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VoL 270, No. 3
Printed in U.SA
1041
0022-3565/94/2703-1041$00.00/0
Tun Jouani. or Piusicoi.ooy AND Exriamnwr*i. Tiizwsuvics
Copyright 0 1994 by The American Society for Pharmaco1o and Experimental Therapeutics
JPET 270.1041-1045, 1994
Pharmacological Characterization of the Novel Discriminative
Stimulus Effects of a Low Dose of Cocaine’
PHIUP TERRY,2 JEFFREY M. WITKIN and JONATHAN L. KATZ
Psychobiology Section, Netlonailnst!tute on Drug Abuse lntramur& Research Program, NaUOn& Institutes of Health, Baltimore, Maryland
Accepted for publication May 6, 1994
ABSTRACT
Twelve rats were trained to press one lever after cocaine injection
(3 mg/kg i.p.) and another lever after saline injection. Once rats
were rdiab, discriminating cocaine from saline, other drugs were
examined for their efficacies In substituting for cocaine. The
dopamine uptake Inhibitors WiN 35,428 [2-$-carbomethoxy-3-fi-
(4-fluorophenyl)tropane-1 ,5-naphthalene-disultonateJ and GBR
12909 1-[2-s(4-fluorophenymethoxy)ethyl-4-[3-phenylpro-
pyI]pIperaEe dihydrochloilde} fully substituted for cocaine (co-
caine responding >80%), whereas the peripherally active co-
caine methiodide and the 5-hydroxytryptamine uptake inhibitor
fluoxetine did not substitute at all. Pentobarbital also fd to
produce any coc&ne-approprlate responding. Two selective
norepinephrine uptake Inhibitors were tested: tomoxetine fully
substituted for the 3-mg/kg dose of cocaine and nisoxetine
approached full substitution (79.7% cocaine responding). The
direct-acting dopamine D-1 agonists SKF 38393 [(±)-7-bromo-8-
hydroxy-3-methyl-1 -phenyl-2,3,4,5-tetrahydro-1 H-3-benzaze-
pine HCI], SKF 77434 [(±)-7,8-dihydroxy-3-aIlyl-1-phenyl-
2,3,4,5-tetrahydro-1 H-benzazepine HCI] and SKF 75670
[3-methyl-7,8-dihydroxy-1 -phenyl-2,3,4,5-tetrahydro-1 H-3-ben-
zazepine HBr] fully substituted for cocaine, whereas the periph-
erally active dopamine D-1 agonist fenoldopam did not. Of four
dopamine D-2 agonists tested, only quinpirole fully substituted;
the others (N-0434 [(±)-2-(N-propyl-N-phenylethylamlno)-5-hy-
droxytetralin], (-)-NPA [R(-)-propylnorapomorphlne HCIJ and
SDZ 208-91 2 N-[(8-)-2,6dmethyIergelIne-8-yIJ-2,2-dImethyI-
propanamide}) produced very limited partial substitution (cocaine
responding < 32%). The results indicate that the 3mg/kg of
cocaine discdmkative stimulus Is centrally mediated and phar-
macologically specific, but different from that produced by the
more commonly used 10-mg/kg training dose. After training at 3
mg/kg of cocaine, there is an enhanced involvement of dopamine
D-1 receptors In comparison with dopamine D-2 receptors. The
results further suggest that not all dopamine D-2 agonists pro-
duce similar effects at a training dose of 3 mg/kg of cocaine.
Finally, these results suggest an involvement of norepinephrlne
in the discriminative stimulus effects of low doses of cocaine.
Mo8t studies assessing drug substitution in rats trained to
discriminate cocaine from saline have used a training dose of
10 mg/kg of cocaine (e.g., McKenna and Ho, 1980; Barrett and
Appel, 1989; Broadbent et aL, 1989, 1991; Callahan et aL, 1991;
Witkin et aL, 1991; Callahan and Cunningham, 1993; Baker et
aL, 1993;). With this procedure, diverse dopamine indirect
agonists, including uptake inhibitors and releasers, consistently
substitute for cocaine in a dose-dependent manner (e.g., Col-
paert et aL, 1976, 1979; McKenna and Ho, 1980; Broadbent et
Received for publication December 27, 1993.
1Animals used in this study were maintained in facilities accredited by the
American Association for the Accreditation of Laboratory Animal Care, and the
experiments were conducted in accordance with the Guide for Care and Use of
Laboratory Animals provided by the National Institutes of Health and adopted
by the National Institute on Drug Abuse.
IPresent address: School of Psychology, University of Birmingham, Edgbas-
ton, Birmingham B15 2TF U.K.
at., 1991; Witkin et at., 1991; Clime et aL, 1992; Baker et at.,
1993; for review, see Woods et at., 1987). However, direct
dopamine D-1 or D-2 receptor agonists (Kebabian and Caine,
1979; Stoof and Kebabian, 1984) yield less consistent results in
drug substitution tests. Selective D-2 receptor agonists typically
substitute in a dose-dependent manner, although that substi-
tution is not always complete; D-1 receptor-subtype agonists
are either similarly or less efficacious than D-2 agonists (Bar-
rett and Appel, 1989; Callahan et aL, 1991; Witkin et at., 1991;
Callahan and Cunningham, 1993; for review, see Woolverton,
1991; Witkin, 1994). These results are congruent with those
obtained using squirrel monkeys (Spealman et aL, 1991; Katz
and Witkim, 1992), but differ somewhat from those obtained
using rhesus monkeys, in which neither the D-1 agonist SKF
38393 nor the D-2 agonist quinpirole engendered cocaime-ap-
propriate responding (Kieven et aL 1990).
