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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
JPET 288:1053–1073, 1999
Vol. 288, No. 3
Printed in U.S.A.
Acquisition of Nicotine Discrimination and Discriminative
Stimulus Effects of Nicotine in Rats Chronically Exposed to
Caffeine1,2
MACIEJ GASIOR3, MOHAMMED SHOAIB4, SEVIL YASAR5, MARIA JASZYNA, and STEVEN R. GOLDBERG
Preclinical Pharmacology Laboratory, National Institute on Drug Abuse, Intramural Research Program, National Institutes of Health,
Baltimore, Maryland
Accepted for publication October 8, 1998
This paper is available online at http://www.jpet.org
Caffeine and nicotine are the main psychoactive ingredients of coffee
and tobacco, with a high frequency of concurrent use in humans. This
study examined the effects of chronic caffeine exposure on 1) rates of
acquisition of a nicotine discrimination (0.1 or 0.4 mg/kg, s.c., training
doses) and 2) the pharmacological characteristics of the established
nicotine discrimination in male Sprague-Dawley rats. Once rats learned
to lever-press reliably under a fixed ratio of 10 schedule for food pellets,
they were randomly divided into two groups; 12 animals were maintained continuously on caffeine added to the drinking water (3 mg/ml)
and another 12 control rats continued to drink tap water. In each group
of water- and caffeine-drinking rats, there were six rats trained to
discriminate 0.1 mg/kg of nicotine from saline and six rats trained to
discriminate 0.4 mg/kg of nicotine from saline. Regardless of the training dose of nicotine, both water- and caffeine-drinking groups required
a comparable number of training sessions to attain reliable stimulus
control, although there was a trend for a slower acquisition in the
caffeine-drinking group trained with 0.1 mg/kg of nicotine. Tests for
generalization to different doses of nicotine revealed no significant
differences in potency of nicotine between water- and caffeine-drinking
groups. The nicotinic-receptor antagonist mecamylamine blocked the
discriminative effects of 0.1 and 0.4 mg/kg nicotine with comparable
potency and efficacy in water- and caffeine-drinking groups. There was
a dose-related generalization to both the 0.1 and 0.4 mg/kg nicotine
cue (maximum average of 51– 83%) in water-drinking rats after i.p.
treatment with d-amphetamine, cocaine, the selective dopamine uptake inhibitor GBR-12909, apomorphine, and the selective dopamine
D1 receptor agonist SKF-82958, but not in caffeine-drinking rats (0 –
22%). There was no generalization to the nicotine cues after i.p. treatment with caffeine or the selective D2 (NPA) and D3 (PD 128,907)
dopamine-receptor agonists in water- and caffeine-drinking rats. The
dopamine-release inhibitor CGS 10746B reduced the discriminative
effects of 0.4 mg/kg nicotine in water-drinking rats, but not in caffeinedrinking rats. There was no evidence of development of tolerance or
sensitization to nicotine’s effects throughout the study. In conclusion,
chronic caffeine exposure (average, 135 mg/kg/day) did not affect the
rate of acquisition of the nicotine discrimination, but it did reduce the
dopaminergic component of the nicotine-discriminative cue. The reduction of the dopaminergic component of the nicotine cue was permanent, as this effect was still evident after the caffeine solution was
replaced with water in caffeine-drinking rats. That nicotine could reliably serve as a discriminative stimulus in the absence of the dopaminergic component of its discriminative cue may differentiate nicotine
from “classical dopaminergic” drugs of abuse such as cocaine and
amphetamine.
Epidemiological surveys in humans show that smokers
tend to smoke more cigarettes while drinking coffee, and they
also drink significantly more coffee than nonsmokers (Istvan
and Matarazzo, 1984; Brown and Benowitz, 1989; Swanson
et al., 1994, 1997). It is now generally accepted that this positive
correlation can be ascribed to interactions between nicotine and
caffeine, the main psychoactive constituents of tobacco and coffee, respectively (Rose and Behm, 1991; Swanson et al., 1994,
1997; Griffiths and Mumford, 1995). Little is known, however,
of the neuropharmacological and psychopharmacological mechanisms or of their behavioral consequences that may contribute
Received for publication July 14, 1998.
1
This work was supported by National Institute on Drug Abuse/National
Institutes of Health, Baltimore MD.
