EFFECTS OF DRUGS AND DRUG COMBINATIONS IN PIGEONS TRAINED TO DISCRIMINATE AMONG PENTOBARBITAL, DIZOCILPINE, A COMBINATION OF THESE DRUGS, AND SALINE

JOURNAL OF THE EXPERIMENTAL ANALYSIS OF BEHAVIOR
2009, 92, 387–412
NUMBER 3 (NOVEMBER)
EFFECTS OF DRUGS AND DRUG COMBINATIONS IN PIGEONS TRAINED TO DISCRIMINATE
AMONG PENTOBARBITAL, DIZOCILPINE, A COMBINATION OF THESE DRUGS, AND SALINE
D. E. MCMILLAN, WILLIAM D. WESSINGER, AND MI LI
UNIVERSITY OF ARKANSAS FOR MEDICAL SCIENCES
Drugs with multiple actions can have complex discriminative-stimulus properties. An approach to
studying such drugs is to train subjects to discriminate among drug combinations and individual drugs
in the combination so that all of the complex discriminative stimuli are present during training. In the
current experiments, a four-choice procedure was used to train pigeons to discriminate among
dizocilpine (noncompetitive NMDA receptor blocker), pentobarbital (GABAA receptor agonist), a
fixed-dose combination of these two drugs, and saline. Following extended training, low doses of
pentobarbital or dizocilpine administered alone produced saline-appropriate responding. Higher doses
of pentobarbital produced responding on the pentobarbital-appropriate key and higher doses of
dizocilpine produced responding on the dizocilpine key. Administering the lowest doses of
pentobarbital and dizocilpine together resulted in responding on the saline-appropriate key. Increasing
the dose of pentobarbital in the presence of low doses of dizocilpine produced responding primarily on
the pentobarbital-appropriate key; increasing the dose of dizocilpine in the presence of the lowest dose
of pentobarbital produced responding primarily on the dizocilpine-appropriate key. Combining the
higher doses of pentobarbital and dizocilpine resulted in responding primarily on the drug-
combination key. Low doses of phencyclidine or ethanol produced responding on the saline-
appropriate key, but intermediate doses resulted in individual subjects responding predominately on
either the pentobarbital key, the dizocilpine key, or the drug-combination key depending on the
subject. After the highest dose of phencyclidine or ethanol, most subjects responded predominantly on
the drug-combination key. Low doses of other drugs tested produced responding on the saline-
appropriate key. With the highest diazepam doses responding was largely confined to the pentobarbital-
appropriate key. The highest doses of dextromethorphan or dextrorphan resulted in responding on the
dizocilpine key more frequently than on other keys. Across a range of doses, morphine produced
responding predominantly on the saline key. The results using the four-key procedure emphasized the
role of both GABAA and NMDA receptors in the complex discriminative stimulus properties of
phencyclidine and of ethanol.
Key words: four-key drug discrimination, drug-combination training, pentobarbital, dizocilpine, drug-
combination tests, phencyclidine, ethanol, diazepam, morphine, dextromethorphan, dextrorphan,
pigeons
_______________________________________________________________________________
Both exteroceptive and interoceptive dis-
duced by drugs can depend on multiple drug
criminative stimuli can vary in more than one
actions. In the present study, we used a new
dimension. For example, visual discriminative
method for the study of the discrimination of
stimuli can vary in color, brightness, shape,
drugs with complex actions using both indi-
size, and position, as well as other attributes. In
vidual drugs and drug mixtures in a four-
establishing discriminative control by visual
choice discrimination procedure in pigeons.
stimuli, it can be difficult to determine to
The focus of these experiments was directed
which of these stimuli the subject is attending.
toward the use of this new procedure to study
Similarly, the development of stimulus control
the effects of drugs purported to act at GABAA
by interoceptive stimuli such as those pro-
receptors and NMDA receptors, receptor
populations which have been implicated in
the effects of benzodiazepines, barbiturates,
This research was supported by NIDA grant DA-2251-25
ethanol, and other drugs. For example, ben-
from the National Institute for Drug Abuse.
zodiazepines and barbiturates are usually
Address correspondence to William D. Wessinger,
considered to act at GABAA receptors al-
Department of Pharmacology and Toxicology, University
of Arkansas for Medical Science, 4301 W. Markham Street,
though at somewhat different sites on the
PO Box 611, Little Rock, Arkansas 72205-7199 (e-mail:
receptor (Charney, Mihic, & Harris, 2001).
wdwessinger@uams.edu).
Dizocilpine is generally considered to act as an
Mi Li is now at the National Center for Toxicological
noncompetitive blocker of NMDA receptors
Research, 3900 NCTR Road, Jefferson, Arkansas 72079-
9502.
(Dingledine, Borges, Bowie, & Traynelis,
doi: 10.1901/jeab.2009.92-387
1999), while ethanol effects have been pro-
387