ABBREVIATION5: 5-HT, 5.hydroxytryptamine; FR, fixed-ratio; NE, norepinephrine; N-0434, (±)-2-(N-propyl-N-phenylethylamlno)-5-hydroxytatraNn
HO; SDZ 208-912, N-[(8+2,6-dmethylergolne-8-yI]-2,2dmethylpropanamide; (-)-NPA, R(-)-propyl-norapomorphlne Ha; WIN 35,428, 2-9-carbo-
methoxy-3--(4-fluorophenylflropane 1,5-naphthalene-dlsultonate; GBR 12909, 1-[2-bI4-fluoropheny)methoxyJeth3-phenyIpropyI]pIperazlne
dIHGI; SKF 38393, (±7romo-8-hydroxy4methyl-1-phenyl-2,3,4.5-tetrahydro-1H-3-benzazepIne HI; SKF 77434. (±)-7,8-dydroxy-3-aUy$-1-
phenyl-2,3,4,5-tetrahydro-1 H-benzazepine Ha; SKF 75670, 3-methyl-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1 H-3-benzazeplne H6r CL, confi-
dance limits.
1042 Trry at ii.
The training dose of 10 mg/kg of cocaine in studies using
rats is considerably higher than the minimum effective training
dose. For example, Jarbe (1978) rapidly trained rats to discrim-
inate 4 mg/kg of cocaine from saline and Emmett-Oglesby et
aL (1983) successfully trained rats to discriminate 1.25 mg/kg
ofcocaine from saline. Colpaert and Janssen (1982) established
that the smallest doses producing criterion performance in
individual rats ranged between 0.31 and 2.5 mg/kg (median,
0.66 mg/kg). They suggested that 2.5 mg/kg of cocaine may
yield the greatest pharmacological specificity, because the gra-
dient of the cocaine generalization curve was steepest at this
training dose, and the ratio of training dose to EDo value was
minimal. Thus, it appears clear that training doses well below
the commonly used 10.0 mg/kg of cocaine can be used success-
fully.
Studies using other compounds often demonstrate qualitative
as well as quantitative differences in the drug substitution
proffles of different training doses, e.g., for caffeine (Mumforci
and Holtzman, 1991), diazepam (Tang and Franklin, 1991),
chiordiazepoxide (De Vry and Slangen, 1986), morphine (Shan-
non and Holtzman, 1979; Young et aL, 1992), d-amphetamine
(Stolerman and D’Mello, 1981) and lysergic acid diethylamide
(Greenberg et aL, 1975; White and Appel, 1982). However, to
date there is little information concerning the low-dose discrim-
inative stimulus effects of cocaine. Emmett-Oglesby et aL
(1983) showed that comparatively high doses ofcocaine (10 and
20 mg/kg) fully substitute in rats trained to discriminate 1.25
mg/kg of cocaine, suggesting that the discriminative stimulus
effects are not qualitatively different after low-dose training’,
d-amphetamine also fully substituted. However, Broadbent et
aL (1989) found that although 3.5, 10 and 20 mg/kg of cocaine
all maintained discriminative control, the amphetamine deny-
ative d-MDA only substituted for the lowest training dose. This
result implies that the discriminative stimulus effects of cocaine
can differ with training dose. The significance of low-dose
training for drug substitution was also illustrated by Colpaert
and Janssen (1982), who showed that apomorphine was 3.4
times more potent than cocaine in rats trained to discriminate
10 mg/kg of cocaine from saline, but it was 17.3 times more
potent in a group trained at 2.5 mg/kg of cocaine. On the other
hand, Katz et at. (1992) showed that there was no difference in
potency of cocaethylene in substituting for cocaine at two
different cocaine training doses (3 and 10 mg/kg), albeit no
significant potency difference for cocaine was obtained in that
study at the two training doses.
The aim of the present experiment was to characterize the
pharmacology of a low-dose cocaine discrimination in reference
to that obtained routinely after training at higher doses. Rats
trained to discriminate 3 mg/kg of cocaine from saline were
tested for substitution with a series of drugs from three classes
(indirect dopamine agonists, dopamine D-1 agonists and do-
pamine D-2 agonists). In addition to centrally active indirect
and direct dopamine agonists, the compounds tested included
the 5-HT uptake inhibitor fluoxetine, the NE uptake inhibitors
tomoxetine and nisoxetine, the peripherally active quaternary
analog of cocaine, (-)-cocaine methiodide (Shriven and Long,
1971), the peripherally active D-1 agonist fenoldopam (SKF
82526-J; Hahn et aL, 1982) and the partial D-2 agonist SDZ
208-912 (Coward et aL, 1990). If peripheral activity becomes
more salient to the discriminative stimulus effects of cocaine
at the lower training dose, then cocaine methiodide and fenol-
dopam might be expected to substitute completely. Similarly,
Vol. 270
based on substitutions obtained with partial agonists acting on
other systems (e.g., Shannon and Holtzman, 1979; Young et
aL, 1992), partial D-1 agomsts (e.g., SKF 38393) and D-2
agonists (e.g., SDZ 208-912) might be expected to substitute
fully in subjects trained at a low dose of cocaine.
