MATERIALS AND METHODS
10% Ethanol Self-Administration with Sucrose Fade
Long-Evans rats (n=12-14 per group) were initially exposed to 10% ethanol as the only
liquid source in their home cages for four days. This forced ethanol exposure has been
postulated to facilitate the initiation of ethanol self-administration by reducing the
influence of neophobia for ethanol during operant training (Nadal et al., 2002). Following
the fourth day of forced ethanol exposure, rats were placed in the operant chambers for a
14 hour overnight session on an FR1 schedule of reinforcement (0.1 ml reinforcer after a
single lever press). The start of the training session was signaled by the illumination of
the house light and extension of the active lever. During this phase, only the active lever
was available for the rat to press to establish lever pressing behavior. Rats were trained to
respond for 10% sucrose in overnight sessions (1-3 nights) and continued on 10% sucrose
until they reached the FR3 stage of training. Initial daily training consisted of 45 minute
FR1 sessions and one-hour daily water access, with water access immediately following
the training sessions. Once responding was established (2-4 days) rats were given free
access to water in the home cage and continued on a 45 minute FR1 schedule for an
additional three to four days. Subsequently, training sessions were reduced to 30 minutes
and the work ratio was increased to an FR3 schedule of reinforcement (3 active lever
presses required for 0.1 ml reinforcer). A second, inactive lever was also introduced at
this time. Upon pressing the inactive lever, no reinforcer, visual (light), or auditory
stimuli were presented and the event was merely recorded as a measure of nonspecific
behavioral activity. Following three sessions of FR3 training with 10% sucrose as the
reinforcer, a modified sucrose-fade technique (Samson, 1986) was initiated. Ten percent
ethanol was added to the 10% sucrose solution and over the next 12 sessions the sucrose
concentration was gradually decreased (10%, 5%, 3%, 1.5% respectively) until rats
responded on an FR3 schedule for 10% ethanol alone. Rats continued on the FR3
protocol with 10% ethanol as the reinforcer for a minimum of 20 sessions. Any animals
not reaching 0.25 g/kg ethanol intake per session were excluded from further study.
5% Sucrose Self-Administration
Long-Evans rats (n=14-15 per group) were trained to self-administer 5% sucrose using
the protocol described above. The training included 1-3 overnight sessions, 2-4 45
minute FR1 sessions with one hour water access, 3-4 45 minute FR1 sessions with full
water, and a minimum of 20 30 minute FR3 sessions. For all sessions, 5% sucrose served
as the reinforcer.
20% Ethanol Self-Administration
After the period of acclimatization, daily 20% ethanol self-administration was initiated in
separate groups of Long-Evans rats (n=5-11 per group). Importantly, food and water were
available ad libitum at all times in the home cage throughout the training. On the first day
of training animals were placed in the operant conditioning chambers for a 14-h
overnight session on an FR1 schedule of reinforcement (0.1 ml after a single lever press)
with 20% ethanol solution as the reinforcer. These FR1 overnight sessions were
performed five days per week for a total of 12 sessions. During this phase, only the active
lever was available for the rat to press to establish lever pressing behavior. Following the
completion of these sessions, rats were then exposed to 45-minute FR1 sessions for a
total of 6 sessions. Subsequently, training sessions were reduced to 30 minutes and the
work ratio was increased to FR3 schedule of reinforcement (three active lever presses
required for 0.1 ml reinforcer). The second (inactive) lever was also introduced at this
time. Upon pressing the inactive lever, no reinforcer, cue light, or auditory stimuli were
presented and the event was merely recorded as a measure of nonspecific behavior. Rats
continued on the FR3 protocol with 20% ethanol as the reinforcer for a minimum of 20
sessions before implantation of cannulae into the central amygdala (CeA) and basolateral
amygdala (BLA). Any animals not reaching 0.25 g/kg ethanol intake per session were
excluded from further study.
Surgery and Amygdala Infusions
Rats trained to self-administer 20% ethanol and 5% sucrose were continuously
anesthetized with isoflurane during the surgery. Four holes were drilled for screws, and
two other holes were drilled for the placement of the cannulae. Single guide cannulae
(C315G, 26 gauge; Plastics One) were bilaterally aimed dorsal to the CeA (n=18; AP 2.12, ML +/- 4.0, DV -6.0) and the BLA (n=10; AP -2.12, ML +/- 5.0, DV -6.0)
according to Paxinos and Watson (Paxinos, 1997) for the ethanol-trained animals.