2
Animals used in these studies were maintained in facilities fully accredited by the American Association for the Accreditation of Laboratory Animal
Care and all experimentation was conducted in accordance with the guidelines
of the Institutional Care and Use Committee of the National Institute on Drug
Abuse, National Institutes of Health, and the Guide for Care and Use of Laboratory Animals (National Research Council, 1996, National Academy Press, Washington, DC). Preliminary findings of this study were presented at the Annual
Meeting of the International Behavioral Neuroscience Society, San Diego, CA,
April 24–27, 1997 [Gasior M, Shoaib M, Yasar S and Goldberg SR (1997) Qualitative changes in the discriminative properties of nicotine produced by chronic
caffeine exposure in rats. Abstract of the International Behavioral Neuroscience
Society 6:54] and during the XIIIth Congress of the Polish Pharmacological
Society, Katowice, Poland, September 13–16, 1998 [Gasior M and Goldberg SR
(1998) Influence of chronic caffeine exposure on the behavioral effects of nicotine:
Implications for abuse potential. Pol J Pharmacol 50 (Suppl):61].
3
A Visiting Fellow in the National Institutes of Health Visiting Program of
the Fogarty International Center, Bethesda, MD. Permanent address: Department of Pharmacology, Medical University School, Lublin, Poland.
4
Present address: Institute of Psychiatry, De Crespigny Park, Denmark
Hill, London, United Kingdom.
5
Present address: Division of Gerontology and Geriatric Medicine, Johns
Hopkins University School of Medicine, Baltimore, MD 21224.
ABBREVIATIONS: ACh, acetylcholine; FI, fixed interval; FR, fixed ratio.
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ABSTRACT
1054
Gasior et al.
ule of food reinforcement (Jaszyna et al., 1998). Chronic
caffeine exposure, however, increased rates of acquisition of
i.v. self-administration of both nicotine (Shoaib et al., 1996)
and cocaine (Horger et al., 1991) relative to control rats.
To our knowledge there are no published reports on how
chronic caffeine exposure might change the discriminative
stimulus properties of psychomotor stimulant drugs. There is
some experimental evidence suggesting that acute presession treatment with caffeine can potentiate the discriminative stimulus effects of cocaine in rats (Harland et al., 1989;
Gauvin et al., 1990), and combinations of caffeine with other
over-the-counter drugs such as phenylethylamine can produce new entities distinct from their component elements
and can mimic the discriminative cue of amphetamine and
cocaine (Holloway et al., 1985; Gauvin et al., 1989). Thus,
there are reasons to speculate that chronic caffeine exposure
may change the subjective effects of psychomotor stimulants,
including nicotine.
In the present study, we adopted from Holtzman (1983)
and Jaszyna et al. (1998) a method of chronically exposing
rats to caffeine in their drinking water to examine possible
changes in the subjective effects of nicotine using a drug
discrimination procedure. Drug discrimination procedures
have been successfully used to examine subjective effects of a
wide range of psychoactive drugs under a variety of experimental conditions in both animals (e.g., Colpaert, 1987;
Samele et al., 1992) and humans (e.g., Kamien et al., 1993).
With these procedures, discriminability of a drug (percentage
of subjects acquiring discrimination) and rate of acquisition
of the discrimination are first established and then qualitative properties of the discriminative cue are assessed in generalization tests with other drugs permitting identification of
receptor(s) mediating discriminative-stimulus properties of
the drug (e.g., Colpaert, 1987; Wiley et al., 1996). All three
characteristics of the discriminative effects of nicotine in rats
chronically exposed to caffeine, in comparison to those of in
control rats, were evaluated in the present study.
Materials and Methods
Subjects. Thirty experimentally naive, male Sprague-Dawley
rats, weighing 250 to 280 g at the beginning of the study, were used.
Rats were acclimated to laboratory conditions and allowed to free
feed for 2 weeks. Their body weights were then reduced to about 80%
of free-feeding by limiting access to food. Rats continued to be kept on
a restricted diet to maintain their weights at about 80 6 5.0% of the
weight of age-matched control rats until the end of the study. Rats
were housed individually in stainless steel cages in a temperatureand humidity-controlled room with a 12-h light/dark cycle (7:00 –
19:00 h lights on).