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388
D. E. MCMILLAN et al.
posed to be due to effects at both GABAA
no-drug condition. In subjects trained to
receptors and NMDA receptors (Fleming,
discriminate a mixture of 8 mg/kg pentobar-
Mihic, & Harris, 2001). Teasing out the
bital and 0.08 mg/kg dizocilpine from no
relative contributions of NMDA and GABAA
drug, very little drug-appropriate responding
receptors to the discriminative stimulus effects
was observed after 3.0 g/kg ethanol. However,
of some of these drugs has been a difficult
after retraining the discrimination using a mix-
task.
ture of 12 mg/kg pentobarbital and 0.04 mg/
Using the usual two-choice discrimination
kg dizocilpine, drug-appropriate responding
procedure (a training drug versus saline) for
after the same dose of ethanol returned to
studying drug discrimination, when the train-
75%. Thus, the ratio of the mixture compo-
ing drug is replaced with a different drug,
nents appeared to be important.
responding can occur predominately on the
An approach to increasing the sensitivity of
drug manipulandum (substitution), predomi-
the drug discrimination procedure has been to
nately on the saline manipulandum (no
train the subjects under three-choice proce-
substitution), or distributed across both ma-
dures. For example, when using dizocilpine,
nipulanda (partial substitution). Even with
ethanol and water as the training drugs (Gatto,
these limited response options, the results
Bowen, & Grant, 1995), phencyclidine, which
can be quite complex. For example, with
substitutes for both ethanol and dizocilpine in
pentobarbital (a GABAA prototype) and the
two-choice studies, only substitutes for dizocil-
drug vehicles as the training drugs, other
pine under the three-choice procedure (Bo-
barbiturates and benzodiazepines substitute
wen & Grant, 1998). In rats trained to
for the training drug (Herling, Valentino, &
discriminate among pentobarbital, ethanol,
Winger, 1980), but ethanol, phencyclidine,
and water, partial substitution for pentobarbi-
dizocilpine and dextrorphan only partially
tal occurs after dizocilpine, diazepam, and
substitute for pentobarbital (Herling et al.,
phencyclidine (Bowen, Gatto & Grant, 1997).
1980; Kline & Young, 1986; Snodgrass &
The partial substitution of diazepam for
McMillan, 1991; Willetts & Balster, 1989).
pentobarbital in three-choice discriminations
Using dizocilpine (a noncompetitive NMDA
is consistent with two-choice discrimination
antagonist) and the drug vehicles as the
data from rats and pigeons, but the partial
training drugs, phencyclidine and dextror-
substitution of diazepam for pentobarbital in
phan substitute for dizocilpine but pentobar-
three-choice discriminations contrasts with the
bital does not substitute (Butelman, France, &
more robust substitution of diazepam for
Woods, 1991), and ethanol only partially
pentobarbital in two-choice discriminations.
substitutes for dizocilpine (Butelman, Baron,
Clearly, providing an additional choice to the
& Woods, 1993). Thus, using two-choice drug
drug-discrimination procedure produces dif-
discrimination procedures, ethanol partially
ferent substitution patterns than are observed
substitutes for both pentobarbital and dizocil-
under two-choice procedures. Drug discrimi-
pine, but while dizocilpine partially substitutes
nation studies which include drug mixtures
for pentobarbital, pentobarbital does not
during training as well as the individual drugs
substitute for dizocilpine. Dextrorphan, which
in the mixtures may well reveal subtle differ-
substitutes for dizocilpine, substitutes only
ences among the discriminative-stimulus ef-
partially for pentobarbital.
fects of drugs that appear to be similar under
Another approach to studying the discrim-
simpler procedures.
inative stimulus effects of depressant drugs
Recently, we have shown that pigeons can
with complex mechanisms of action such as
come under stimulus control of a four-choice
ethanol is by training subjects to discriminate
procedure in which subjects are trained to
drug mixtures. Stolerman and Olufsen (2001)
discriminate among three active drugs and
found that 3 g/kg (intragastric) ethanol pro-
saline (Li & McMillan, 2001). Not only can
duced a high level of drug-appropriate re-
pigeons perform such discriminations, but
sponding (76%) in rats trained under a two-
they are also able to discriminate among two
choice procedure to discriminate a mixture
drugs, a combination of the two drugs, and the
of 5 mg/kg chlordiazepoxide (a GABAA re-
drug vehicle using four-choice drug discrimi-
ceptor modulator) and 0.08 mg/kg dizocil-
nation procedures (McMillan & Li, 2002).
pine (NMDA receptor antagonist) from the
Training subjects to learn a discrimination

