Materials and Methods (Cocaine)

Subjects
Male C57BL/6J mice (8-10 weeks old) were purchased from Jackson
Laboratories (Bar Harbor, Maine). Mice were housed in groups of 5/cage with food and
water ad-libitum and were acclimatized to the vivarium for one week before experiments
began. Animal care was in accordance with the Guide for the Care and Use of Laboratory
Animals (National Research Council, National Academy Press, 1996) and was approved
by the University of Miami Animal Care and Use Committee.
Drugs
Cocaine HCl and (+)-MK801 hydrogen maleate (dizocilpine) were dissolved in
saline (0.9% NaCl). The NR2B antagonist ifenprodil tartrate salt (Williams, 1993) was
dissolved in distilled water. The CRH-receptor subtype 1 (CRH-R1) antagonist
antalarmin (Webster et al., 1996) was dissolved in 15% DMSO, 25% polyethylene glycol
and 60% distilled water and the nNOS inhibitor 7-nitroindazole (7-NI) was dissolved in a
1:3:6 mixture of DMSO, polyethylene glycol and distilled water, respectively. All drugs
were purchased from Sigma-Aldrich (St. Louis, MO, USA). Drugs and vehicles were
administered intraperitoneally (i.p.) in a volume of 0.1ml/10g.
Conditioned place preference apparatus
Place preference was monitored using custom-designed Plexiglas cages (Opto-
Max Activity Meter v2.16; Columbus Instruments, Columbus, OH) as previously
described (Liddie et al., 2012). The training context consists of two compartments
separated by an opaque removable divider. One compartment has black and white striped
walls and a white floor covered with stainless steel grid; the other compartment has black
walls and smooth black floor, thus providing distinct visual and tactile cues. Time spent
in each compartment was recorded and analyzed by the Opto-max interface and software.
Conditioning procedure
On the first day, mice were habituated (20 min) to the training context;
preconditioning compartment-preference/aversion was determined. Half the subjects
showed slight preference for one side or the other. Accordingly, mice were paired with
cocaine in the less preferred compartment; half were paired with cocaine in the black
compartment and the other half were paired with cocaine in the black and white-striped
compartment. Following habituation, mice were conditioned by saline in the morning and
by cocaine a) 11.25mg/kg X 4 days (Fix-C) or b) 3,6,12 and 24mg/kg; one dose per day
(Esc-C) in the afternoon. Doses were chosen to ensure that the total amount of cocaine
administered during the 4 days was equal between Fix-C and Esc-C mice. Hence, the Fix-
C schedule for each day represents the average dose of the Esc-C schedule. All
experiments comprised four training days (days 2-5), two sessions per day (30 min each),
separated by 2-3h interval.
Extinction
Seventy-two hours after the final cocaine administration, mice were tested for the
acquisition of CPP (day 8). Following the acquisition of Fix-C and Esc-C memory, for
the next 4 days mice were subjected to extinction sessions to eliminate the acquired
cocaine-associated memory. For extinction training, mice were confined to the previously
saline-paired compartment during the morning session and to the previously cocainepaired
compartment during the afternoon session following administration of a saline
injection. A test for extinction was carried out 72h after the final saline reconditioning
session (day 15).


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