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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
JPET 280:1250 –1260, 1997
Vol. 280, No. 3
Printed in U.S.A.
Effects of g-Aminobutyric Acid Agonists and N-Methyl-Daspartate Antagonists on a Multiple Schedule of Ethanol and
Saccharin Self-administration in Rats1
KEITH L. SHELTON2,3 and ROBERT L. BALSTER
Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia
Accepted for publication November 19, 1996
A diverse group of neurotransmitter systems are affected
by ethanol, and the roles of these systems in the production
of the pharmacological and behavioral effects of ethanol are
beginning to be understood. Relatively recently, the actions
of ethanol on the NMDA subtype of glutamate receptors and
the GABAa/benzodiazepine receptor system have received
considerable attention (Grant, 1994). It is known that ethanol potentiates the effect of GABA and the GABAa agonist
muscimol in several in vitro functional assays (Mehta and
Ticku, 1988; Takada et al., 1989). There is also emerging
evidence that ethanol has a number of effects on the NMDA
receptor complex at physiologically relevant concentrations
(Gonzales and Woodward, 1990; Lovenger et al., 1989; Nie et
al., 1994).
At the behavioral level, ethanol intoxication, tolerance and
Received for publication June 25, 1996.
1
This research was supported by National Institute on Alcoholism and
Alcohol Abuse Grant AA08437 and National Institute on Drug Abuse Grant
DA01442.
2
Supported by National Institutes of Health Training Fellowship AA05357.
3
Present address: Department of Psychiatry and Behavioral Sciences,
M.S.I., University of Texas Health Science Center at Houston, 1300 Moursund,
Houston, TX 77030.
ethanol selectively decreased ethanol self-administration without altering saccharin self-administration. The competitive
NMDA antagonist CPPene (D-3-(2-carboxypiperazine-4-yl)-1propenyl-1-phosphonic acid [SDZ EAA 494]) and the noncompetitive NMDA antagonist phencyclidine decreased both ethanol and saccharin self-administration. The GABA agonists
pentobarbital and diazepam also failed to reduce ethanol selfadministration, relative to saccharin. Although these results do
not support the hypothesis that antagonism of the NMDA receptor system or activation of the GABA receptor system can
selectively modify ethanol-reinforced responding, they identify
important issues for designing the best strategies to be used to
assess selective drug effects on ethanol self-administration.
dependence may also have both a GABAergic component and
a NMDA component. GABA agonists potentiate ethanol-induced sedation (Lilijequist and Engel, 1982) and, in a similar
fashion, NMDA antagonists increase the potency of ethanol
for inhibiting the righting reflex in mice (Daniell, 1990) and
decreasing locomotor activity in rats (Robledo et al., 1991).
Barbiturates and benzodiazepine also exhibit cross-tolerance
(Rosenberg et al., 1983) and cross-dependence (Cooper et al.,
1979) with ethanol, whereas noncompetitive NMDA antagonists attenuate alcohol withdrawal seizures (Morrisett et al.,
1990) and enhance the behavioral and toxic effects of ethanol
(Stone and Forney, 1977; Wessinger and Balster, 1987). In
yet another line of investigation, drug discrimination studies
have shown that ethanol and certain GABA agonists and
NMDA antagonists have similar interoceptive stimulus properties (Grant and Colombo, 1992; Grant et al., 1991; Kubena
and Barry, 1969; Sanger, 1993; Shelton and Balster, 1994;
Winter, 1975) .
Both the GABAa/benzodiazepine receptor system and, to a
lesser extent, the NMDA receptor system have been implicated as modulatory substrates for ethanol self-administration. GABA-transaminase inhibitors, GABAb agonists
ABBREVIATIONS: AP-5, 2-amino-5-phosphonopentanoic acid; AP-7, 2-amino-7-phosphonoheptanoic acid; CPPene, D-3-(2-carboxypiperazine4-yl)-1-propenyl-1-[phosphonic acid [SDZ EAA 494]; FR, fixed ratio; GABA, g-aminobutyric acid; NMDA, N-methyl-d-aspartate; PCP, phencyclidine.
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ABSTRACT
Recently, it has been shown at both the cellular and behavioral
levels that ethanol has effects on the N-methyl-D-aspartate
(NMDA) and g-aminobutyric acid (GABA)a receptor systems,
leading to the possibility that the reinforcing effects of ethanol
may be, at least partially, mediated via these receptor ionophores. In this study, a multiple schedule of ethanol and saccharin self-administration was used to study that possibility.
