Male Sprague Dawley rats weighing 225-250 g at the start of the experiment will be housed individually in a climate-controlled colony under a 12-h reverse light/dark cycle (lights off at 6:00 am). They will be acclimated to handling for at least 5 days prior to surgery. Catheters will be implanted intravenously under isoflurane anesthesia. The catheters will run subcutaneously from the neck to an incision across the skull and will be secured to the top of the skull using dental acrylic and anchor screws as described by Neisewander et al (2000; also see Vertebrate animals section). Canulae aimed at brain regions of interest will also be implanted to a depth 1 mm above the intended infusion sites and later, viruses constructed and/or purchased by Dr. Bizzozero will be infused into the targeted brain regions prior to behavioral testing in Dr. Neisewanders lab. The viruses are designed to over-express miR-495 and HuD so that we can determine the effects of these chemicals on motivation for cocaine. After at least 5 days of recovery from surgery, rats will be randomly divided into Cocaine and Control groups. The Cocaine group will be trained to press a lever reinforced by cocaine infusions (0.75 mg/kg/0.1 ml, IV) on a fixed ratio 1 schedule. The Control group will receive an equal volume of saline yoked to the schedule completions made by a rat in the Cocaine group. Training sessions will occur daily for 2-4 h at the same time of day across 20 consecutive days in operant conditioning chambers equipped with two levers mounted on the front wall, a cue light above one lever, a tone generator (500 Hz, 10 db above background), and a house light mounted on the center of the back wall. The lever with the cue light will be designated as the active lever and the other as the inactive lever. Schedule completions by a Cocaine rat on the active lever will simultaneously activate the cue light, house light, and tone, followed one second later by a cocaine infusion. Control rats will simultaneously be presented the same stimulus complex and a saline infusion (0.1 ml, IV) contingent upon responses of their Cocaine rat counterpart. Upon completion of the 6-s infusion, the cue light, tone, and infusion pump will be inactivated. The house light will remain on for a 20-s timeout period during which lever presses have no scheduled consequences. Responses on the inactive lever will be recorded but will have no scheduled consequences. Lever presses by Control rats will have no programmed consequences. Once the rats exhibit stable response rates, they will be test for break points on a progressive ratio schedule, for which the response requirement for reinforcement increases exponentially with each reinforcer earned. Break points are the point at which animals fail to complete a ratio schedule within 1 h and are measured as the final ratio completed within 1 h. Following this self-administration training, all rats will receive daily 1 h extinction training sessions during which their responses on either lever will have no consequences. Once responding has dropped by 80% or more of initial extinction response rates, the rats will be tested for reinstatement of cocaine-seeking behavior either following a priming injection of cocaine (10 mg/kg, IP) or by response-contingent presentation of the light/tone cues that had been previously paired with cocaine during self-administration training. At the conclusion of the experiments, the rats will be sacrificed by decapitation, their brains will be harvested and rapidly frozen. The frozen brains will be shipped on dry ice to Dr. Bizzozeros laboratory at the University of New Mexico for mRNA analyses.
|Effective start/end date||6/1/12 → 4/30/18|
- HHS: National Institutes of Health (NIH): $988,153.00