Methods
Subjects. Twelve male Sprague-Dawley rats (Charles Rivers, Wil-
mington MA), weighing 310 to 385 g, were individually housed with
free access to water under a 12 hr light/dark cycle (lights on 7:00A.M.).
The rats had not been tested previously. All testing was between 2.-00
to 3:00 P.M.. Rats were fed daily 15 g of standard laboratory chow 30
mm after testing. Three rats died toward the end ofthe study, although
this only affected the sample size for substitution tests of WIN 35,428
and niaoxetine see “Results”).
Apparatus. Rats were tested in two-lever operant-conditioning
chambers (BRS/LVE, model RTC-022, Laurel, MD) housed within
light- and sound-attenuatingboxes with white noise present throughout
testing. Ambient illumination was by a lamp in the top center of the
front panel. Levers were set 17 cm apart, with a pair ofred lamps above
the left lever and a pair of white lamps above the right. A force of 0.4
Nthrough 1 mm was required to register a lever press; reinforced
presses activated an audible click and dispensed one 45-mg pellet
(BioServe, Frenchtown NJ) into the centrally located food tray.
Procedure. Rats were trained initially to press both levers under a
FR schedule of food reinforcement. Responding on each lever was
trained separately in random order, with the active lever on a given
training session indicated by illumination of the lamps directly above
it. Rats were then trained to discriminate i.p. injections of cocaine (10
mg/kg) from i.p. injections of saline. After cocaine, responses on only
one lever were reinforced after saline, responses on the other lever
were reinforceci The assignment of cocaine- and saline-appropriate
levers was counterbalanced across rats. Immediately after injection,
rats were placed inside the experimental chambers and a 5-rain timeout
period was initiated, during which all lamps were extinguished and
responding was not reinforcecL All lamps were then illuminated and
responses on the appropriate lever were reinforced. The FR value was
increased to 20 (FR 20) over several training sessions. Responses on
the inappropriate lever reset the FR response requirement on the
appropriate lever. Each food presentation was followed by a 20-sec
timeout period during which all lamps were off, and responding had no
scheduled consequences. Sessions ended after 20 food presentations or
15 rain, whichever occurred first. As the FR value reached 20, training
sessions for which cocaine (C) and saline (S) injections were adminia-
tered were ordered in an SCCS sequence, with test sessions conducted
after consecutive SC or CS training sessions.
In two subsets of six rats, a fading procedure (Overton, 1979) was
then used to establish discriminative performance on a low dose of
cocaine. Six rats (subset 1) were trained for an average of 18 sessions
at the lower dose, and then the dose was reduced further to 1 mg/kg.
Despite extensive training at this dose (average, 18 sessions), perform-
ance never became reliable; therefore the training dose was increased
to 1.7 mg/kg of cocaine. Performance at this dose was consistent after
an average of only 10 sessions, but after testing at several different
doses of cocaine there was a deterioration in discriminative perform-
ance. The training dose was subsequently increased back to 3 mg/kg
of cocaine for the substitution tests. In subset 2, the training dose was
first reduced to 5.6 mg/kg of cocaine (average, 11 sessions) and then to
3 mg/kgofcocaine (average, 18 sessions), 1.7 mg/kgofcocaine (average,
21 sessions) and back to 3 mg/kg of cocaine before drug substitution
testing.
On test sessions, different doses of cocaine or doses of the novel
compounds were substituted for cocaine or saline to produce dose-effect
curves. A test session was run for a given rat if it attained criterion
performance on both of the immediately preceding saline and cocaine
training sessions. The criteria were atleast 85% appropriate responding
overall and during the first FR of the session. Test sessions were
100 -
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of cocaine:
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010 mg/kg
1994 Stimulus Efficts of Cocains 1043
identical to training sessions, with the exception that 20 consecutive
responses on either lever were reinforced.
Drugs. The drugs tested were: (-)-cocaine HC1 (National Institute
on Drug Abuse, Rockville MD); WIN 35,428, GBR 12909, nisoxetine
HC1, (±)-SKF 38393, (±)-SKF 77434, (-)-quinpirole HC1, and R-(-)-
NPA (all Research Biochemicala Inc., Wayland, MA); N-0434 HC1
(Horn et aL, 1984; Nelson Research Laboratories); fenoldopam mono-
methanesulfonate (SKF 82526-J) and SKF 75670 (SmithKline Bee-
cham, King of Prussia, PA); SDZ 208-912 base (Coward et at., 1990;
Sandoz Research Institute, Berne, Switzerland), fluoxetine HC1 and
tomoxetine HC1 (Eli Lilly and Company, indianapOlis IN); (-)-cocaine
methiodide (Shriver and Long, 1971; National Institute on Drug Abuse,
synthesized by Research Triangle institute, Research Triangle Park,
NC); and pentoberbital Na (Abbott Laboratories, N. Chicago IL). All
drugs were dissolved in sterile water with the exceptions of cocaine,
WIN 35,428 and pentobarbital, which were dissolved in 0.9% saline.
