of scintillant (Research Products International Corp., Mount Prospect, IL),
and were monitored 6 h later for tritium in a Beckman LS5000TA scintillation spectrometer at
55% efficiency. The specific binding of [3H]domperidone was defined as total binding minus that
in the presence of 10 µM S-sulpiride.
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It is important to note that control experiments have been done on rat homogenized striata
showing that D3 receptors do not contribute to the population of sites detected by
[3H]domperidone. This has been done by comparing the competition of dopamine versus
[3H]domperidone in the presence and absence of 75 nM FAUC 365, which is a D3-selective
drug (Seeman et al., 2006). The amount of [3H]domperidone bound and the pattern of the two
competition curves were identical, indicating no significant contribution from D3 receptors under
these conditions.
E3. Surgery for fast-scan cyclic voltammetry
One week after arrival at the University of Washington (Seattle, Washington), bred rats were
implanted with carbon fiber microelectrodes in the core of the nucleus accumbens for in vivo
voltammetry. Surgical preparation used an aseptic technique, following the University of
Washington Institutional Animal Care and Use Committee guidelines. Briefly, rats were
anesthetized with isoflurane, administered the long-acting, non-steroidal anti-inflammatory,
carprofen (5 mg/kg, subcutaneously) and placed in a stereotaxic frame. The scalp was swabbed
with 10% povidone iodine, bathed with a mixture of lidocaine (0.5 mg/kg) and bupivicaine (0.5
mg/kg), and incised to expose the cranium. Holes were drilled and cleared of dura mater above
the nucleus accumbens (1.3 mm lateral and 1.3 mm rostral from bregma) and at convenient
locations for a reference electrode and three anchor screws. The reference electrode and
anchor screws were then positioned and secured with cranioplastic cement. A recording
microelectrode was attached to the voltammetric amplifier and lowered into the target recording
region (7.0 mm ventral of dura mater for the core of the nucleus accumbens). The voltammetric
waveform was switched on and dopamine monitored. Finally, cranioplastic cement was applied
to the exposed part of the cranium to secure the recording electrode. Placement of the
microelectrode was verified histologically upon completion of the experiment.
After recovery from surgery, rats were food restricted and maintained at 90% of free-feeding
body weight for the duration of the experiment. . Electrochemical recordings were made in
standard Med Associates chambers equipped with a food cup located at the center of the side
wall. All rats had been previously trained to retrieve pellets from the tray using procedures
identical to those specified for experiment B1.
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Electrochemical detection of dopamine
Changes in dopamine concentration were assessed with FSCV in awake, behaving rats using
methods adapted from (Phillips et al., 2003). Briefly, in FSCV the potential at a microelectrode is
held at -0.4 V, vs. a Ag/AgCl reference electrode, and then ramped to +1.3 V and back to -0.4 V
(400 V/s) every 100 ms. Each one of these sweeps is one scan, and these scans are cycled at
a rate of 10 Hz. When dopamine is present at the surface of the electrode during one of these
scans, it is oxidized during the positive portion of a sweep to form dopamine-o-quinone (peak
reaction at approximately +0.7 V) which is reduced back to dopamine in the negative portion of
the sweep (peak reaction at approximately -0.3 V). During the oxidation reaction of dopamine,
electrons are transferred between these molecules and the microelectrode. This change (flow)
of electrons is measured as current and is directly proportional to the number of molecules that
undergo the reaction. For the chemical identification of dopamine, current during a voltammetric
scan can be plotted against the applied potential to yield a cyclic voltammogram. The cyclic
voltammogram provides a chemical signature that is unique and thus allows resolution of
dopamine from other substances. Waveform generation, data collection and analysis were
carried out on a PC-based data acquisition system (National Instruments, Austin, TX) coupled to
a miniaturized head-mounted voltammetric amplifier.
For each animal, a peak amplitude score was generated by averaging the food-evoked
response obtained at the beginning of each of 4 sessions. The number of spontaneous events
for each animal was determined by averaging 8 separate 1-min samples from the 4 sessions
that generated the food-evoked data. These samples were taken at the end of each session and
were identical across animals.
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Titles and Legends to Supplemental Figures
Supplemental Figure 1. A) Locomotor response to novelty for bHR and bLR rats from 18
rounds of selective breeding (n=80-120 per phenotype). Data represent the mean locomotor
activity score ± standard deviation. Locomotor scores diverge dramatically between the selected
lines with increasing rounds of selection (Effect of Generation, F=44.10, P<0.0001; Effect of
Phenotype, F=6379.48, P<0.0001; Generation x Phenotype, F=68.37, P<0.0001). B)
Predictability of bHR and bLR traits based on breeding. Data represent the percentage of rats
that were correctly classified as bHR or bLR based on locomotor response to novelty for 18
generations. By the 10th generation of breeding we could predict with over 95% certainty the
classification of our rats as bHR or bLR based on parental lineage.
Supplemental Figure 2. Histological verification of recording sites for fast-scan cyclic
voltammetry. A) Electrolytic lesions were used to confirm that all recording sites (●) were within
the nucleus accumbens core. The numbers on each plate indicate the distance in millimeters
anterior from bregma (Paxinos and Watson, 2005). B) A representative lesion (→) produced
with the carbon fiber microelectrode for histological verification.
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