Drugs. The drugs and their sources were as follows: (2)-nicotine
hydrogen tartrate (Sigma Chemical Company, St. Louis, MO), caffeine
base (Sigma), mecamylamine HCl (Research Biochemicals International, RBI, Natick, MA), CGS 10746B (5-(4-methyl-1-piperazinyl)imidazo[2,1-b][1,3,5]-benzothiadiazepine maleate; a gift from Novartis Pharmaceutical Corp., Summit, NJ), d-amphetamine (National
Institute on Drug Abuse, Rockville, MD), cocaine HCl (National
Institute on Drug Abuse), GBR-12909 2zHCl (1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-[3-phenylpropyl]piperazine dichloride; RBI),
R(2)-apomorphine HCL (R(2)-10,11-dihydroxyapomorphine hydrochloride; RBI), (6)-SKF-82958 HBr ((6)-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide;
RBI), R(2)-NPA HCl (R(2)-10,11-dihydroxy-N-n-propylnoraporphine hydrochloride; RBI), S(1)-PD 128,907 HCl (S(1)-(4aR,10bR)-
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to the concurrent use of nicotine and caffeine in humans. Pharmacokinetic factors such as a shorter half-life of caffeine or
nicotine in coffee-drinking smokers or behavioral factors such
as stress or anxiety can only partially explain their concurrent
use (Brown and Benowitz, 1989; Swanson et al., 1994, 1997),
suggesting that other factors are involved (Istvan and Matarazzo, 1984).
Both nicotine and caffeine, when administered alone, can
have qualitatively comparable, often biphasic, dose-dependent effects on a variety of nonoperant and operant measures
of behavior (for review, see Carney et al., 1985; Stolerman,
1990; Nehlig et al., 1992). These behavioral effects include,
for example, increases in locomotor activity (Holtzman, 1983;
Lee et al., 1987; Nikodijevićc et al., 1993) and rates of schedule-controlled responding (White, 1988; Goldberg et al., 1989;
Newland and Brown, 1997; Jaszyna et al., 1998) followed by
decreases as doses of the drugs increase. Moreover, both
nicotine and caffeine can each serve as discriminative stimuli
(Winter, 1981; Stolerman et al., 1984; Carney et al., 1985;
Rosecrans, 1989; Griffiths et al., 1990) and reinforcing stimuli under certain conditions (Goldberg and Henningfield,
1988; Heishman and Henningfield, 1992; Griffiths and Mumford, 1995; Rose and Corrigall, 1997) in laboratory animals
and human subjects. Nicotine and caffeine, when coadministered acutely, have been shown to produce additive-in-nature
stimulation of locomotor activity and increases in rates of
operant responding maintained under a fixed-interval schedule of food reinforcement in rats (Lee et al., 1987; White,
1988) and monkeys (Howell and Landrum, 1997). Acute pretreatment with caffeine also produced significant increases
in rates of responding for i.v. nicotine self-administration in
squirrel monkeys (Yasar et al., 1997). Caffeine, however, is
consumed chronically by humans, and behavioral responses
to caffeine can be altered by repeated administration (see
review by Jacobson et al., 1996), as observed with other
psychomotor stimulant drugs (Goudie and Emmett-Oglesby,
1989; Stewart and Badiani, 1993), suggesting the need for
more studies of the effects of chronic caffeine exposure on
nicotine’s behavioral actions.
It has been well documented that daily exposure to “physiological” doses of caffeine (equivalent to the caffeine content
in two to three cups of coffee) results in the development of
tolerance to the diuretic, cardiovascular, and some, but not
all, of the behavioral effects of caffeine in humans (Benowitz,
1990; James, 1991; Nehlig et al., 1992). Likewise, chronic
caffeine exposure in rodents results in the development of
tolerance to the stimulant effects of caffeine on locomotor
activity and rates of food-reinforced responding, and its effects as a discriminative stimulus (Holtzman and Finn, 1988;
Nikodijevićc et al., 1993; Lau and Falk, 1994; Newland and
Brown, 1997; Jaszyna et al., 1998). There is no clear predictive pattern of change in the behavioral effects of psychomotor-stimulant drugs that are produced by chronic caffeine
exposure in experimental animals. For example, chronic caffeine exposure markedly potentiated the stimulatory effects
of nicotine on locomotor activity (Shoaib et al., 1996),
whereas the response to amphetamine and cocaine remained
unchanged (Holtzman, 1983; Finn and Holtzman, 1987;
Holtzman and Finn, 1988; Nikodijevićc et al., 1993). In contrast, chronic caffeine exposure potentiated the response-rate
increases produced by amphetamine and cocaine, but not by
nicotine, in rats responding under a fixed interval (FI) sched-
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1999
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when it completed eight consecutive sessions in which at least 90% of
responses during the session were on the correct lever and no more
than four responses occurred on the incorrect lever during the first
trial. The number of sessions required to reach this criterion for
stimulus control was calculated for each rat. Test sessions with other
doses of nicotine or other drugs were not started until all rats in the
two groups (caffeine- and water-drinking) trained to discriminate 0.4
mg/kg nicotine from saline met the criteria for stimulus control, to
assure the same duration of caffeine exposure within the group. The
same criterion was applied for the rats trained to discriminate 0.1
mg/kg of nicotine from saline.
