Surgical procedures

Cannula implantation surgery

All animals were anesthetized with sodium pentobarbital (i.p. 50mg/kg) and given atropine (s.c. 0.54mg/kg). Using standard stereotaxic procedures, the implantation of a unilateral ICV 9mm guide cannula (23 gauge; Small Parts, FL, USA), positioned 1.0mm above the lateral ventricle, according to the following coordinates: 1.0mm caudal to bregma, 1.5mm lateral to the midsagittal sinus, and 3.0mm ventral to dura, below the surface of the skull at the point of entry (Febo et al. 2005). The cannulae were secured to the skull using stainless steel screws (Plastic One, VA, USA) and dental cement and light-curable resin (Caribbean Dental Corporation Inc, San Juan, PR, USA). To ensure clean and functional cannulae, wire stylets were inserted until the microinjections were made. Antibiotic ointment was placed on the incision and then sutured. Animals were allowed between 5 and 7days for postsurgery recovery, before the experimental procedures.

Catheter surgery

Rats were surgically implanted with a chronic indwelling catheter made of silastic tubing (0.03cm ID×0.06cm OD; Dow Corning, Midland, MI, USA) as previously described (Weiss et al. 2000). The catheters consisted of polyethylene tubing glued to a guide canulae bent at a 90° angle and encased in dental cement anchored with a 4-cm square of durable mesh. Catheter patency was maintained flushing 0.1ml of sterile heparin/saline (1:25) solution before and after the self-administration training. Animals with compromised catheters were excluded from experiments.

Intracerebroventricular microinjections

For microinjections, the wire stylets were removed from the guide cannulae and 11.0mm injector cannulae (30 gauge; Small Parts) were inserted. Microinfusions of OT, OTA, or TGOT in a 10-µl volume were performed using polyethylene tubing (PE-10, Harvard Apparatus, CA, USA) connected to a syringe (Harvard Apparatus). At the end of each microinfusion, the injector was removed and stylets replaced. All animals received the ICV microinfusions 5min prior to behavioral testing.

Self-administration behavioral methods

Self-administration apparatus

Rats were trained to self-administer cocaine in standard operant conditioning chambers (Coulbourn Instruments, Allentown, PA, USA). They were equipped with a retractable lever and a white cue light located 3cm above the lever. The auditory stimulus was a white noise presented via a speaker located in the middle of the chambers. Intravenous infusions were delivered via a syringe pump (Razel, Stamford, CT, USA) through a metal swivel that was connected to the external guide cannula on the rats' back. A cue light above the lever turned on once the animal made a lever press.

Self-administration training

Once the animals recovered from surgery, they were trained to self-administer cocaine using a cue-induced cocaine seeking behavior protocol. Sessions consisted of three different stages: acquisition, extinction, and reinstatement. Separate groups of animals were used during self-administration procedures for either the extinction or the reinstatement experiments. Our behavioral protocol was adapted from cocaine-seeking behavior paradigms previously described in Ramos-Ortolaza et al. (2009).

Acquisition

Each session (2h) was initiated by the extension of a lever and activation of a white noise that remained active throughout the session. Upon lever pressing, a pump was activated and a cocaine infusion of 0.25mg was delivered over a 3-s period. Each infusion was followed by a time-out period (TO) of 17s, signaled by the illumination of the cue light. During this TO period, lever pressing was recorded but had no consequences. The animals were trained on a fixed ratio (FR) schedule of reinforcement. Upon stable responses (less than ~10% variation in responses between three consecutive sessions), the response requirement was raised from FR1, to FR5 and, to FR10. Both the cue light and the white noise represent the environmental cues associated with the drug infusion.

Extinction

Once the animals reached stable behavioral responding at FR10 in the acquisition phase, they were then exposed to extinction conditions, where no cocaine related cues were presented. In this extinction stage, lever pressing was recorded but it had no consequences. One-hour sessions were conducted until the extinction criterion was reached (the extinction criterion is ≥85% reduction in lever pressing during five consecutive sessions).

