Male B6129S adult mice (8-10 weeks old; weighing 25-35g) were supplied frombreeding 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 thecurrent study. ‘Litter effect’ was negated by grouping mice from 4-5 different litters intoeach 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 adlib except during training and testing. Animal care was in accordance with the Guide forthe Care and Use of Laboratory Animals (National Research Council, National AcademyPress, 1996) and approved by the University of Miami Animal Care and Use Committee.Measurement of cyclic nucleotide levelsOur studies focused on the effects of the PDEi on cyclic nucleotides levelsparticularly in hippocampus and amygdala because these brain regions are involved inassociative learning (Robbins et al., 2008). Papaverine (Sigma, St. Louis, MO) weredissolved 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 in10% Solutol® HS 15 (BASF, Ludwigshafen, Germany) solution (vehicle). To determinethe dose effects of the PDE inhibitors, mice received single intraperitoneal (IP) injectionsof (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 brainswere immediately removed and placed in ice-cold saline. The hippocampus andamygdala 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 withdirect EIA kits (Enzo Life Sciences International, Inc.
, PA) as described in themanufacturer’s protocols and determined by spectrophotometry (OD450nm). The optimaldoses of the PDE inhibitors for the behavioral studies were determined from theseexperiments.Place conditioning apparatusAn Opto-Max Activity Meter (Colombus Instruments, Columbus, Ohio, USA)was used to monitor place preference. The training context consisted of twocompartments separated by a removable divider. One compartment had black walls andsmooth black floor while the other compartment had white walls with a floor coveredwith sandpaper (fine grit 150C, Norton; Stephenville, TX) thus providing distinct visualand tactile cues.
Each compartment was scanned by seven infrared beams at a rate of10Hz (2.54 cm intervals). The horizontal sensors were mounted alongside opposinglengths of each compartment. A null zone of 8 cm was assigned at the interface of thetwo compartments to ensure that only full entry into one compartment or the other wasregistered as distinct time spent on each side. Time spent in each compartment andlocomotor activity were recorded and analyzed by the Opto-max interface and software.ConditioningMice were trained and tested in a room separate from the housing room asdescribed previously (Itzhak & Anderson, 2012). The testing room was equipped with afluorescent lamp strategically positioned to create a dimmed lighting environment.
On thefirst day of each experiment, between 12:00-14:00 hours, mice were habituated for 20minutes to the training context; time spent in each compartment was recorded. Extremelybiased mice (about 10%) were eliminated from the study. About 50% of the remaininganimals had slight preference for the black compartment and the other 50% had slightpreference for the white compartment. Accordingly, the assignment criterion was suchthat mice were conditioned by cocaine in their least preferred compartment. Thus, thetraining procedure was counterbalanced where half the mice were trained with cocaine inthe black compartment while the other half was trained with cocaine in the whitecompartment.
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. Eachconditioning session lasted 30 minutes.Mice were conditioned with ascending dosages (3, 6, 12, 24mg/kg) of cocaineover four days (Table 3.1).
This regimen of escalating dosage caused a CPP that was ofhigher 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 ofrobust conditioning is more significant to the real-life human situation of escalating druguse than the typical model of conditioning (fixed daily dose of cocaine) which affordsrelatively quick extinction.Cocaine was administered immediately before the animal was placed into itsrespective compartment. To maintain a consistent environment for each mouse, thesandpaper was removed and the cages thoroughly cleaned with dilute laboratory-gradedetergent followed by water and then dried, following each training session. Thelocomotor activity in response to the different doses of cocaine was recorded daily.