Male Sprague-Dawley rats (Harlan, Israel) weighing 250-350g were maintained on a 12-hr light/12-hr dark reversed cycle, with food and water available ad libitum. Rats were housed 2 per cage with a metal divider between them. Experiments were conducted during the dark cycle. Rats were anesthetized with xylazine and ketamine (10mg/kg and 100mg/kg, respectively). Jugular vein catheterization: Immediately after cannula implantation, rats were implanted with intravenous silastic catheters (ID 0.55 mm, OD 0.94 mm, Dow Corning, MI) into the right jugular vein. The catheter was secured to the vein with silk sutures and was passed subcutaneously to the top of the skull, where it exited into a connector (a modified 22-gauge cannula; Plastics One, VA, USA) mounted to the skull with MX-80 screws (Small Parts, Inc., FL, USA) and dental cement (Yates and Bird, IL, USA). Cocaine self-administration: Rats were trained to self-administer cocaine for 6 hrs per day, over 10 days. The self-administration chambers (Med-Associates, Inc.; St Albans, VT, USA) had two levers, one active and one inactive. An active lever press generated a cocaine infusion (0.75 mg/kg, 0.13 ml, 5 sec/infusion; cocaine obtained from the National Institutes on Drug Abuse, MD, USA) through the i.v. catheter, and also activated a light located above the lever, which was lit for 40 sec. Active lever presses during the last 35 sec of the light cue did not result in additional cocaine infusions. Presses on the inactive lever did not activate the infusion pump and light. Control rats were subjected to saline self-administration under the same conditions. The number of active lever responses, infusions, and inactive lever responses were recorded for both treatment and control groups. Rats were returned to their home cages at the end of the daily sessions. Cocaine withdrawal (forced abstinence): After the 10-day period of self-administration training sessions, rats were subjected to either a 1-day period of withdrawal (for cocaine-trained and saline-trained rats) or 30-day period of withdrawal (cocaine-trained rats). During the withdrawal periods, rats were left in their home cages and handled 3 times a week. Extinction test : Rats were placed in the self-administration chambers, and only the contingent light cue appeared during active lever presses, without cocaine reinforcement. Cocaine-seeking behavior was assessed for 60 min. For examining DNA methylation alterations, brains were removed one hour after completion of the session, and the NAc was isolated for further epigenetic analysis.
Extracted molecule
total RNA
Extraction protocol
Double stranded cDNA was synthesized from total RNA from the NAc.
Label
biotin
Label protocol
In vitro transcription was performed to produce biotin-labeled cDNA using Affymetrix Gene Chip 3’ IVT Express reagent kit according to manufacturer’s instructions (Affymetrix, Santa Clara, CA)
Hybridization protocol
After fragmentation, 12.5ug of cDNA was hybridized to Affymetrix Rat Gene 1.0 ST microarrays
Scan protocol
GeneChips were then scanned with the GeneChip scanner 3000 (Affymetrix)
Data processing
Microarray probe intensities were normalized to each other using Robust Multi-array Average (RMA).
