Electronic Supplemental Materials, Schramm-Sapyta et al.
Detailed Methods not included in Manuscript
Novel Object Exploration Task
Twenty-four hours before testing, an object was placed into the rats’ home cages (the “familiar object”). On the morning of the test day, rats were habituated to a dimly lit testing room (20-25 lux in the testing area) for at least one hour and then videotaped in their home cages for 5 minutes after removal of their cagemate and the introduction of a second, novel object into the cage. Experimental objects included an aluminum foil ball, a glass holiday ornament ball, and a pyramid made from Legos (all approximately 3.5 x 3.5 x 3.5 cm). The order of placement of the objects was counterbalanced across the group. Time spent exploring both objects was scored by an observer who was blinded to the order of presentation of the objects. The measure of interest is the time spent interacting with the novel object, and is referred to as “novel object time.” Preliminary experiments indicated that the amount of time spent interacting with the familiar object was consistently very low or zero. ANOVA revealed no effect of order of testing within a cage (first vs. second), and no effect of object (legos vs. ornament ball vs. foil ball). This method is based on a combination of published techniques (Douglas et al., 2003; Heyser et al., 2004; Sproson et al., 2001), modified so that the test could be performed without extensive habituation sessions and could therefore be used as a rapid prescreen.
Blood Collection from Saphenous Vein
Blood was collected from the saphenous (leg) vein for analysis of corticosterone levels (Hem et al., 1998). Immediately after completion of the light-dark task, rats were placed in cages containing pre-warmed bedding on a heating pad on medium setting for 5-7 minutes to stimulate blood flow. Rats were then removed from the cage and restrained in a Decapi-cone (Braintree Scientific) during the 1-2 minute blood collection procedure. We chose this time of blood collection because corticosterone levels which were stimulated by the stress of being placed in the light-dark apparatus would be maximal (Brown and Martin, 1974; Seggie and Brown, 1975). Approximately 250 microliters of blood was collected from each animal. All collections were performed between 09.00 and 12.00.
Radioimmunoassay for determination of Corticosterone levels in Serum
Serum was separated by low-speed centrifugation and frozen at -80oC until analysis. Corticosterone levels were determined using the Coat-a-count kit from Diagnostic Products Corporation for rat corticosterone. Radioactivity was counted on a Packard Gamma counter. Within- and between-assay coefficients of variation were less than 5% and 10%, respectively.
Surgery to implant jugular catheter
After completing food training through the FR-5 session, rats were anesthetized with either a ketamine/domitor cocktail (0.60 mg/kg ketamine and 0.15 mg/kg domitor, administered intraperitoneally) or the inhalant isoflurane (2 – 2.5% with 1.25 L oxygen) and placed on an insulated heating pad. The fur was shaven from the intrascapular area, right lateral neck, ventral neck, and chest. The areas were sterilized using a 10% povidone-iodine solution. A small longitudinal incision was made in the skin just over the anterior right jugular vein. The jugular vein was isolated from surrounding connective tissues. Two strands of 4-0 silk suture were passed under the vein with a separation of ~5 to 7mm. The anterior suture was ligated to prevent bleeding. A small incision was made in the vein below the ligature. A heparin and antimicrobial-infused catheter (Instech Solomon, 3 Fr.) was inserted. The catheter was secured in place with the caudal suture, the cranial suture, and a third suture strand, placed midway between the previous two. Two to three drops of 0.25% Bupivacaine were instilled for analgesia. The rat was placed in ventral recumbency and a small skin incision made on the intrascapular area. Using a metal trocar, the catheter was passed under the skin, out the dorsal incision, and attached to the port (Instech, Plastic SoloPort, 16mm). The ventral incision was closed using 4-0 prolene suture. Two to three drops of 0.25% Bupivacaine were instilled along the suture line. The access port was implanted subcutaneously and attached to the trapezius muscle using 4-0 silk suture. The incision was closed with 4-0 prolene suture. Two to three drops of 0.25% Bupivacaine were instilled along the suture line. The total duration of the surgical procedure was ~ 50 min. Rats were placed on a post-surgical warming pad until fully awake. They were allowed to recover for 2-4 days with food and water ad libidum before initiating self-administration training. Catheters were flushed daily with a 0.3 mL sterile lock solution containing 25 IU/mL heparinized saline and 0.4 mg Gentamicin. Catheter patency was tested 24 – 48 hrs post-operatively, mid-way in the self-administration period, and at the end of the self-administration period. Patency was also tested when dramatic changes in lever pressing behavior occurred. To test catheter patency, each rat received a 0.3mL Brevital solution (5mg/mL in sterile saline) via the port. Rats that did not respond to Brevital immediately were excluded from further study.