The drugs were administered i.p. at 1mi/kg b.wt. All drugs were
injected immediately before testing, with the exceptions of niaoxetine
and tomoxetine (30 rain before test) and fluoxetine (15 rain before
test). These pretest intervals were selected to maximize drug concen-
tration in the brain at the time of testing.
Data analysis. On-line experimental control and data collection
were by aPDP11I73 minicomputer operating SKED software (State
Systems Inc., Kalamazoo MI). For each rat, the overall response rate
and the percentage of responses occurring on the cocaine-appropriate
lever were calculated. The mean values were Calculated for each meas-
*ire at each drug dose tested. Data from any rat which failed to produce
at least 20 responses were not included in the calculation of mean
cocaine-appropriate responding at that dose. Ifless than three rats met
the response rate requirement, no mean value was Calculated for
percentage of cocaine-appropriate responding at that dose. At least
20% cocaine-appropriate responding was adopted as a conservative
criterion at which to assume a significant difference from saline; 80%
or higher cocaine-appropriate responding was taken as similar to the
training dose ofcocaine, and intermediate levels of cocaine-appropriate
responding were considered partial substitution (Witkin et aL, 1991).
Standard analysis of variance and linear regression techniques were
uaedto calculate EDovalues and their 95% CL (Snedecor and Cochran,
1967). A significance level of P <.05 was assumed throughout. Relative
potency estimates were obtained by parallel line bioasssay techniques
(Finney, 1964), the values representing the dose of the standard drug
(milligrams per kilogram) equal to 1 mg/kg of the comparison drug. A
significant difference was assumed if the 95% CL did not include 1.0.
Results
The 3-mg/kg training doses of cocaine reliably maintained
cocaine-appropriate responding (>95%) throughout the period
during which substitution tests were conducted. There was no
apparent difference between the two subsets of rats trained at
3 mg/kg ofcocaine in terms ofthe potency ofcocaine’s discrim-
inative stimulus effects; EDso values for the two subsets were
1.1 mg/kg (95% CL: 0.7-1.6 mg/kg) and 1.3 mg/kg (95% CL,
0.8-2.2 mg/kg) of cocaine. Taking all rats together, dose-re-
spouse functions for cocaine were CalCulated after initial train-
ing at 10 mg/kg of cocaine and after training at 3 mg/kg of
cocaine. At both stages cocaine produced dose-dependent in-
creases in cocaine-lever responses (fig. 1). The ED values at
10 and 3 mg/kg of cocaine were, respectively, 2.6 mg/kg (95%
CL: 2.3-3.0 mg/kg) and 1.2 mg/kg (95% CL: 0.9-1.6 mg/kg).
This corresponds to asignificant change in potency (estimated
relative potency: 2.2; 95% CL: 1.6-3.0). Response rates did not
differ significantly between cocaine and saline training see-
sions.
Two dopamine uptake inhibitors produced a dose-dependent
substitution for the training dose ofcocaine. The cocaine analog
0.3 1 3 10
COCAINE DOSE (mg/kg)
Fig. 1. Effects of cocaine after Initial training at 10 mgftg and later
training at 3 mg/kg (same rats) on the percentages of responses on the
cocaine-correlated lever (top panel) and on rates of responding ex-
-th respect to saline response rates (bottom panel). Each poit
represents performance Wi 12 rats tested at each dose. Bars extend I
S.E.M. above the mean.
WIN 35,428, fully substituted for cocaine with an EDso value
of 0.65 mg/kg, and was approximately 1.8 times more potent
than cocaine (fig. 2; table 1). The selective dopamime uptake
inhibitor, GBR 12909, also fully substituted but was less potent
than cocaine (fig. 2; table 1). In contrast, the cocaine analog,
(-)-cocaine methiodide, showed negligible substitution up to a
dose (56 mg/kg) that decreased response rates to approximately
60% of control (fig. 2). Higher doses were not tested because
this drug is toxic at doses only slightly higher than toxic doses
of (-)-cocaine (cf., Witkin et at., 1993).
Neither the 5-HT uptake inhibitor, fluoxetine, nor the sed-
ative, pentobarbital, produced cocaine-appropriate responding
(fig. 3). Each of these drugs was tested across a range of doses,
from those that were inactive to those that decreased response
rates to less than 20% of control. On the other hand, both NE
uptake inhibitors, tomoxetine and nisoxetine, produced co-
caine-like discriminative stimulus effects (fig. 3). Tomoxetine
fully substituted for cocaine (>80% cocaine-appropriate re-
sponding), whereas the maximal effect of nisoxetine (79.7%
cocaine-appropriate responding) was just below the criterion
for full substitution. The slope of the dose-effect curve for
tomoxetine was significantly different from that of the cocaine
dose-effect curve, preventing a reliable estimate of relative
potency (table 1). There was no significant deviation from
parallelism between the cocaine and nisoxetine dose-effect
curves.
