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  • Experiments were conducted in accordance with the Guidelines for the Care and Use of Mammals in Neuroscience issued by the National Academies. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Texas at Austin (current Animal Use Protocol #2011-00069). Male Long Evans rats weighing 200–225 g were allowed one week of habituation and daily handling after arriving at the Animal Resources Center of the University of Texas at Austin from Charles River Laboratories. Rats were group housed until commencement of behavioral training (after which they were individually housed) in a temperature-controlled room (72±4°F). Food and water was available ad libitum, except for as described under Behavioral training and assessment. Rats were weighed prior to any procedure, all of which occurred during the light phase of a 12 hour light/dark cycle. In total, 180 rats were used in connection with the research reported here. Eighteen rats were used for the pilot experiments described in the Supporting Information Text S1. Of the 162 rats used for the extended and limited training experiments, 25 were excluded from final analysis of the data, because they did not complete training or their data were unreliable. For the extended training experiment, one VI and three VR rats were excluded for insufficient acquisition of 10S10E-reinforced behavior; four 10S10E VI and two 10S VI were excluded because of experimenter error. In the limited training experiment, two VI and two VR rats were excluded for low responding for 10S10E, eight were excluded for insufficient acquisition of 10S reinforced behavior, and one from each of the three groups was excluded because of experimenter error. LiCl (Sigma-Aldrich) was dissolved in sterile saline (0.9% NaCl; Hospira) for injection of 125 mg LiCl/ml solution/kg body weight. Drinking solutions, 10% sucrose (w/v), 10% ethanol (v/v), or 10% sucrose (w/v):10% ethanol (v/v), were prepared using the appropriate proportions of ultra-pure sucrose (MP Biomedicals, LLC, Solon, OH), 95% ethanol (AAPER Alcohol and Chemical Co., Shelbyville, KY), and distilled water. Drinking solutions were stored at 4°C and prepared fresh approximately every three days. Instrumental training sessions were conducted in rat operant conditioning chambers (30.5 cm×24.1 cm×21 cm interior dimensions) with metal bar floors connected to lickometer circuits, housed inside sound attenuating cubicles in a dedicated behavioral testing room (chambers and cubicles from MedAssociates, Inc., Vermont, USA). The cubicles, modified by removal of the doors, were equipped with exhaust fans that provided ambient noise during all operant sessions. Med-PC IV software (MedAssociates, Inc.) controlled all chamber components. For the entire duration of all operant chamber sessions, a house light (at the top center of the left wall) was lit, and a 4.6 cm-wide retractable lever remained inserted into the chamber (6.35 cm above the grid floor, on the distal portion of the right wall). A retractable bottle assembly on the outside of the proximal panel of the right chamber wall held a bottle containing a drinking solution, with the sipper tube of the bottle positioned to be inaccessible from inside the chamber. When a lever press earned reinforcement (as determined by the programmed schedule in effect), the bottle assembly inserted, and then retracted 10 seconds later, the sipper tube through a hole into the chamber to allow the rat brief access to the drinking solution. Conditioning of instrumental lever pressing: Water deprivation was initiated 22 hours prior to the first session in the operant chamber. In this habituation session only, both the lever and the sipper tube were inserted into the chamber for the entire session. Lever presses had no consequence, and the sipper tube contained 20 ml of 10S for ad libitum consumption. Approximately 24 hours later, rats received a conditioning session in which lever presses were reinforced according to a fixed ratio 1 schedule: each lever press yielded insertion of the sipper tube containing 10S for 10 seconds. Conditioning sessions were repeated daily until the lever press response was acquired. Sessions were usually 20 minutes in length, but were sometimes extended (to a maximum of 40 minutes) if a rat appeared to be on the verge of learning the lever press-10S reinforcer contingency. At the end of each conditioning session, rats were returned to their home cages and given free access to food and water for a minimum of two hours before water bottles were removed. Rats not acquiring lever pressing behavior after five conditioning sessions were excluded from the study. Instrumental training with VI or VR reinforcement schedules: After acquiring the operant lever press response, rats were no longer water deprived, and began baseline instrumental training, receiving one 20 minute operant session per day, five-seven days per week (Figure 1). Table 1 shows the progression of reinforcement schedules and drinking solutions across training sessions. Slight variations to the protocol outlined in Table 1 occurred because of the a priori-determined requirement that the dose of ethanol be at least 0.3 g/kg for a minimum of two of the last three baseline sessions before advancement to the next phase of the experiment. For the extended training 10S10E VI and VR groups, additional sessions were conducted until reaching this criterion, and the number of sessions the 10S10E VI group received was matched by the 10S VI group. The limited training groups received a maximum of one additional session. Figure data removed from full text. Figure identifier and caption: 10.1371/journal.pone.0042886.g001 Overview of experimental phases and design.Pre-LiCl training and assessment: Initial training (detailed in Table 1) was followed by one (limited training) or more (extended training) cycles of extinction and retraining. Data from the last extinction and retraining sessions were used for pre-LiCl measures. LiCl treatment: The day after the last retraining session, all animals received the appropriate drinking solution in their home cage. LiCl injections were given either at the end of the home cage drinking period (paired treatment condition), or given 24 hours later (unpaired treatment condition). Post-LiCl assessment: The test for habitual behavior occurred 24 hours after LiCl injection, and was followed 24 hours later by the reacquisition test. Table data removed from full text. Table identifier and caption: 10.1371/journal.pone.0042886.t001 Experimental groups and representative sequence of training parameters. aValues listed for the reinforcement schedules are the average of the programmed values used to determine the time interval (seconds) before a single press yielded reinforcement for the VI groups, or the number