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FOUR-CHOICE DRUG DISCRIMINATION
389
among two drugs that produce discriminative
(MED Associates, Inc., St. Albans, VT) en-
stimuli by different mechanisms, a combina-
closed in a Gerbrands Model G7211 sound-
tion of these two drugs, and the absence of all
and light-attenuating enclosure. A 28-V DC
three drug states (vehicle) might permit a
light mounted near the ceiling illuminated
separation of the relative contribution of
each test chamber during behavioral sessions,
different mechanisms to the discriminative
except during food presentations. The cham-
stimulus effects of a drug whose effects were
ber was equipped with four pigeon response
a blend of the effects of the two mechanisms.
keys that could be transilluminated with
For example, if drug C produces responding
different colored lights (MED Associates,
on the key associated with training drug A, but
Model ENV-124AM). These were mounted
not the key associated with training drug B or
on the front panel in a row 21 cm above the
the drug-combination key, this would suggest
grid floor, each spaced 6 cm apart. When
that drug A mechanisms mediate the discrim-
operative, the left key was red, the left–center
inative stimuli produced by drug C. In
key was white, the right–center key was green,
contrast, responding on the drug-combination
and the right key was blue. Beneath the left–
key would suggest that elements of both drug
center key, 2 cm above the grid floor was an
A and drug B mechanisms were contributing
opening through which pigeon chow could be
to the discriminative stimulus effects of drug
presented when schedule contingencies were
C. Toward this end pigeons were trained to
met. During food presentations the houselight
discriminate among pentobarbital (a drug
was extinguished and the food opening was
acting at GABA
illuminated.
A
desktop
computer,
pro-
A receptors), dizocilpine (an
uncompetitive NMDA receptor blocker), a
grammed using MED-PC software (MED Asso-
combination of pentobarbital and dizocilpine,
ciates), controlled the experimental contin-
and saline. Subsequently, the effects of other
gencies and recorded the data through an
drugs were studied with a particular emphasis
interface (MED Associates).
on ethanol, which is purported to act at both
Procedure
GABAA receptors and NMDA receptors.
The methods for training subjects have been
discussed in detail (Li, Wessinger, & McMillan,
METHOD
2005). Initially, pecks on a single illuminated
Subjects
response key were reinforced by food presen-
tation (4-s access to pigeon chow) under a
Six adult male White Carneau pigeons
fixed-ratio 1 schedule (FR1). Once responding
(Palmetto Pigeon Plant, Sumter, SC) served
was established the schedule of reinforcement
as subjects in these experiments. All were
was gradually incremented across sessions to
experimentally na?¨ve at the beginning of these
FR 20. Then a second key was illuminated and
experiments. Pigeons were housed individually
responding on it was reinforced by food
with free access to water and grit in a
presentation in a similar manner. After re-
temperature- and humidity-controlled room
sponding on both keys under an FR 20
that was maintained under a 12-h normal-
schedule was established, responding during
phase lighting cycle. During the study, the
subsequent sessions was differentially rein-
pigeons were maintained at 80–85% (410–
forced depending upon whether an i.m.
530 g) of their free-feeding weights by food
injection of saline or 5 mg/kg pentobarbital
earned during the experimental sessions and
was administered 10 min before the session.
postsession supplemental feeding (Purina Pi-
Thus, if saline had been administered before
geon Chow Checkers 5405, Purina Mills, LLC,
the session, only responding on one of the
St. Louis, MO). Procedures used during these
keys was reinforced under the FR 20 schedule.
experiments were in accord with the Institu-
If 5 mg/kg pentobarbital had been adminis-
tional Animal Care and Use Committee of the
tered 10 min before the session, only respond-
University of Arkansas for Medical Sciences.
ing on the other key was reinforced under the
FR 20 schedule. After the two-key drug
Apparatus
discrimination had been established, a third
The experimental chamber was a MED
key was introduced by illuminating it also.
Associates ENV-009A Modular Test Cage
Responding on the third key was first estab-