0.1
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AWIN 35,428
.GBR 12909
.Cocaine Methiodide
panel).
Of the four D-2 agonists tested, only quinpirole fully substi-
tuted for cocaine (fig. 5, top panel). The compounds (-)-NPA
and N-0434 produced maximal cocaine-appropriate responding
at levels just above the saline range (26.9 and 32.1% cocaine
responding, respectively), whereas the effects of SDZ 208-912
did not differ from saline. The curves for both (-)-NPA and
N-0434 appeared to reach asymptote, with negligible increases
in cocaine-appropriate responding over the two highest doses
tested. ED50 values and relative potency estimates could only
be CalCUlated foi quinpirole, which proved to be the most potent
ofall the compounds reproducing the stimulus effects of cocaine
(table 1). All of the D-2 agonists dose-dependently reduced
response rates (fig. 5, lower panel). The rank order of potencies
for suppression of responding was: (-)-NPA >quinpirole > N-
0434 SDZ 208-912.
Discussion
Reliable discriminative control was established at a training
dose of 3 mg/kg of cocaine. It may seem surprising that it was
not possible to reduce the training dose further, given the lower
doses achieved by Colpaert and Janssen (1982) and Emmett-
Oglesby et at. (1983). However, it should be noted that, in
1044 ‘r.ny at al Vol. 270
0.1 1 10 100
DRUG DOSE (mg/kg)
Ag. 2. Effects of cocaine, WiN 35,428, GBR 12909 and (-)-cocaine
methkdkie on the percantages of responses on the cocaine-correlated
lever (top panel) and on rates of responding expressed with respect to
saline response rates (bottom panel). Each point represents performance
In at least four(top panel)or six (bottom panel) rats of the six rats tested
at each dose. For cocaine each point represents the average of twelve
rats (top and bottom panels), and for WIN 35,428 each point represents
performance k at least four(top panel) or five (bottom panel) rats of the
five rats tested at each dose.
TABLE 1
EDes valuss (milligrams per kilogram) and relative potsncles for
compounds which fully substituted for the 3.0 mg/kg cocaine
dlecdninedvs smukis
%
ED(95 cL PotencyReistiveto
Coceine
Cocaine I.2 (0.9-1 .6) 1.00
WIN 35,428 0.65 (0.35-1 .20) 1.79(0.98-3.25)
GBR 12909 8.36 (5.99-1 I.67) 0.14(0.09-0.22)
Tomoxetine
Nisoxetine 0.75 (0.31-1 .80) -
0.17(0.08-0.32)
SKF 77434
SKF 38393 0.24 (0.14-0.41)
9.10 (7.84-10.56) 4.27(2.17-8.12)
0.12(0.07-0.18)
SKF 75670 0.84(0.38-1.93)
Quinpirole 0.046 (0.02-0.1 1) 24.61 (8.4-49.62)
aSignfficant devlatlon from p&alstism prevents estimate.
5Nston.
CInsuffident data to catoute.
All three centrally acting D-1 agonists fully substituted for
cocaine (fig. 4, top panel). The rank order of potencies was
SKF 77434 > SKF 75670 > SKY 38393, and all dose-response
functions were parallel to the cocaine curve (table 1). The
peripherally active D-1 agonist fenoldopam showed only partial
substitution, and failed to present a clear dose-response func-
tion. Dose-effect curves for response rates revealed an inverse
1/
0.1 0.3 1 3 10 30
0.1 0.3 1 3 10 30
DRUG DOSE (mg/kg)
Fig. 3. Effects of tomoxetine, nisoxetine, fluoxetine and pentobarbital on
the percentages of responses on the cocaine-correlated Iever(top panel)
and on rates of responding expressed with respect to saline response
rates (bottom panel). Each point represents performance In at least four
(top panel) or six (bottom panel) rats of the six rats tested at each dose
with the following exceptions: 0.1 and 0.3 mg/kg tomoxetine n =5; 10
and 17 mg/kg nisoxetine n -5; 3 and 5.6 mg/kg nisoxetine n =4; 30
mg/kg nisoxetine n -3; 17 mg/kg fluoxetine n =3. Bars extend 1
S.E.M. above the mean.
relationship between test dose and response rate (fig. 4, lower
I
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I.