Adult male Long-Evans rats were trained during 1-hr sessions
to press on two different levers for 10% (w/v) ethanol and 0.1%
(w/v) saccharin solutions, under an alternating 5-min, fixedratio-4 schedule of liquid availability. After training, tests were
conducted with ethanol, NMDA antagonists and GABA agonists given before six consecutive sessions. Pretreatment with
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assess the ability of the procedure to detect selective drug
effects, because one might predict that noncontingent ethanol would selectively decrease ethanol-maintained responding.
Methods
Subjects. Twelve experimentally naive, adult, male, Long-Evans
hooded rats (Harlan Sprague Dawley, Indianapolis, IN), weighing
275 to 300 g at the beginning of the study, were used as experimental
subjects. The rats were individually housed in standard hanging
wire rodent cages. A 12-hr light/dark cycle was in effect for the
duration of the study. The rats were food-restricted to 15 g of Agway
rodent chow per day, which was available after the experimental
sessions, with the exceptions noted for the ethanol-induction phase
of the study. Rats reached an average weight of 350 g by the end of
the experiment.
Apparatus. Experimental sessions were conducted in a separate
room in two-lever operant conditioning chambers (BRS/LVE, Beltsville, MD), each of which had been fitted with a custom-built front
panel (Fabco, Albany, TX) containing two solenoid-operated liquiddelivery devices with two associated rodent response levers and 8-W
stimulus lights (Coulbourn Instruments, Allentown, PA). The solenoid value system allowed small amounts of liquid (0.05 ml/delivery)
to be accurately metered into 0.15-ml liquid cups located on the front
interior wall of each cage. Liquid was supplied to each solenoid value
through 3/8-inch Tygon tubing (Norton Performance Plastics, Akron,
OH) attached to suspended 250-ml aspirator bottles (Corning, Corning, NY). The chambers were individually housed in sound-attenuated and ventilated cubicles (BRS/LVE). Experimental sessions and
data recording were accomplished using an IBM-compatible 486DX2 computer system (Win Laboratories, Manassas, VA), running
Med-state operant conditioning software. Smart-control interface
equipment linked the computer and operant chambers (Med Associates, Albans, VT).
Drugs. Dehydrated 100% ethyl alcohol (Pharmco, Bayonne, NJ)
was obtained from the Medical College of Virginia hospital pharmacy
and diluted with tap water into concentrations of 1, 3, 6 and 10%
(w/v) for the drinking solutions. The same 100% ethanol was diluted
in saline for the pretreatment injections. Saccharin HCl (Sigma
Chemicals) was diluted in tap water to a concentration of 0.1% (w/v).
PCP HCl (National Institute on Drug Abuse, Rockville, MD) was
dissolved to injection concentrations with sterile saline. Sodium pentobarbital (Anthony Products, Arcadia, CA) and diazepam HCl (Elkins-Sinn, Inc., Cherry Hill, NJ) were diluted from a commercial
injection solution into a vehicle consisting of 50% sterile water, 40%
propylene glycol and 10% ethanol. CPPene (Sandoz Research Institute, Berne, Switzerland) was dissolved in sterile saline, and the pH
was adjusted to between 6 and 7 by the addition of sodium hydroxide.
All drugs and vehicles were sterile-filtered (Millipore filters, 0.2 mm;
Gellman) before use.
Presession injections were given i.p. at a volume of 1 ml/kg, with
the exception of ethanol, which was administered i.p. at a volume of
10 ml/kg. Ethanol (180, 560, 1000 and 1560 mg/kg), PCP (1, 2 and 4
mg/kg) and pentobarbital (3, 10 and 20 mg/kg) were injected 15 min
before the start of the session. Diazepam (1, 3 and 5.6 mg/kg) was
injected 30 min before the session, and CPPene (1, 3 and 5.6 mg/kg)
was injected 60 min before the session.
Training and testing procedure. The animals were tested 5
days per week (Monday through Friday), between 8:00 A.M. and 12:00
noon. To promote operant responding for liquid, the animals were
initially maintained in a water-deprived state for 20 hr before the
start of each session. In addition, 5 g of each animal’s daily food
allotment was placed in the home cage 15 min before each session.
Over the course of 20 sessions, the animals were trained to respond
for water under a multiple schedule for 0.05-ml water deliveries.