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390
D. E. MCMILLAN et al.
lished as before, and in subsequent sessions
dose was reached that eliminated responding
responses on the third key were reinforced
for the 15-min response period. Similar dosing
under an FR 1 schedule of food presentation
procedures were used to determine dose–
only if 0.13 mg/kg dizocilpine had been
response curves for all drugs. Drug substitu-
administered 10 min before the session. Once
tion tests were generally conducted once a
responding was established on the third key,
week, with training sessions continuing on 4 or
the response requirement was gradually in-
5 other days of the week. One cumulative
creased to FR 20. After the three-key discrim-
dose–response curve was conducted in each of
ination had been established, the fourth key
the 6 pigeons for every drug or drug-combi-
was introduced and responding was estab-
nation condition.
lished as before. In this case, responses on
After determining the dose–response curves
the fourth key were reinforced only if both
for pentobarbital and dizocilpine alone, vari-
5 mg/kg pentobarbital and 0.13 mg/kg dizo-
ous combinations of doses of pentobarbital
cilpine had been administered 10 min before
and dizocilpine were studied. To study dose
the session. The assignment of drug-appropri-
combinations, a fixed dose of pentobarbital
ate keys was varied randomly across subjects.
was administered along with the lowest dose of
Training sessions ended after 20 reinforcer
dizocilpine and the first test trial was conduct-
presentations or 40 min, whichever occurred
ed. In subsequent test trials, the cumulative
first. Training sessions were conducted 5 or 6
dose of dizocilpine was incremented in the
days a week with at least one session under
same manner as when dizocilpine was tested
each stimulus condition occurring each week.
alone. Single ascending doses of pentobarbital
Training the subjects to make this four-choice
were tested (1.0, 3.0, 5.6 and 10.0 mg/kg) in
discrimination required about 13 months
combination with cumulative doses of dizocil-
before responding was stable, accurate and
pine. Pentobarbital doses ranged from low
showed no further improvement.
doses that produced exclusively saline-appro-
During subsequent test sessions, cumulative
priate responding when tested alone up to
doses of pentobarbital or dizocilpine were
doses that exceeded the pentobarbital-training
administered to establish dose–response rela-
dose. Upon completion of these dose–re-
tionships. Test sessions were similar to training
sponse curves, a series of experiments using
sessions, except that they were made up of
cumulative dosing was performed with drugs
several test trials and responding on any of the
other than pentobarbital and dizocilpine.
four keys was reinforced. Each test trial began
Cumulative dosing with these drugs was
with the administration of a test dose of drug.
conducted as previously described for pento-
Following a presession period (10 min; except,
barbital and dizocilpine alone.
15 min for ethanol) to allow time for drug
absorption, a response period ensued during
Data Analysis
which responding on any key was reinforced
under an FR 20 schedule of 4-s access to food.
The percentage of responses on each key
The trial ended immediately after the food
was calculated by dividing the number of
presentation, or after 15 min, whichever oc-
responses on each key by the total number of
curred first; then the next cumulative dose was
responses on all four keys and converting to a
administered and the next test trial began. For
percentage. Response keys are identified by
example, to determine the pentobarbital
the training drugs that were associated with
dose–response curve a 1.0 mg/kg dose of
reinforced responses on the respective keys.
pentobarbital was administered i.m. 10 min
The total number of responses on all keys was
before the first test trial. After 20 responses
divided by the session time to calculate the
had been made on any key the food reinforcer
overall rate of responding during the session
was presented and the trial ended; or if the
(responses/s). Response-rate data are report-
subject failed to complete an FR 20 schedule
ed in the Appendix as mean rates of respond-
within 15 min the trial was ended. The subject
ing in tabular form.
then received a 2.0 mg/kg dose of pentobar-
bital for a cumulative dose of 3.0 mg/kg and a
Drugs
second response period ensued 10 min later.
The drugs used were purchased from
Cumulative dosing trials continued until a
commercial sources or obtained from the