I.SDZ 208-912
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DRUG DOSE (mg/kg)
Stimulus Effscts of Cocaine 1045
Fig. 4. Effects of the D-1 agonists SKF 77434, SKF 75670, SKF 38393
and ferddopam on the percentages of responses on the cocaine-
correlated lever (top panel) and on rates of respoMng expressed with
respect to saline response rates (bottom panel). Each point represents
performance In at least three(top panel) or six (bottom panel) rats of the
six rats tested at each dose. Bars extend I S.E.M. above the mean.
training rats to discriminate lower doses, both previous studies
eliminated rats from their original samples. This suggests that
to achieve a greater separation between training doses in the
future, the most sensitive subjects could be selected out of a
larger sample for extended training (although this may also
select for other factors which might influence drug substitu-
tion). Even so, Colpaert and Janseen (1982) changed the po-
tency of apomorphine significantly by using a training dose not
dissimilar from that reported here: 2.5 mg/kg of cocaine. The
half log-unit difference between the present training dose of
cocaine and the dose most commonly used also equals or
exceeds the difference in training doses successfully used by
others to identify differences in substitution profiles, albeit
with other classes of compound (e.g., Stolerman and D’Mello,
1981; Shannon and Herling, 1983; Broadbent et aL 1989; Zhang
and Barrett, 1991).
At the reduced training dose, all three D-1 agonists fully
substituted for cocaine, whereas only one of four D-2 agonists
(quinpirole) substituted. In no previous experiment using a
training dose of 10 mg/kg of cocaine, have D-1 agonists fully
substituted or substituted for cocaine more completely than D-
2agonists. Witkin et at. (1991) trained rats in this laboratory
to discriminate 10 mg/kg of cocaine from saline, and tested two
of the three D-1 agonists examined in the present study (SKF
38393 and SKY 75670). In that study, as well as in others with
SKF 38393 (Barrett and Appel, 1989; Callahan et aL, 1991;
Ukai et aL, 1993), the D-1 agonists only substituted in part for
cocaine, in contrast with the full substitution obtained in this
0.001 0.01 0.1 1
DRUG DOSE (mg/kg)
Fig. 5. Effects of the D-2 agonists (-)-NPA, N-0434, quinpirole and SDZ
208-912 on the percentages of responses on the cocaine-correlated
lever (top panel) and on rates of respordng expressed with respect to
saline response rates(bottom panel). Each point represents performance
in at least three (top panel) or aix (bottom pannel) rats of the six rats
tested at each dose. Bars extend 1 S.E.M. above the mean.
study (see table 2 for a comparison of results). Unpublished
data from this laboratory also show partial substitution of the
other D-1 agonist tested in the present study, SKF 77434, in
rats trained to discriminate 10 mg/kg of cocaine (table 2). D-1
agonists also have failed to substitute for the dopamine indirect
agonist d-amphetamine at training doses of 0.5 and 1.0 mg/kg
(Arnt, 1988; Nielsen et aL, 1989; Callahan et aL, 1991; Furmidge
et aL, 1991; van Groll and Appal, 1992). Thus, the present
results are the first to demonstrate, in rats, full substitution of
direct D-1 agomst8 for compounds whose discriminative stini-
ulus effects are mediated by dopamine indirect agonist actions.
That all of the D-1 agonists presently studied fully substi-
tuted for cocaine may seem surprising given the differences
among these compounds with regard to in vitro efficacy at
stimulating adenylyl cyclase (SKY 75670, 24%; SKF 38393,
45%; and SKY 77434, 55% of that for dopamine; Andersen and
Jansen, 1990). In this respect, the present results are in accord-
ance with those suggesting that other behavioral effects and
electrophysiological effects of D-1 agonists show no obvious
relationship with cyclase activity (e.g., Arnt et aL, 1987, 1988;
Johansen et aL, 1991). However, at a relatively low training
dose, as in the present study, partial agonists would be expected
to substitute more fully than at a higher dose of the training
drug in discrimination experiments (e.g., Shannon and Holtz-
man, 1979). Thus, in that respect the present results together
with previous results using a higher training dose (e.g., Witkin
et aL, 1991) suggest a role for efficacy in the actions of D-1
agomsts in the substitution for the discriminative stimulus
1046 Terry St al Vol. 270
TABLE 2
Maximum cocaine-appropdate responding Obtained for substitution tests of D-1 and D-2 agonists at two different training doses of
cocaine: 3 mg/kg (present study) and 10 mg/kg (WitkIn at al., 1991)
Training Dose (mg/kg):
% Coceine Responding Dose Range Tested
3.0 100-’ 3.0 10.0-’
D-1 agonists
SKF 77434
SKF 75670
SKF 38393
Fenoldopam
D-2 agonists
(-)-NPA
N-0434
Quinpirole
SDZ 208-912
100 (0)1
89.9 (7.5)
97.6 (1 .0)
50.1 (20.4)
26.9 (12.8)
32.1 (18.5)
88.3 (9.5)
18.9 (15.5)
54C (20.3)
79.0(12.3)
51 .4 (18.3)
30.2 (14.2)
69.8 (8.0)
67.0 (26.1)
71 .1 (9.6)
19.3’(13.4)
0.1-1 .0
1.0-3.0
5.6-17.0
3.0-17.0
0.001-0.01
0.03-0.3
0.01-1 .0
0.1-1.0
0.13.0c
0.1-3.0
3.0-17.0
1.0-17.0
0.003-0.10
0.01-0.3
0.003-1.0
0.1-3.0#{176}
.
avaiues reanalyzed to incorporate the crerIa adopted in the present study.