Each session lasted for 1 hr and consisted of 12 (5-min) periods of
water availability. Every 5 min, the active liquid delivery device was
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(Daoust et al., 1987), picrotoxin-site ligands (Rassnick et al.,
1993a) and GABA metabolites (Fadda et al., 1983; Gallimberti et al., 1989) have all been shown to decrease ethanol
intake in a number of models. Several benzodiazepines have
also been tested for their ability to affect ethanol self-administration; the findings from these studies are mixed, with
both positive (Samson and Grant, 1985) and negative
(Daoust et al., 1987; Rassnick et al., 1993a) results being
obtained. The benzodiazepine partial inverse agonist Ro15–
4513 decreases ethanol intake in choice as well as in forced
drinking studies (June et al., 1992; McBride et al., 1988;
Rassnick et al., 1993a; Samson et al., 1987, 1989).
The role of the NMDA receptor site in modulating ethanol
self-administration has not been well characterized. To date,
there are only two published reports concerning the ability of
NMDA antagonists to attenuate ethanol self-administration.
Both of these studies came from the same laboratory group,
and they used similar experimental designs. These preliminary results indicate that injections of the competitive
NMDA antagonists 2-amino-5-phosphonopentanoic acid
(AP-5) and 2-amino-7-phosphonoheptanoic acid (AP-7) directly into the nucleus accumbens attenuate operant responding for 10% ethanol without affecting baseline levels of
water self-administration (Rassnick et al., 1992a,b).
Many studies have examined drug effects on ethanol selfadministration behavior; however, relatively few experiments have attempted to assess the selectivity of pretreatment drug effects for ethanol self-administration, compared
with other reinforcers. Clearly, pharmacologically useful
treatment drugs for ethanol abuse should exhibit some selectivity for attenuating drug-reinforced responding without altering other behaviors. The more commonly used methods of
assessing the effects of pretreatment drugs on a self-administration base line, such as bottle drinking and simple operant schedules, are generally of limited utility in separating
any attenuation of reinforcing effects from nonspecific behavioral disruption, which is often caused by these pretreatment
compounds. A number of methods have been devised to overcome this limitation, such as the use of a second control group
of subjects self-administering a non-drug reinforcer (Hubner
and Koob, 1990; Rassnick et al., 1993a; Samson et al., 1989)
or the concurrent availability of water along with drug
(Rassnick et al., 1993b). In studies using the latter method,
rates of water self-administration are often very low compared with drug; therefore, rate-disruptive drug effects
would be difficult to assess due to the lack of a similar
operant baseline. Overall, these strategies provide some assessment of selectivity but are far from ideal controls.
A number of i.v. drug self-administration studies in nonhuman primates have addressed the issue of pretreatment
selectivity by training animals under multiple schedules in
which alternating periods of drug and food availability are
used to assess nonspecific drug effects (Aigner and Balster,
1979; Mello et al., 1993; Woolverton and Virus, 1989). It was
our goal to develop a multiple schedule of oral ethanol and
saccharin self-administration in rats similar to that previously used to assess selectivity of drug effects on i.v. selfadministration in monkeys. We then tested a number of
drugs acting as GABAa/benzodiazepine receptor agonists or
NMDA antagonists for their ability to alter ethanol selfadministration at doses that did not effect saccharin selfadministration. Ethanol pretreatment was also studied to
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Results
Ethanol and water self-administration. The data from
the 10 sessions of 10% ethanol and water self-administration
are shown in figure 1. During the first two self-administration sessions, rates of ethanol and water self-administration
were similar. Over the next several two-session blocks, ethanol self-administration steadily increased to a high of 36
deliveries, whereas water self-administration decreased
nearly to zero levels. These results clearly show that the
delivery of 10% ethanol served as a reinforcer under the
multiple schedule.
Effects of ethanol pretreatment on ethanol and saccharin self-administration. The results of ethanol pretreatment on total session rates of ethanol and saccharin
self-administration are shown in figure 2. A total of nine rats
met the ethanol and saccharin delivery performance criteria
for inclusion in this dose-effect curve. Ethanol pretreatment
dose-dependently suppressed ethanol self-administration
(fig. 2, top left). The 1000 mg/kg and 1560 mg/kg doses of
ethanol significantly [t(8) 5 4.95 and t(8) 5 3.558, respectively] reduced ethanol responding relative to saline control sessions. Ethanol preinjections also significantly affected saccharin responding; however, the effects did not appear to be
dose-related (fig. 2, top right). At the 180 mg/kg and 560
mg/kg doses of ethanol, small but statistically significant
increases [t(8) 5 22.90 and t(8) 5 22.75, respectively] in
saccharin self-administration were observed. The 1000
mg/kg ethanol dose significantly decreased [t(8) 5 6.38] saccharin deliveries relative to the preceding saline control. No
effect on saccharin self-administration was observed at the
1560 mg/kg ethanol pretreatment dose.
