Supplementary Methods (doc 46K)

126 Male Wistar rats (Charles River Co., Wilmington, MA) weighing 250-275 g at the
beginning of the experiment and 450-550g at the time of testing were used. Rats were pairhoused and maintained on a 12h/12h reverse light-dark cycle in a temperature and humidity
controlled vivarium. All training and testing were conducted during the dark phase of the cycle.
Standard laboratory rat chow and water were available ad libitum throughout the experiment. All
procedures were conducted in accordance to the National Institutes of Health Guide for the Care
and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use
Committee of the Scripps Research Institute.
Behavioral testing apparatus
Self-administration training and testing were conducted inside sound-attenuated operant
conditioning chambers (Med Associates, St. Albans, VT) located inside sound-attenuating
cubicles equipped with exhaust fans as previously described (Weiss et al, 1993). Each chamber
was equipped with a 0.15 ml drinking reservoir positioned 4 cm above the grid floor in the center
of the front panel of the chamber, and two retractable levers located 4.5 cm on either side of the
drinking reservoir. Responses on the right (active) lever resulted in activation of the infusion
pump for 0.5 s, resulting in delivery of 0.1 ml liquid reinforcer into the drinking reservoir, while
responses on the left (inactive) lever were recorded but had no other scheduled consequences.
Each lever required a 0.01 N force to register a response. Auditory stimuli were presented via a
speaker mounted on the rear wall. A microcomputer controlled the delivery of fluids,
presentation of auditory stimuli and recording of behavioral data.
Locomotor activity was measured in 16 identical metal wire hanging cages, each
measuring 36 cm (L) × 25 cm (W) × 20 cm (H). Each cage contained two sets of infrared
emitter-detector photocells positioned along the long axis, 1 cm above the grid floor and 8 cm
from the front and back of the cage. Movement within the cages produced photocell beam breaks
that were automatically recorded by computer.
Pre-training procedure for operant self-administration
Rats (N = 96) were trained to self-administer ethanol in daily 30 min sessions on a
schedule of continuous reinforcement, using the “supersac” sweet solution fading procedure to
(Ji et al, 2008; Walker et al, 2008) to establish operant responding for ethanol reinforcement
without using food or water deprivation. During the first five days of self-administration training,
lever responses resulted in delivery of a solution containing 3% glucose and 0.125% saccharin
(“supersac”). Starting on day six, training continued with 10% (w/v) ethanol added to the
supersac solution for four days. During the following four training days, glucose was removed
from the solution, followed by removal of saccharin after completion of 14 total training days.
Ethanol withdrawal ratings
After every 3-day intoxication cycle (RW rats) or at the end of the 12-day intoxication
period (SW rats), withdrawal from ethanol was evaluated in the rats using five well-characterized
behavioral signs and a rating scale adapted from Macey et al. (Macey et al, 1996): ventromedial
limb retraction, impaired gait, vocalization, tail rigidity and tremors were examined 12h after
removal from the ethanol vapor chambers. Each sign was assigned a score of 0 to 2, based on the
following severity scale: 0 = no apparent symptom, 1 = moderate, 2 = severe. The sum of the
five observation scores (0 to 10) was used as a quantitative measure of withdrawal severity. For
these behavioral observations, rats were individually transferred from their home cages to a quiet
observation room. Rats in the SW group were evaluated for withdrawal once before ethanol
vapor exposure and 12h following the last exposure period. Rats in the RW group were measured
12h after each 3-day intoxication cycle. Randomly selected rats of the CTRL group were
evaluated during the corresponding time periods for comparison.
Calculation of fractional inhibition indices
For all LY379268-pretreated rats in each intoxication group, responses recorded during
the S+ reinstatement tests were subtracted from, and normalized to, the corresponding responses
of vehicle-treated rats in the same intoxication group. The resulting indices of fractional
inhibition were then fit to an ordinary pharmacokinetic model y = 1 / (1 − 10A − x), where the
parameter A estimated the logarithm of EC50, the effective dose for half of the population.
Significant differences were assessed by t-tests between rats from different intoxication groups
pretreated with the same dose of LY379268.
Membrane preparation for the [35S]GTPγS binding assay
Brain punches were homogenized and then centrifuged at 3000 rpm for 10 min in lysis
buffer [0.32 M sucrose, 2 mM EGTA, 0.002% SDS, protease inhibitor (Roche Diagnostics,
Rahway, NJ), pH 7.4]. The tissue pellet was re-suspended in 2mM EGTA, protease inhibitor, pH
7.4 and incubated for 15 minutes at 37 °C. The homogenate was centrifuged at 1400 rpm for 20
min. Membranes were re-suspended in storage buffer at −80 °C (20mM HEPES, 0.1mM EGTA,
protease inhibitor, pH 7.4). All steps were processed at 4 °C unless noted differently. Protein
concentrations were determined using a bicinchoninic acid assay (Thermo Scientific, Rockford,
Calculation of binding parameters in [35S]GTPγS binding data
The specific [35S]GTPγS binding rate data were fit to a one-site compartment kinetic
model for specific binding y = BMAX x / (KD + x), where the parameter BMAX estimated the
maximum specific binding and KD estimated the equilibrium binding constant. KD was also the
estimated radioligand concentration needed to achieve a half-maximum binding at equilibrium.
Significant differences were assessed by t-tests between parameters associated with rats from
different intoxication groups.