CS.E. in parentheses.
0Lhipublished data collected by using methods identical to those reported wi this papar.
effects of cocaine. Alternatively, the present results also may
reflect the actions of these compounds at some other site,
perhaps the dopamine D-5 receptor (Sunahara et at., 1991) or
a subtype of the dopamine D-1 receptor (e.g., Mailman et at.,
1986; Dearry et at., 1990) that may have a greater role in
mediating the effects of lower doses of cocaine.
The poor substitution by three of four D-2 agonists for the
discriminative stimulus effects of cocaine conflicts with many
studies in which rats were trained at 10 mg/kg of cocaine.
However, many of those studies (e.g., Barrett and Appel, 1989;
Callahan et aL 1991; Ukai et aL, 1993) only tested single
compounds, most commonly quinpirole, the only D-2 agonist
to fully substitute here. Broadbent et at. (1991) reported that
the D-2 agonist bromocriptine did not substitute for cocaine
(as did Colpaert et at., 1979), and questioned whether the results
obtained by using quinpirole might be related to the specific
pharmacology of this drug. Since then, however, Callahan and
Cunningham (1993) have reported full substitution of bromo-
criptine for cocaine. Those authors attributed the lack of full
substitution by D-2 agonists in other studies (e.g., Colpaert et
aL, 1979; Broadhent et aL, 1991; Witkin et aL, 1991) to differ-
ences in drug pretreatment times and/or doses. The possibility
that changes in pretreatment times might increase the efficacies
of (-)-NPA, N-0434 and SDZ 208-912 cannot be discounted.
It also might be argued that the failure to obtain greater than
32% cocaine-appropriate responding with (-)-NPA and N-0434
may be related to the maximum doses at which responses were
reliably emitted. However, similar doses were examined by
Witkin et aL (1991) by using comparable procedures. In that
study, cocaine-appropriate responding was always greater than
that obtained in the present study in rats trained with a low
dose of cocaine (table 2). Also at the lower training dose,
cocaine-appropriate responding showed a distinct plateau over
the two highest doses of N-0434 and (-)-NPA tested. At a
training dose of 10 mg/kg of cocaine (Witkin et at., 1991), the
dose-effect function for N-0434 also appeared to reach asymp-
tote below the full-substitution criterion. These results suggest
that no difference in outcome would be obtained if higher doses
could be tested, or if pretreatment times could be varied to
allow for greater effectiveness of the drugs.
In other assays the behavioral effects of (-)-NPA have been
reported to be very similar to those of quinpirole (e.g., Arnt
and Hyttel, 1990; Widzowski and Cory-Slechta, 1993). Simi-
larly, although behavioral data for N-0434 are limited, the drug
clearly presents the characteristics of a potent, selective and
efficacious agonist at the D-2 receptor (Horn et at., 1984; van
der Weide et at., 1986). Nevertheless, there are some character-
istics which differentiate these two drugs from quinpirole. First,
(-)-NPA stimulates adenylyl cyclase in the manner of a D-1
agonist. Secondly, Arnt et at. (1988) reported it to be excep-
tional among D-2 agonists in inducing oral stereotypies; also,
unlike quinpirole, it only substituted in part for the discrimi-
native stimulus effects of d-amphetamine (Arnt, 1988). As
regards N-0434, Witkin et at. (1991) reported that, of six D-2
agonists tested for generalization to the discriminative stimulus
effects of 10 mg/kg of cocaine, only this compound and its
structural analog N-0437 produced significant discriminative
stimulus effects at doses that did not disrupt responding. Quin-
pirole may also be unique in having dopamine releasing prop-
erties within the dose range and temporal parameters adopted
here (Sharp et aL, 1987). A clearer understanding of the differ-
ences between these compounds may await a full characteriza-
tion of the effects of N-0434 and (-)-NPA at dopamine D-3,
D-4 and D-5 receptor subtypes (Sokoloff et at., 1990; van Tol
et at., 1990; Sunahara et at., 1991). Given that quinpirole has a
higher affinity for the D-3 than the D-2 receptor (Sokoloff et
at., 1990), differential selectivities at subtypes of “D-2-like”
receptors may explain the differences obtained here.
The other D-2 agonist tested, SDZ 208-912, is characterized
as a partial agonist, and such drugs have shown a different
pharmacological profile from putative full agonists (e.g., Arnt
and Hyttel, 1990; Coward et at., 1990; Clark et aL, 1991; Svens-
son et aL, 1991). Partial D-2 agonists, including SDZ 208-912,
demonstrate very limited substitution for cocaine or d-amphet-
amine (Arnt and Hyttel, 1990; Exner and Clark, 1992; Callahan
and Cunningham, 1993;). Thus, there is a clear pharmacological
basis for a difference in effect between quinpirole and SDZ
208-912. A reduction in cocaine training dose failed to affect
the substitution of SDZ 208-912 (see table 2).