Reinstatement

Animals underwent both acquisition and extinction training prior to reinstatement. Once the animals reached the extinction criterion, they were re-exposed to environmental cues previously related to the acquisition session of the paradigm. Upon lever pressing, the illumination of the cue light above the lever lasted for 17s, and the cue light response requirement was FR1. During this TO period, lever pressing was recorded but had no consequences. During the reinstatement testing, the infusion pump was not activated, but lever pressing was recorded without consequences. One 1-h reinstatement session was conducted for three consecutive testing days. This testing period of 3days was selected to examine the saliency of the cues even after persistent re-exposure following long-term abstinence. In addition, locomotor activity during reinstatement was also measured.

ICV infusions of OT prior to the reinstatement phase

In the reinstatement experiments, separate groups of animals were trained in the cocaine seeking behavior protocol until they reached the extinction criteria. Once the animals reached this criterion they were re-exposed to the cues previously paired with cocaine self-administration in the reinstatement phase. Five minutes prior to the reinstatement test, all animals were pretreated with one ICV injections of either aCSF, OT, TgOT, or OTA. This procedure was repeated for the 3days of the reinstatement stage. At the end of the reinstatement testing, animals were killed and their brains were collected for histology.

Elevated plus maze protocol

In all EPM experiments, animals were tested in an apparatus consisting of four black Plexiglas arms with dimensions of 50cm long×10cm wide (Coulburn Instruments). The EPM was localized in the center of the testing room with regular lighting and quiet sound conditions. A videotape camera was placed inside the room to record behavior. Two of the EPM arms were open, while the other two were closed arms, making the shape of a plus sign. During testing, each animal was placed by the experimenter in the center of the apparatus facing an open arm. Rats were allowed to freely explore the maze for 5min. At the end of each EPM session, animals were returned to their home cage. The following EPM behaviors are reported: (a) the total time spent in the open arms as a measurement of anxiety, (b) number of entries into the open, and (c) the total time spent in closed arms as a locomotor control measurement. Sessions were taped and later evaluated by an experimenter that was blind to treatment.

OT treatment during reinstatement procedures followed by the EPM testing

In this experiment, animals were first trained in the cue-induced cocaine-seeking behavior paradigm as previously described. Once the animals reached the extinction criterion, they were exposed to reinstatement procedures for one 1-h daily session for three consecutive testing days. Five minutes prior to each day of testing, separate groups of animals were pretreated with either aCSF or OT ICV microinfusions and immediately placed in the EPM for 5min. In this procedure, rats were allowed to freely explore the maze during the time of testing.

Conditioned locomotion methods

Activity chamber apparatus

TruScan Photobeam Scanning System activity boxes (Coulbourn Instruments) were used for the conditioning experiments. The boxes were 41cm×41cm×41cm on the inside and had a 53-cm-×-53-cm base plate. There were 16 photobeams in each box to track animal movement. The boxes were located with visual and olfactory environmental cues in a room different from the room of their home cages. The chambers used for the cocaine-paired group were surrounded by a 61-cm-×-61-cm-×-61-cm black plexiglass box. In addition, California orange oil (Sigma-Aldrich, St. Louis, MO, USA) was released in the room to provide an olfactory cue to the chamber. For the saline (control group), the chambers were localized in a different room, surrounded by a 61-×-61-×-61-cm black and white (checkers) Plexiglas box and the chamber exposed to a Nutmeg East Indian oil scent (Sigma-Aldrich). The present protocol is adapted from our conditioning paradigm previously reported in Martínez-Rivera et al. (2013).

Training sessions

The training consisted of a conditioning protocol of ten consecutive days, one session per day for 90min. Just before each training session started, rats assigned to the cocaine-paired group were injected i.p. with 10mg/kg of cocaine-hydrochloride (Rodriguez-Borrero et al. 2010; Martínez-Rivera et al. 2013). In addition, at the end of each training session, animals received an i.p. injection of saline and were returned to their home cages. The control group received i.p. saline injections before and after each training session. Animals completed their training sessions in the same activity chamber throughout all the experiments. Ambulatory distance (AD) was measured as distance traveled in the floor plane minus stereotypic movements.

Testing session

This session was completed 2days (day 12) after the last training session (day 10). On this day, animals were returned to the activity chambers with the environmental cues for a 30-min period but without cocaine or saline injections. Five minutes before placing the rats in the chambers, animals received a microinfusion of either OT or aCSF. No cocaine or saline i.p. injections were given before or after testing. At the end of the 30-min testing session, animals from the cocaine or saline groups were immediately placed in the elevated plus maze for 5min.