The daily means of ethanol and saccharin deliveries during
the ethanol pretreatment dose-effect determination are also
shown in figure 2. Saccharin self-administration did not
reach a stable baseline level until well into the ethanol doseeffect curve (fig. 2, bottom right). When the data are shown in
this way, it is clear that the ethanol-produced increases in
saccharin self-administration are probably an artifact of the
steady increases in rates of saccharin-reinforced responding
over this period, independent of ethanol pretreatment. Eth-
Fig. 1. Comparison of ethanol (EtOH) and water self-administration
under a multiple schedule of alternating 5-min periods of ethanol and
water availability. Deliveries (mean 6 S.E.M.) of 10% (w/v) ethanol (f)
and water (E) over the course of five two-session blocks are shown.
Each data point is a group mean and is composed of the mean
deliveries over two sessions for each subject.
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alternated (i.e., left, right, left, etc.). Each period of access from a
device was signaled by the illumination of an amber stimulus light
directly above the spout cup. Only responses on the lever associated
with that device produced fluid deliveries. Responding on the other
lever was recorded but had no scheduled consequences. The first
delivery device to be active (left or right) was alternated daily.
When responding had stabilized under a FR-1 schedule for water
delivery, the FR size was increased over a period of 10 days to FR-4.
After FR-4 responding for water was obtained, increasing concentrations of ethanol were introduced into both delivery devices. Every 10
sessions, the ethanol concentration was increased in a stepwise
fashion (1, 3, 6 and 10%, w/v) until the rats were responding for a
10% ethanol solution. At that point, the water restriction was gradually discontinued over a period of 10 days. After that, the presession
feeding was also discontinued over a period of 10 sessions. The 10%
ethanol in one of the two delivery systems was then replaced with
water for 10 sessions to assess the reinforcing effects of ethanol in
alternate components.
After this initial test for ethanol reinforced responding, a 0.1%
saccharin solution replaced water in alternating components of each
daily self-administration session. Because saccharin is a potent reinforcer in rodents, no training was necessary to initiate operant
responding for saccharin deliveries. The sides from which ethanol
and saccharin were available remained constant throughout the
study. To balance the time periods of ethanol and saccharin access,
the side that was active at the start of the session was alternated
daily (i.e., left, right, left, right), resulting in ethanol being available
first in half of the sessions and saccharin being available first in half
of the sessions. The multiple schedule of 10% ethanol and 0.1%
saccharin was then tested for 10 sessions, to allow the self-administration of both solutions to stabilize. Pretreatment tests were then
conducted with various doses of ethanol, PCP, CPPene, diazepam
and pentobarbital, in that order. Each drug test block consisted of six
daily drug injections at each dose. Every block of drug sessions was
preceded by six sessions of daily vehicle injections. Drug doses were
administered in ascending order for all of the drugs tested.
Data analysis and inclusion criteria. The first two sessions of
each six-session block of drug pretreatments were discarded from the
data analysis based on the a priori assumption that these sessions
were likely to show behavior in transition. The final four sessions
were used for data analysis purposes. Both correct and incorrect
responses and liquid deliveries were collected for each 5-min segment, as well as for the total session. Changes in overall rates of
ethanol and saccharin self-administration, as well as changes in the
within-session distribution of responding, were determined from the
number of fluid deliveries. Separate two-tailed paired t tests were
performed for each drug dose, comparing each drug dose to the saline
control block immediately preceding that dose. Individual data
points for these statistical analyses consisted of the mean of each
animal’s last 4 days at each dose of pretreament drug and the mean
of the last 4 days of each saline control block. Separate t tests were
performed for both ethanol and saccharin self-administration data.
The criterion for statistically significant effects was set at the P , .05
level.
Only animals that exhibited a mean of at least 20 ethanol and 20
saccharin deliveries during the final four saline-injection control
sessions before each dose-effect curve were used for that dose-effect
curve. Animals that exhibited some ethanol self-administration but
failed to reach the inclusion criteria received additional training in
the event that their level of ethanol self-administration increased
sufficiently to reach the inclusion criteria for the next dose-effect
curve. Animals that consistently exhibited no responding or very low
levels of responding for ethanol were removed from the study.