of lever presses required to yield reinforcement for the VR groups. From session 7 on, the average ratio was 5 for some VR animals (n = 2 extended training, 11 limited training).bDrinking solutions were always 10% sucrose (w/v), with either 0% or 10% ethanol (vol/vol), as shown. In the limited training 10S10E groups, some animals received one additional session with 10S before transitioning to 10S10E.cExtended training continued with the same parameters for an additional 9 sessions, and limited training with no more than 1 additional session, before extinction and retraining (depicted in Figure 1 and described in Behavioral methods). Pre-LiCl behavioral assessment and retraining: Following Samson et al. [14], extinction responding was assessed prior to LiCl treatment. On the day after the last session of the initial baseline training period, an 8 minute extinction session was conducted. During this session, a bottle containing the appropriate solution was present outside the self-administration chamber in the retracted bottle holder, but lever presses did not yield access to the sipper tube. This was followed by two (limited training) or three (extended training) sessions of retraining with the appropriate reinforcement schedule and drinking solution. Consumption of at least 0.3 g ethanol per kg body weight during at least two of the retraining sessions was required before advancement to the next phase of the experiment. Regardless of the number of baseline extinction sessions, the (final) extinction session prior to LiCl treatment was used for comparison with the extinction test conducted after LiCl treatment. Samson and colleagues [14] suggested that repeated extinction and retraining sessions could give a better estimate of pretreatment extinction responding; thus the initial experiments utilized two cycles of extinction and retraining. The results from earlier pilot studies and the VI extended training animals showed that approximately 1/3 of animals exhibited more than ±40% change from the first to second extinction session (data not shown). Because of a concern that such variability in pretreatment extinction responding could result in reduced power to observe a devaluation effect, some animals in the VR extended training group received additional cycles of extinction and retraining until stable pressing (less than ±40% change between extinction session) was observed. However, subsequent statistical analysis of the data from all extended training groups did not indicate that stability of extinction behavior across multiple sessions was a factor influencing whether or not a devaluation effect was observed. Therefore, all limited training animals received only one cycle of extinction and retraining before LiCl treatment. Regardless of the number of baseline extinction sessions, the (final) extinction session prior to LiCl treatment was used for comparison with the extinction test conducted after LiCl treatment. LiCl treatment commenced 24 hours after the final retraining session (Figure 1). Each animal was weighed and then returned to its home cage, after which the water bottle was replaced by a bottle containing the drinking solution that previously had been used as the operant reinforcer. The dose (of ethanol) and/or amount (of sucrose) that could be consumed during this procedure was limited by filling the bottle to a volume equal to the maximum the individual rat had consumed within any of the last two-three retraining sessions, plus one additional ml to compensate for loss/leakage of fluid. Animals had 20 minutes of access to the bottle, and ‘Paired’ LiCl treatment (125 mg/ml/kg, i.p. injection) was administered at the end of this period. The exception to this was the 10S limited training group, which had a maximum of 10 minutes of access before receiving paired LiCl treatment. Any rat in this group that consumed the entire volume of 10S in the bottle in less than 10 minutes was injected with LiCl immediately. For all groups, ‘Unpaired’ LiCl injections were given exactly 24 hours after the home cage consumption. Refer to the Supporting Information Text S1 for description and discussion of the pilot experiments in which the LiCl treatment procedure was optimized to elicit outcome specific devaluation by paired injections only. For all groups, one 8 minute extinction test (lever presses did not yield sipper tube access) was conducted exactly 24 hours after the LiCl injection (Figure 1). Twenty-four hours after the extinction test was the test for reacquisition of operant behavior. In this 20 minute operant session, animals received response-contingent reinforcement with the appropriate solution according to the schedule used prior to LiCl treatment. Data collection, representation, and analysis: During any session in the operant chamber, Med-PC IV software recorded the occurrence and time of event for each lever press, insertion of the sipper tube (reinforcer delivery), and lickometer circuit completion (one lick of the sipper tube). Occasionally, it was noted that the lickometer circuit appeared to not record all licks during a session (due to faulty wiring or improper placement of the sipper tube and bottle in the holder). Any animal for which this was noted was not included in lick analyses (n = 3 extended, 11 limited). At the end of every session in the operant chamber, the total volume of remaining drinking solution (leaked solution collected by a plastic tray placed under the bottle assembly plus solution in the bottle and sipper tube) was manually measured and recorded. For the home cage presentation, the volume of any solution remaining in the tube and bottle was measured manually, but we could not recover any solution that leaked from the tube into the bedding below. Estimates of ethanol (g) consumption were calculated by subtracting the recovered volume from the initial volume of the drinking solution, and multiplying this difference by 0.0774. Raw data from MedPC output files and paper training logs were imported, copied, or entered into Excel (Microsoft Office 2007). Excel, Prism 5 (GraphPad Software Inc.), and Adobe Illustrator CS5 (version 15.0.0) were used to create graphical representations of data (depicted as mean ± s.e.m). SPSS Statistics (versions 17.0 and 19; IBM) was used to perform general linear model procedures as appropriate. Behavioral measures from the post-LiCl tests were analyzed in two ways. Each measure was expressed as % of the pre-LiCl session for between-groups comparisons of LiCl treatment conditions (paired vs. unpaired). Additionally, raw data collected during pre- and post-LiCl sessions were used in mixed model repeated measures analyses that tested for an interaction of session (pre or post LiCl) with LiCl treatment condition (paired or unpaired).
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