Materials and Methods – Animals

Male B6129S adult mice (8-10 weeks old; weighing 25-35g) were supplied from
breeding colonies in our facilities at the University of Miami, Miller School of Medicine,
Miami FL as previously described in detail (Balda et al., 2006). Following weaning
(postnatal day 21), mice were housed in single-sex groups; males were used for the
current study. ‘Litter effect’ was negated by grouping mice from 4-5 different litters into
each cage. Animals were housed in a temperature (22 ± 0.5 °C) and humidity (50%)
controlled room and maintained on a 12-hour light/dark schedule with food and water ad
lib except during training and testing. Animal care was in accordance with the Guide for
the Care and Use of Laboratory Animals (National Research Council, National Academy
Press, 1996) and approved by the University of Miami Animal Care and Use Committee.
Measurement of cyclic nucleotide levels
Our studies focused on the effects of the PDEi on cyclic nucleotides levels
particularly in hippocampus and amygdala because these brain regions are involved in
associative learning (Robbins et al., 2008). Papaverine (Sigma, St. Louis, MO) were
dissolved in saline (0.9% NaCl); rolipram (Sigma, St. Louis, MO) was dissolved in 2%
DMSO (Sigma, St. Louis, MO); BAY-73-6691 (Sigma, St. Louis, MO) was dissolved in
10% Solutol® HS 15 (BASF, Ludwigshafen, Germany) solution (vehicle). To determine
the dose effects of the PDE inhibitors, mice received single intraperitoneal (IP) injections
of (a) rolipram (0.05, 0.25, 1mg/kg; n=3/group) (b) BAY-73-6691 (0.03, 0.3 and 3mg/kg;
n=3/group), (c) papaverine (10, 20, 40mg/kg; n=3-4/group) or (d) saline/vehicle
(n=3/group). Thirty minutes later, mice were sacrificed by cervical dislocation and brains
were immediately removed and placed in ice-cold saline. The hippocampus and
amygdala were dissected on an ice-cold surface according to The Mouse Brain atlas
(Paxinos & Franklin, 2001), snap-frozen on dry ice and stored at -80°C. Levels of cGMP
(sensitivity, 25fmol/mL) and cAMP (sensitivity, 0.39pmol/mL) were quantified with
direct EIA kits (Enzo Life Sciences International, Inc., PA) as described in the
manufacturer’s protocols and determined by spectrophotometry (OD450nm). The optimal
doses of the PDE inhibitors for the behavioral studies were determined from these
Place conditioning apparatus
An Opto-Max Activity Meter (Colombus Instruments, Columbus, Ohio, USA)
was used to monitor place preference. The training context consisted of two
compartments separated by a removable divider. One compartment had black walls and
smooth black floor while the other compartment had white walls with a floor covered
with sandpaper (fine grit 150C, Norton; Stephenville, TX) thus providing distinct visual
and tactile cues. Each compartment was scanned by seven infrared beams at a rate of
10Hz (2.54 cm intervals). The horizontal sensors were mounted alongside opposing
lengths of each compartment. A null zone of 8 cm was assigned at the interface of the
two compartments to ensure that only full entry into one compartment or the other was
registered as distinct time spent on each side. Time spent in each compartment and
locomotor activity were recorded and analyzed by the Opto-max interface and software.
Mice were trained and tested in a room separate from the housing room as
described previously (Itzhak & Anderson, 2012). The testing room was equipped with a
fluorescent lamp strategically positioned to create a dimmed lighting environment. On the
first day of each experiment, between 12:00-14:00 hours, mice were habituated for 20
minutes to the training context; time spent in each compartment was recorded. Extremely
biased mice (about 10%) were eliminated from the study. About 50% of the remaining
animals had slight preference for the black compartment and the other 50% had slight
preference for the white compartment. Accordingly, the assignment criterion was such
that mice were conditioned by cocaine in their least preferred compartment. Thus, the
training procedure was counterbalanced where half the mice were trained with cocaine in
the black compartment while the other half was trained with cocaine in the white
compartment. Mice were trained with IP injections of saline during the morning (10:00-
12:00 hours) session and cocaine during the afternoon (14:00-16:00 hours) session. Each
conditioning session lasted 30 minutes.
Mice were conditioned with ascending dosages (3, 6, 12, 24mg/kg) of cocaine
over four days (Table 3.1). This regimen of escalating dosage caused a CPP that was of
higher magnitude and longer-lasting than the CPP that resulted from a fixed daily dose;
this enhanced CPP was also resistant to extinction by free exploration (Itzhak &
Anderson, 2012). Hence we hypothesized that investigating extinction in a model of
robust conditioning is more significant to the real-life human situation of escalating drug
use than the typical model of conditioning (fixed daily dose of cocaine) which affords
relatively quick extinction.
Cocaine was administered immediately before the animal was placed into its
respective compartment. To maintain a consistent environment for each mouse, the
sandpaper was removed and the cages thoroughly cleaned with dilute laboratory-grade
detergent followed by water and then dried, following each training session. The
locomotor activity in response to the different doses of cocaine was recorded daily.

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