Additionally, one group of animals trained to self-administer 5% sucrose (n=12) was
cannulated in the CeA as a control for the ethanol experiments. Animals were given a
minimum of five days to recover from surgery after which they were returned to selfadministration training for two weeks followed by extinction. During extinction the rats
were habituated to handling and the microinjection procedure. Drug or vehicle was
infused into the CeA or BLA via injection cannulae extending 2.2 mm beyond the guide
cannula tip. Mifepristone or vehicle was infused via a 10 μl Hamilton syringe 10 minutes
before yohimbine or yohimbine vehicle administration. Due to the small size of the CeA
and the BLA, and to limit the possible diffusion of mifepristone, a volume of 0.3 μl was
microinfused over 2 min. The injectors remained in position for an additional 1 min. The
order of mifepristone doses infused was counterbalanced across all subjects. Mifepristone
and vehicle infusions into the BLA caused a dramatic decrease in reinstatement when
compared with the baseline reinstatement level. Therefore, in order to verify that the drop
in responding was not caused by the DMSO vehicle, in the fourth week of reinstatement
testing phosphate buffered saline (PBS) was infused before yohimbine vehicle and
yohimbine administration.
Effect of Mifepristone on 10% Ethanol and 5% Sucrose Self-Administration and
Yohimbine-induced Increases in 10% Ethanol and 5% Sucrose Self-Administration
The effects of mifepristone were assessed on maintained responding for 10% ethanol
(n=12) and 5% sucrose (n=15), and yohimbine-induced increases in responding for 10%
ethanol (n=11) and 5% sucrose (n=14), following a minimum of 20 FR3 operant sessions.
In addition to its ability to reinstate ethanol-seeking following extinction, yohimbine has
been shown to increase lever pressing and ethanol consumption in the maintenance phase
of operant self-administration paradigms (Le et al., 2005; Le et al., 2009; Marinelli et al.,
2007). Animals were administered mifepristone (5, 10, and 30 mg/kg i.p.) or vehicle 30
minutes prior to the onset of regular, reinforced operant sessions or pretreated with
mifepristone (5 and 30 mg/kg) or vehicle 30 minutes prior to a yohimbine challenge
(2mg/kg) and placed in the operant chambers for a reinforced operant session 30 minutes
following yohimbine treatment. Each test session was given seven days apart in a Latinsquare design, thus each animal served as its own control. Between the injection days, the
rats were exposed to their normal training schedule.
Progesterone Measurements Following Yohimbine Administration
To examine the effects of yohimbine on circulating progesterone levels, blood was
collected from extinguished rats trained to respond for 10% ethanol (n=12). Animals
were divided into two groups (n=6 per group) matched based on previous ethanol selfadministration levels. One group received a yohimbine vehicle injection while the other
received a yohimbine (2 mg/kg) injection. Thirty minutes after the yohimbine or vehicle
treatment, the animals were anesthetized with isoflurane and blood was collected from
the lateral tail vein. Samples were centrifuged at 4°C for 13 minutes at 8000 rpm then
stored at -80°C. Serum progesterone concentrations were determined by ELISA (Assay
Designs Inc., Ann Arbor, MI). Data were analyzed using an unpaired t-test.
Histology
Locations of cannulae were verified in 30-μm coronal sections stained with cresyl violet.
Only data from subjects with injectors located in the CeA (Fig. 2B) and BLA (Fig. 2D)
were included in the analysis. Specifically, 4 rats were removed from the CeA ethanol
group due to incorrect cannula placement. To examine brain tissue for signs of cell death
following drug or vehicle delivery, a group of rats was cannulated in the CeA (n=3) and
administered six infusions of either vehicle (100% DMSO), mifepristone (10μg)
dissolved in 100% DMSO, or phosphate buffered saline (PBS) over three weeks. Animals
were euthanized 24 hours after the last infusion and brains were collected for analysis.
Tissue was then stained with Hoescht 33,342 (Invitrogen, Carlsbad, CA) for labeling
DNA according to a previously described method (Lin et al., 2006). Viable nuclei were
then visualized using a Zeiss LSM 510 META laser confocal microscope (Zeiss
MicroImaging, Thornwood, NY, US) using Plan-Neofluar 5x/0.15 and 10X/0.30
objectives. The nuclei were quantified using the Imaris Neuroscience software pack
(v.7.1.1, Andor Technology, Belfast, Northern Ireland). Comparisons between treated
cells groups were performed using one-way ANOVA following by Newman-Kleus
comparison test where statistical significance was p < 0.05.
RESULTS
Progesterone Measurements Following Yohimbine Administration
An unpaired t-test comparing the progesterone levels in animals trained to self-administer
10% ethanol then extinguished revealed that yohimbine treatment caused a nonsignificant increase in progesterone levels (p=0.067, Supplemental Fig 1).
SUPPLEMENTAL FIGURE 1
Progesterone Measurements Following Yohimbine Administration
Progesterone (ng/ml)
4
n.s.
3
2
1
0
Vehicle
Yohimbine