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anol pretreatment at both the 560 mg/kg and 1000 mg/kg
doses resulted in a progressive decrease in ethanol self-administration over sessions (fig. 2, bottom left). The data also
show that ethanol deliveries quickly returned to baseline
levels after cessation of ethanol pretreatment.
Figure 3 illustrates the within-session pattern of responding for ethanol and saccharin. Plotted in figure 3 are the
mean ethanol deliveries (fig. 3, left) and saccharin deliveries
(fig. 3, right) in each 5-min session component, averaged
across the last 4 days of each 6-day block of ethanol and
saline pretreatment. The within-session patterns of control
ethanol and saccharin self-administration show similar numbers of ethanol and saccharin deliveries during the initial
5-min period of availability, followed by a marked decrease in
ethanol self-administration in later components. Saccharin
self-administration also decreased over the course of the session, but to a much lesser degree than ethanol self-administration. As was the case with the session totals, ethanol
pretreatment dose-dependently suppressed ethanol drinking
during the first 5-min component (fig. 3, left). Conversely,
ethanol pretreatment had little effect on saccharin intake
during the first 5-min bin. Ethanol pretreatment at doses of
560 and 1560 mg/kg did, however, have rate-increasing effects on saccharin self-administration, which were more apparent in later bins. These increases may also be a result of
the steadily increasing saccharin baseline and not a consequence of ethanol pretreament itself.
Effects of PCP pretreatment on ethanol and saccharin self-administration. Seven rats met criteria for inclusion into the PCP dose-effect curve. The effects of PCP pretreatment on session totals for ethanol and saccharin selfadministration are shown in figure 4. Doses of 1 and 2 mg/kg
PCP did not have any significant effect on either ethanol or
saccharin deliveries, whereas 4 mg/kg decreased both. The 4
mg/kg dose of PCP significantly [t(6) 5 2.802] suppressed
ethanol responding (fig. 4, top left). Saccharin self-administration was likewise significantly [t(6) 5 2.735] decreased by
4 mg/kg PCP (fig. 4, top right).
Figure 4 also shows the daily means for both ethanol (fig.
4, bottom left) and saccharin (fig. 4, bottom right) deliveries.
There were no apparent trends over days for control rates of
either ethanol or saccharin self-administration across this
phase of the study. In addition, there were no noticeable
trends within blocks of PCP pretreatment at 1 and 2 mg/kg
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Fig. 2. Effects of noncontingent ethanol (EtOH) pretreatment on ethanol and saccharin self-administration. Top, ethanol (left) and saccharin (right)
deliveries after pretreatment with ethanol (f) and deliveries during pretest saline control sessions (M). Bottom, left, daily means of ethanol
deliveries during noncontingent ethanol administration (f) and during pretest saline control sessions (M); right, daily means of saccharin deliveries
during noncontingent ethanol administration (F) and during pretest saline control sessions (E). Points shown are the number of deliveries (mean 6
S.E.M.) of ethanol and saccharin received in the last four sessions of a six-session block at each condition. *, statistically significant effects (P ,
.05).
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PCP, nor was there any evidence of suppression of ethanol or
saccharin self-administration until pretreatment with the 4
mg/kg dose of PCP. At this dose, rates of both ethanol and
saccharin self-administration decreased over days of PCP
pretreatment.
Effects of CPPene pretreatment on ethanol and saccharin self-administration. A total of 10 rats were used for
the determination of the CPPene dose-effect curve. The results of CPPene pretreatment on the four-session means of
ethanol and saccharin deliveries are shown in figure 5 (top).
The 1 mg/kg dose of CPPene had no effect on ethanol intake
(fig. 5, top left); however, this dose of CPPene slightly but
significantly [t(9) 5 2.29] suppressed saccharin self-administration (fig. 5, top right). The 3 mg/kg dose of CPPene had a
nonselective effect on ethanol and saccharin self-administration, significantly suppressing ethanol [t(9) 5 4.63] and saccharin [t(9) 5 6.28] self-administration. The effects of 5.6
mg/kg CPPene were similar to, but more pronounced than,
those of 3 mg/kg. Ethanol deliveries were significantly suppressed [t(9) 5 5.16] to ,25% of control levels. Saccharin
deliveries were also greatly reduced [t(9) 5 5.08, P , .05], by
.64%, compared with pretest saline control levels.
The single-session means of ethanol and saccharin deliveries over successive days also clearly show the pronounced
dose-related effects of CPPene on both saccharin and ethanol
self-administration (fig. 5, bottom). Although there was some
variability in the daily data, the effects of CPPene on ethanol
and saccharin self-administration were consistent, in that
the lowest number of both ethanol and saccharin deliveries
under saline control conditions were higher than the greatest
number of deliveries at the 3 and 5.6 mg/kg doses of CPPene.
