2.7. Intravenous Catheterization and Self-Administration.

Rats were anesthetized with an isoflurane/oxygen vapor mixture (isoflurane 5% induction, 1–3% maintenance) and prepared with chronic intravenous catheters as described (Miller et al, 2013; Nguyen et al, 2017b) on week 4 of the vaccination protocol. The catheters consisted of a 14.5-cm length polyurethane based tubing (MicroRenathane®, Braintree Scientific, Inc, Braintree MA, USA) fitted to a guide cannula (Plastics one, Roanoke, VA) curved at an angle and encased in dental cement anchored to an ~3-cm circle of durable mesh. Catheter tubing was passed subcutaneously from the animal’s back to the right jugular vein. Catheter tubing was inserted into the vein and secured gently with suture thread. A liquid tissue adhesive was used to close the incisions (3M™ Vetbond™ Tissue Adhesive; 1469S B). A minimum of 4 days was allowed for surgical recovery prior to starting an experiment. For the first three days of the recovery period, an antibiotic (cephazolin) and an analgesic (flunixin) were administered daily. During testing and training, intravenous catheters were flushed with ~0.2–0.3 ml heparinized (32.3 USP/ml) saline before sessions and ~0.2–0.3 ml heparinized saline containing cefazolin (100 mg/ml) after sessions. Catheter patency was assessed weekly after the first two weeks via administration through the catheter of ~0.2 ml (10 mg/ml) of the ultra-short-acting barbiturate anesthetic, Brevital sodium (1% methohexital sodium; Eli Lilly, Indianapolis, IN). Animals with patent catheters exhibit prominent signs of anesthesia (pronounced loss of muscle tone) within 3 s after infusion. Animals that failed to display these signs were considered to have faulty catheters and were discontinued from the study. Data that was taken prior to failing this test and after the previous passing of this test were excluded from analysis.

Drug self-administration was conducted in operant boxes (Med Associates) located inside sound-attenuating chambers located in an experimental room (ambient temperature 22±1°C; illuminated by red light) outside of the housing vivarium. To begin a session, the catheter fittings on the animals’ backs were connected to polyethylene tubing contained inside a protective spring suspended into the operant chamber from a liquid swivel attached to a balance arm. Each operant session started with the extension of two retractable levers into the chamber. Following each completion of the response requirement (response ratio), a white stimulus light (located above the reinforced lever) signaled delivery of the reinforcer and remained on during a 20 s post-infusion timeout, during which responses were recorded but had no scheduled consequences. Drug infusions were delivered via syringe pump. The training doses (Cohort 1: 0.06 mg/kg/infusion; Cohort 2: 0.15 mg/kg/infusion) and session duration of 1 h were selected from a prior self-administration study (Wade et al. 2014). Rats were allowed to self-administer under a Fixed Ratio 1 (FR1) contingency for 18 sessions. Successful acquisition in the first 18 sessions was defined as an average of 7 or more infusions across two sequential days; the day of acquisition was defined as the first day. Significant individual differences were shown to contribute to outcome in human clinical trials of cocaine and nicotine vaccines (Hoogsteder et al, 2014; Martell et al, 2009). Likewise, there are significant individual differences in drug use trajectories, since problematic drug use is a minority outcome among those who sample drugs (Anthony et al, 1994; Schramm-Sapyta et al, 2009; Taffe, 2015). thus a median split analysis was planned a priori to address the possibility of individual differences of outcome in this study. Median splits were determined by ranking animals on their mean number of infusions obtained across 18 sessions, and are referred to as Upper and Lower. The 19^th session was a PR session with the respective training dose for all animals. In the PR paradigm, the required response ratio is increased after each reinforcer delivery within a session (Hodos, 1961; Segal and Mandell, 1974) as determined by the following equation (rounded to the nearest integer): Response Ratio=5ê(injection number*j)–5 (Richardson and Roberts, 1996). The j value is as specified for a given experiment, i.e., PR^J2 refers to a session with the j value set to 0.2.

Sessions 20–21 were FR1/training dose and Session 22 featured saline substitution. Training conditions were restored for Sessions 28–30 (i.e., FR1) and thereafter the schedule was FR5 for Sessions 31–44 and FR10 for Sessions 45–63. The PR^J2 schedule was in effect for Sessions 64–70 and FR1 for Sessions 71–73. Mean infusions in Sessions 28–30 were used to re-rank individuals for the median split for this part of the experiment to determine effects of schedule changes on current, rather than acquisition, drug preference phenotype. This resulted in 3 individuals in the original lower half of the TT group (TT Lower) switching with Upper half individuals and one individual in the original Oxy-TT Lower half switching with an Upper half individual.

Sessions 20–23 were PR^J2 sessions with doses (0.0, 0.06, 0.15, 0.3 mg/kg/inf) presented in randomized order. There was negligible difference attributable to dose and the subsequent experiments focused on FR/PR transitions at a fixed per-infusion dose. Sessions 24–25 restored the training dose under FR1. Sessions 26–30 were PR^J2 and sessions 31–35 were PR^J3 (0.15 mg/kg/inf). Sessions 36–37 were FR1 (0.15 mg/kg/inf). Sessions 38–39 were FR1 (0.06 mg/kg/inf) and session 40 was PR^J3 (0.06 mg/kg/inf).