Overall, the results for D-1 and D-2 agonist substitution
reported here and by Witkin et at. (1991) suggest caution in
concluding that D-2 receptors are more critical to the discrim-
inative stimulus effects of cocaine (Callahan et at., 1991; Cal-
lahan and Cunningham, 1993), at least across a range of train-
ing doses in rats. This is supported by studies in primates (e.g.,
Kieven et at., 1990; Spealman et at., 1991). The present finding
1047
1994
that D-1 agonists can fully mimic the discriminative stimulus
effects of cocaine is also congruent with the ability of D-1
antagonists to attenuate these effects both in rats and monkeys
(Kieven et at., 1988; Barrett and Appel, 1989; Callahan et aL,
1991; Spealman et aL, 1991; Witkin et aL, 1991).
Full substitution of the cocaine analog WIN 35,428 and the
selective dopamine uptake inhibitor GBR 12909 reproduces
results obtained in rats trained at a higher dose of cocaine
(Broadbent et aL, 1991; Witkin et aL, 1991; Clime et at., 1992;
Baker et at., 1993) and also in monkeys (Kleven et at., 1990;
Spealman et aL, 1991). The failure of the peripherally active
compounds, cocaine methiodide and fenoldopam, to substitute
at the lower training dose implies that peripheral cocaine or D-
1 stimuli are no more significant at the low training dose than
at a higher training dose, further highlighting the importance
of central mechanisms in the discriminative stimulus effects of
cocaine (cf., Wood et at., 1986; Witkin et at., 1991). The failure
of fluoxetine and pentobarbital to substitute is in accordance
with results indicating that 5-HT and GABAergic mechanisms
are involved minimally in the discriminative stimulus effects
of cocaine (McKenna and Ho, 1980; Kleven et aL, 1990; Cun-
ningham and Callahan, 1991; Witkin et at., 1991; Baker et aL,
1993), and atest to the pharmacological specificity this lower
cocaine training dose.
Full substitution ofthe NE uptake inhibitors tomoxetine and
nisoxetine was unexpected. Both drugs are highly selective
blockers of NE uptake, with inhibition constants for dopamine
and 5-HT uptake at least two orders of magnitude higher than
for NE uptake (Wong and Bymaster, 1976; Wong et at., 1982).
The doses tested here were in line with those used by others
for selective NE effects in various species (e.g., Snoddy and
Tessel, 1985; Woolverton et aL, 1987; Silverman and Schulz,
1989; Zhang and Barrett, 1991). Studies in rats trained to
discriminate 10 mg/kg of cocaine from saline have shown only
limited substitution by drugs interactingwith central NE mech-
anisms (Colpaert et aL, 1979; Silverman and Schulz, 1989;
Broadbent et aL, 1991; Cunningham and Callahan, 1991; Baker
et at., 1993;). Tomoxetine has been reported not to substitute
in monkeys (Kleven et aL, 1990).
Although studies at the higher training dose in rats have not
indicated a significant role for NE, nisoxetine has been shown
to cross-substitute with d-amphetamine and cocaine in mice
(Snoddy and Tessel, 1983, 1985), and tomoxetine (but not
fluoxetine) fully substituted for cocaine in pigeons (Johanson
and Barrett, 1993). Furthermore, the a4,ha-1 adrenergic antag-
onist prazosin blocked cocaine’s effects in pigeons (Johanson
and Barrett, 1993), and has been reported to reverse the effects
ofcocaine on schedule-controlledbehavior in pigeons and squir-
rel monkeys (Teasel and Barrett, 1986). Thus, there is some
support for the contribution of NE to the discriminative stim-
ulus effects of cocaine in other species.
It is notable that the dose-response function of tomoxetine
differed significantly from parallelism with the cocaine curve,
consistent with its noradrenergic rather than dopaminergic
mechanism of action. Nevertheless, this demonstration of com-
plete substitution of NE uptake inhibitors in rats trained to
discriminate cocaine from saline suggests further that the low-
dose discriminative stimulus effects ofcocaine differ from those
obtained at a higher training dose. The cause of the differences
between training doses cannot be attributed to reduced discrim-
inative control at the lower dose, inasmuch as discriminative
control remained at least as high throughout testing as is
Stimulus Eftects of Cocaine
typically achieved at higher training doses. Nonspecific substi-
tution also is ruled out by the failures of fluoxetine and pento-
barbital, in particular, to substitute even partially for cocaine
(and also by the divergent profiles of the DA D-2 agonists).
In conclusion, training rats to discriminate a low dose of
cocaine from saline can alter the profile of dopamine receptor-
subtype agonist substitution, with D-1 agonists substituting
fully and D-2 agonists showing either full or minimal substi-
tution. The present results, along with those from previous
studies, suggest a potential preferential activity of relatively
high doses of cocaine (10 mg/kg) for the D-2 receptor subtype,
with increased D-1 involvement at lower training doses. The
results also suggest that NE-mediated effects become more
significant as the training dose of cocaine is reduced.
Acknowledgments
The authors are grateful for gifts of compounds from SmithKline Beecham,
Sandoz and Nelson Research Laboratories, and for technical assistance from
Catherine Harlow and Sean Carter.
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