The effects of CPPene on ethanol self-administration progressively increased over days of CPPene administration,
whereas the effects of CPPene on saccharin self-administration did not show a trend across days. As was the case with
PCP, baseline levels of ethanol and saccharin self-adminis-
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Fig. 3. Within-session time-course showing the results of ethanol (EtOH) pretreatments on successive 5-min segments of ethanol (left) and saccharin
(right) self-administration, averaged
across the last four of six test sessions.
u, ethanol and saccharin deliveries
(mean 6 S.E.M.) during ethanol pretreatment testing. M, ethanol and saccharin
deliveries (mean 6 S.E.M.) during saline
control testing. From top to bottom,
each panel shows increasing pretreatment doses of ethanol.
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tration were quickly recovered after the 1.0 and 3 mg/kg
doses of CPPene. In fact, on the final day of saline pretreatment before both the 3 and 5.6 mg/kg doses of CPPene,
ethanol self-administration was considerably greater than in
previous sessions (fig. 5, bottom left). These increases did not,
however, appear to be part of a trend, because responding in
each of the other three sessions appeared to be stable.
Effects of diazepam pretreatment on ethanol and
saccharin self-administration. A total of eight animals
met the inclusion criteria for the diazepam dose-effect curve
determination. The effects of diazepam on session totals of
ethanol and saccharin self-administration are shown in figure 6 (top). Only the 5.6 mg/kg dose of diazepam produced a
statistically significant [t(7) 5 4.25] decrease in ethanol deliveries. This dose of diazepam also significantly [t(7) 5 5.42]
suppressed saccharin responding (fig. 6, top right), resulting
in a 71% decrease in saccharin deliveries compared with
saline control levels.
The daily means of ethanol and saccharin self-administration during diazepam pretreatment are also shown in figure
6 (bottom). The 3 mg/kg dose of diazepam resulted in a
gradual decrease in ethanol responding over the final 4 days
of treatment (fig. 6, bottom left), but the overall change in
ethanol self-administration at this dose was not statistically
significant. Saccharin responding during the same period
remained stable (fig. 6, bottom right). The 5.6 mg/kg dose of
diazepam completely abolished ethanol responding during
the first 2 days, with a gradual recovery occurring on the last
2 days of diazepam pretreatment. A similar pattern of modest recovery occurred for saccharin self-administration.
Effects of pentobarbital pretreatment on ethanol
and saccharin self-administration. A total of 11 rats
reached criteria for inclusion in the pentobarbital dose-effect
curve. Pentobarbital, at a dose of 3 mg/kg, had no effect on
either ethanol (fig. 7, top left) or saccharin (fig. 7, top right)
self-administration. However, subsequent baseline levels of
ethanol self-administration were lower after this dose of pentobarbital (fig. 7, top left). Saccharin deliveries were not
affected in this manner and, in fact, increased somewhat
from the first to the second saline control block. The 10 mg/kg
dose of pentobarbital differentially affected ethanol and saccharin deliveries, in that only saccharin deliveries were significantly reduced from baseline rates (fig. 7, top right) [t(10)
5 3.375]. Similarly, although the 20 mg/kg dose of pentobarbital somewhat reduced ethanol deliveries, saccharin selfadministration was decreased significantly [t(10) 5 6.56] and
to a much greater degree. At a gross observational level, the
20 mg/kg dose of pentobarbital resulted in sedation and loss
of righting reflex at the start of the session in the majority of
the animals tested.
Daily totals of ethanol and saccharin deliveries showed
substantial variability over days. This fact was most evident
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Fig. 4. Effects of noncontingent PCP pretreatment on ethanol and saccharin self-administration. Top, ethanol (left) and saccharin (right) deliveries
after pretreatment with PCP (f) and deliveries during pretest saline control sessions (M). Bottom, left, daily means of ethanol deliveries during
noncontingent PCP administration (f) and during pretest saline control sessions (M); right, daily means of saccharin deliveries during noncontingent PCP administration (F) and during pretest saline control sessions (E). Points shown are the number of deliveries (mean 6 S.E.M.) of ethanol
and saccharin received in the last four sessions of a six-session block at each condition. *, statistically significant effects (P , .05).