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FOUR-CHOICE DRUG DISCRIMINATION
391
Table 1
Percentage of responses on each key after each training drug (first four rows of data) and the
mean rate of responding across all keys in responses/sec (last row of data) for subjects trained
under the FR 20 schedule.
Training Drug
Saline Key
Pentobarbital Key
Dizocilpine Key
Combination Key
Saline
96.6 (1.0)
2.5 (0.8)
1.2 (0.6)
0.1 (0.1)
Pentobarbital
4.5 (1.1)
92.9 (1.4)
1.0 (0.6)
0.5 (0.4)
Dizocilpine
10.9 (2.1)
0.8 (0.4)
87.5 (2.2)
0.8 (0.6)
Combination
4.0 (1.1)
5.7 (1.5)
3.4 (1.0)
87.1 (1.6)
Responses/ Second
1.35
1.37
0.74
0.26
Note. Values in parentheses are standard errors.
National Institute on Drug Abuse. The drugs
After training doses of pentobarbital and
were pentobarbital sodium, dizocilpine hydro-
dizocilpine, most of the incorrect responses
gen maleate, phencyclidine hydrochloride,
occurred on the saline key. During training
morphine sulfate, dextrorphan tartrate, dex-
sessions following administration of saline or
tromethorphan hydrobromide, diazepam as
the combination of pentobarbital and dizocil-
the commercial preparation for injection, and
pine, incorrect responses were more equally
10% (w/v) ethanol. Doses are expressed in
distributed across two or three keys. Overall
these forms and were administered as i.m.
rates of responding on the saline and pento-
injections 10 min before the training sessions
barbital keys were almost two times higher
or test sessions in a volume of 1.0 ml/kg of
than the rate of responding on the dizocilpine
body weight, except ethanol which was admin-
key, which in turn was about three times
istered orally through a rubber tube 15 min
higher than the rate of responding on the
before the beginning of the session. After drug
drug-combination key.
administration, the pigeons were placed in the
Figure 1 shows the dose–response curves for
darkened test chamber until the session
pentobarbital in individual subjects. Responses
began. When cumulative dosing was used,
were distributed to the saline-appropriate key
subsequent i.m. injections were made into
at the lowest dose of pentobarbital. At inter-
alternating sides of the breast muscle. When
mediate doses of pentobarbital (3 or 5.6 mg/
drug combinations were studied, pentobarbi-
kg) the subjects switched from responding on
tal was given into one side of the breast
the saline key to responding on the pentobar-
muscle, followed by injection of the first of a
bital key; the dose at which they switched keys
series of cumulative doses of dizocilpine into
varied among subjects. Almost no responses
the opposite breast muscle. Subsequent cumu-
were made on the dizocilpine-appropriate key
lative doses alternated injection sites.
or the drug-combination key after any dose of
pentobarbital.
Figure 2 shows the dose-response curves for
RESULTS
dizocilpine in individual subjects. At low doses
Table 1 shows the percentage of responses
of dizocilpine, responding occurred primarily
on each key and rates of responding after
on the saline key. As the dose of dizocilpine
responding stabilized under training condi-
increased, the subjects switched to responding
tions. Across all four training conditions the
on the dizocilpine-appropriate key and made
pigeons made an average of 91% of their
relatively few responses on the pentobarbital
responses on the appropriate key. The per-
key or the drug-combination key. P460 re-
centage of correct responses on the saline key
sponded primarily on the dizocilpine-appro-
after saline administration was slightly greater
priate key after 0.056 mg/kg, while the re-
than the percentage of pentobarbital-appro-
maining 5 subjects discriminated dizocilpine
priate responses after a training dose of
after 0.1 mg/kg.
pentobarbital, which also was slightly higher
Figures 3–6 show the effects of combina-
than the nearly equal percentage of drug-
tions of different doses of pentobarbital with
appropriate responses on the dizocilpine and
cumulative doses of dizocilpine. Figure 3
the drug-combination keys after these drugs.
shows that when 1.0 mg/kg pentobarbital was