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at the 20 mg/kg dose of pentobarbital, at which ethanol
self-administration on day 2 (day 4 of testing) was very low,
compared with the other 3 days at this dose (fig. 7, bottom
left). Saccharin deliveries, at doses of 3.0 and 10.0 mg/kg
pentobarbital, showed a gradual decrease over days (fig. 7,
bottom right), not unlike that seen for ethanol self-administration after low doses of noncontingent ethanol.
Discussion
This examination of the effects of GABA agonists and
NMDA antagonists on a multiple schedule of ethanol and
saccharin self-administration resulted in a number of findings. Clearly, the study showed that it is possible to train rats
to self-administer ethanol and saccharin under a multiple
schedule. Moreover, it was shown that ethanol could serve as
a reinforcer, relative to water, in the absence of any experimental manipulations other than food restriction. Responding for ethanol was robust, and a clear separation between
responding for ethanol and water quickly developed over the
course of the 10 test sessions. This crucial demonstration of
ethanol reinforcement, relative to vehicle, is implicit in most
operant ethanol self-administration studies but is rarely
closely examined or emphasized.
Pretreament of the rats with ethanol produced differential,
dose-dependent effects on ethanol and saccharin self-administration. The 1000 mg/kg pretreatment dose of ethanol suppressed both ethanol and saccharin self-administration, although ethanol was decreased to a much greater degree than
was saccharin. The 1560 mg/kg dose of ethanol selectively
and significantly suppressed ethanol self-administration relative to saccharin. At this dose, ethanol deliveries were
greatly decreased, whereas saccharin deliveries were not altered. The results are complicated by the fact that saccharin
self-administration increased during both the ethanol and
saline pretreatment sessions and continued to increase during determination of the ethanol pretreatment dose-effect
curve. Based on this continuous increase, it is likely that
saccharin self-administration had not reached a stable level
before the beginning of the ethanol pretreatment curve. Nevertheless, the possibility that ethanol injections caused the
increases in saccharin self-administration cannot be ruled
out.
There are a number of possible mechanisms through which
noncontingent ethanol might selectively reduce ethanol intake. One possibility relates to food restriction. It is possible
that noncontingent ethanol pretreatment replaced the calories provided by self-administered ethanol and thereby reduced ethanol self-administration. This hypothesis is un-
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Fig. 5. Effects of noncontingent CPPene pretreatment on ethanol and saccharin self-administration. Top, ethanol (left) and saccharin (right)
deliveries after pretreatment with CPPene (f) and deliveries during pretest saline control sessions (M). Bottom, left, daily means of ethanol
deliveries during noncontingent CPPene administration (f) and during pretest saline control sessions (M); right, daily means of saccharin deliveries
during noncontingent CPPene administration (F) and during pretest saline control sessions (E). Points shown are the number of deliveries (mean 6
S.E.M.) of ethanol and saccharin received in the last four sessions of a six-session block at each condition. *, statistically significant effects (P ,
.05).
1997
Ethanol Self-Administration
1257
likely for a number of reasons. Firstly, self-administration of
other drugs of abuse that have no caloric or anorectic properties is increased by food deprivation (Macenski and Meisch,
1994; Meisch, 1987). Although not conclusive proof, the fact
that the self-administration of other drugs is also affected in
this manner does suggest that the rats were not drinking
ethanol primarily for its caloric value. In addition, ethanol
self-administration increased during acquisition even though
the level of food restriction remained constant. Finally, the
finding that ethanol drinking decreased when presession
feeding was discontinued, rather than increased because of
this loss of presession food, also supports the contention that
caloric restriction increased ethanol drinking by enhancing
the reinforcing properties of ethanol.
A second possible explanation for the selective effect of
ethanol on ethanol self-administration is that ethanol pretreatment produced effects similar to those of self-administered ethanol, thereby reducing the reinforcing effects of
ethanol. This conclusion is necessarily tentative, but it is
supported by two other studies using different species, routes
of ethanol administration and experimental methodologies
(Karoly et al., 1978; Petry, 1995). It is interesting to note that
ethanol is the only drug that has been shown to produce this
effect (Karoly et al., 1978; Petry, 1995). For example, noncon-
tingent cocaine does not suppress cocaine self-administration
(Skjoldager et al., 1993), dizocilpine is not effective in selectively reducing oral PCP self-administration (Carroll et al.,
1994) and methadone does not selectively attenuate alfentanil self-administration (Mello et al., 1983) .
Neither the noncompetitive NMDA antagonist PCP nor the
competitive NMDA antagonist CPPene selectively suppressed ethanol self-administration, compared with saccharin self-administration. The inability of PCP or CPPene to
selectively decrease ethanol self-administration is in contrast
to other literature reports. Only two other studies have directly examined the effects of NMDA antagonists on ethanol
self-administration. Both of those studies found that intraaccumbens injections of the competitive NMDA antagonist
AP-5 decreased ethanol self-administration, compared with
water (Rassnick et al., 1992a,b). There are a number of methodological differences between these experiments that may
account for the different findings. Firstly, the cited studies
injected AP-5 directly into the nucleus accumbens, rather
than systemically. A second relevant difference between the
present study and past experiments is the use of a saccharin
baseline rather than a water baseline. Water responding is
typically very low in water-satiated animals and is therefore
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 6, 2017
Fig. 6. Effects of noncontingent diazepam pretreatment on ethanol and saccharin self-administration. Top, ethanol (left) and saccharin (right)
deliveries after pretreatment with diazepam (f) and deliveries during pretest saline control sessions (M). Bottom, left, daily means of ethanol
deliveries during noncontingent diazepam administration (f) and during pretest saline control sessions (M); right, daily means of saccharin
deliveries during noncontingent diazepam administration (F) and during pretest saline control sessions (E). Points shown are the number of
deliveries (mean 6 S.E.M.) of ethanol and saccharin received in the last four sessions of a six-session block at each condition. *, statistically
significant effects (P , .05).
1258
Shelton and Balster
Vol. 280
probably not as sensitive to drug-induced rate decreases as
the saccharin baseline in the present studies.
As was the case with NMDA antagonists, pretreatment of
rats with the indirectly acting GABAa agonists diazepam and
pentobarbital also failed to selectively suppress ethanol selfadministration relative to saccharin responding. Other laboratories have shown both increases (Barrett and Weinberg,
1975; Petry, 1995) and decreases (Chan et al., 1983a,b; Roehrs et al., 1984; Samson and Grant, 1985) in ethanol selfadministration after benzodiazepine administration. Direct
comparison of our findings and those in previous studies is
difficult because of the different methodologies. Among the
literature reports, only three studies have used operant techniques and, of these, only two have examined the specificity
of benzodiazepines for decreasing ethanol self-administration relative to another reinforcer (Petry, 1995; Samson and
Grant, 1985). However, there are a number of possible reasons for the divergence in findings between our study and
previous investigations. One possibility is that there are differences among benzodiazepines in their ability to selectively
reduce ethanol self-administration. Another difference is in
the choice of reporting measures used. We reported actual
numbers of ethanol and saccharin deliveries, whereas the
other studies used either percentages of baseline (Samson et
al., 1982) or responses per second under a variable-interval
5-sec schedule, in which decreases in responding may not
directly affect rates of reinforcement (Petry, 1995).
Overall, the general lack of specificity for both NMDA
antagonists and GABA agonists reported here could have
been the result of a number of factors. It has been shown that
there is a great deal of uniformity of drug effects on operant
behavior, regardless of the reinforcer used to maintain that
behavior. For instance, d-amphetamine can increase both
food- and shock-maintained behavior when administered under proper conditions (Barrett, 1977), even though the two
maintaining events are radically different. Therefore, it is
not surprising that the majority of the pretreatment drugs in
the present study produced nonselective effects. If all maintaining events (drug or another reinforcer) are fundamentally similar, it may be difficult to infer specific neurochemical processes based on self-administration data. It is also
possible that selective decreases in ethanol self-administration were masked by the combined pharmacological and behavioral effects of pretreatment and self-administered drugs.
This hypothesis could partially account for the fact that the
pretreatment drugs decreased saccharin self-administration
more than ethanol self-administration. It is conceivable that
the pretreatment injection was additive with early-session
ethanol self-administration and thereby produced greater
effects on saccharin responding that occurred later in the
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 6, 2017
Fig. 7. Effects of noncontingent pentobarbital pretreatment on ethanol and saccharin self-administration. Top, ethanol (left) and saccharin (right)
deliveries after pretreatment with pentobarbital (f) and deliveries during pretest saline control sessions (M). Bottom, left, daily means of ethanol
deliveries during noncontingent pentobarbital administration (f) and during pretest saline control sessions (M); right, daily means of saccharin
deliveries during noncontingent pentobarbital administration (F) and during pretest saline control sessions (E). Points shown are the number of
deliveries (mean 6 S.E.M.) of ethanol and saccharin received in the last four sessions of a six-session block at each condition. *, statistically
significant effects (P , .05).
1997
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