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392
D. E. MCMILLAN et al.
Fig. 1.
Discrimination of pentobarbital in individual pigeons trained to discriminate among pentobarbital,
dizocilpine, a combination of these drugs and saline. Filled circles represent responding on the saline key, unfilled
circles represent responding on the pentobarbital key, filled triangles represent responding on the dizocilpine key, and
unfilled triangles represent responding on the drug-combination key. Each point is a single observation in each of
6 pigeons.

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FOUR-CHOICE DRUG DISCRIMINATION
393
Fig. 2.
Discrimination of dizocilpine in individual pigeons trained to discriminate among pentobarbital, dizocilpine,
a combination of these drugs and saline. Filled circles represent responding on the saline key, unfilled circles represent
responding on the pentobarbital key, filled triangles represent responding on the dizocilpine key, and unfilled triangles
represent responding on the drug-combination key. Each point is a single observation in each of 6 pigeons.

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394
D. E. MCMILLAN et al.
Fig. 3.
Discrimination of combinations of 1 mg/kg pentobarbital with increasing doses of dizocilpine in individual
pigeons trained to discriminate among pentobarbital, dizocilpine, a combination of these drugs and saline. Filled circles
represent responding on the saline key, unfilled circles represent responding on the pentobarbital key, filled triangles
represent responding on the dizocilpine key, and unfilled triangles represent responding on the drug-combination key.
Each point is a single observation in each of 6 pigeons.

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FOUR-CHOICE DRUG DISCRIMINATION
395
Fig. 4.
Discrimination of combinations of 3 mg/kg pentobarbital with increasing doses of dizocilpine in individual
pigeons trained to discriminate among pentobarbital, dizocilpine, a combination of these drugs and saline. Filled circles
represent responding on the saline key, unfilled circles represent responding on the pentobarbital key, filled triangles
represent responding on the dizocilpine key, and unfilled triangles represent responding on the drug-combination key.
Each point is a single observation in each of 6 pigeons.

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396
D. E. MCMILLAN et al.
Fig. 5.
Discrimination of combinations of 5.6 mg/kg pentobarbital with increasing doses of dizocilpine in individual
pigeons trained to discriminate among pentobarbital, dizocilpine, a combination of these drugs and saline. Filled circles
represent responding on the saline key, unfilled circles represent responding on the pentobarbital key, filled triangles
represent responding on the dizocilpine key, and unfilled triangles represent responding on the drug-combination key.
Each point is a single observation in each of 6 pigeons.

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