Tests for Generalization or Antagonism. Test sessions were
identical to training sessions with the exception that 10 responses on
either lever resulted in delivery of a food pellet. There were no more
than two test sessions conducted per week (usually on Tuesdays and
Fridays) and there were regular training sessions with either nicotine or saline injections conducted on the other days to ensure robust
stimulus control. Only rats that continued to meet the above criteria
for stimulus control were tested. If a rat failed to meet criteria for
stimulus control during one of the training sessions, it remained in
the training condition until at least five consecutive sessions were
completed in which criteria were met.
In generalization tests, rats were injected with different doses of a
drug (including injection with drug vehicle) to determine the degree
to which the drug generalized to nicotine. In antagonism tests, the
ability of a drug to block the discriminative stimulus properties of
nicotine was determined. To do so, rats were injected with a putative
blocking agent in different doses (or its vehicle) first, at a time
appropriate to its onset of action, and then with the training dose of
nicotine (or saline) 10 min before the session. All drugs were given in
doses that ranged from those without behavioral effects to doses
which decreased response rates (the choice of doses was additionally
confirmed by literature searches). Each dose of a respective drug was
tested in a randomized order. After all doses of one drug were tested,
a 1-week washout period was allowed before the next drug was
tested. During this one-week period, rats continued under the training condition for maintenance of stimulus control.
Sequence of Testing. The following parameters, in order of their
assessment, were compared in water- and caffeine-drinking rats: 1)
rates of acquisition of the nicotine discrimination, 2) discrimination
of different doses of nicotine (0.025– 0.8 mg/kg; 10 min before test
session) to establish dose-response functions, 3) ability of the nicotinic-receptor antagonist, mecamylamine (0.01–1.0 mg/kg; 20 min
before test sessions), to block the discriminative stimulus actions of
nicotine, and 4) abilities of a variety of drugs that act at different
receptors to generalize to the nicotine-discriminative stimulus. The
drugs studied (doses and pretreatment times in parentheses) were:
caffeine (1.0 –56 mg/kg; 15 min), d-amphetamine (0.3–3.0 mg/kg; 10
min), cocaine (0.3–17 mg/kg; 10 min), the selective dopamine-uptake
inhibitor GBR-12909 (3.0 –17 mg/kg; 15 min), the nonselective dopamine agonist apomorphine (0.1– 0.3 mg/kg; 10 min), the selective D1,
D2, and D3 dopamine-receptor agonists, SKF-82958 (0.01– 0.17 mg/
kg; 10 min), NPA (0.001– 0.01 mg/kg; 10 min), and PD 128,907
(0.03– 0.3 mg/kg; 15 min), respectively, and 5) ability of the dopamine-release inhibitor, CGS 10746B (3.0 –30 mg/kg; 30 min before test
sessions), to block the discriminative-stimulus actions of nicotine.
Dose-response functions for nicotine were redetermined after the
above tests to assess whether any tolerance or sensitivity to nicotine
had developed over time or as a result of different treatments.
Finally, the ability of amphetamine (0.3–3.0 mg/kg; 10 min) to generalize to the nicotine discriminative stimulus was assessed in rats
subjected to a double crossover design in which caffeine was either
removed or added to the drinking water of caffeine- and waterdrinking rats, respectively.
Measurement of Plasma Caffeine Concentration. A separate
group of six rats was maintained on a restricted diet as above and
was continuously exposed to caffeine (3.0 mg/ml) added to their
drinking water. Daily caffeine intake was monitored once per week.
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3,4,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano-[4,3-b]-1,4oxazin-9-ol hydrochloride; RBI). GBR-12909 was suspended in 40%
(w/v) hydroxypropyl-g-cyclodextrin (RBI), CGS 10746B was dissolved in sterile water, and the remaining drugs were dissolved in
sterile 0.9% NaCl saline. The pH of nicotine solutions was adjusted
to 7.0 with dilute NaOH. When necessary, mild heat and sonication
were applied to prepare solutions of the drugs. Except for nicotine
and caffeine, doses of drugs are expressed as milligrams of salt per
kilogram of body weight measured before the start of the session.
Doses of nicotine and caffeine refer to the free base forms. The drugs
were administered either s.c. (nicotine and mecamylamine) or i.p.
(the remaining drugs) in a volume of 1.0 ml/kg of body weight.
Apparatus. Standard operant chambers (Coulbourn Instruments
Inc., Lehigh Valley, PA) located singly in sound-attenuating plastic
cubicles were used. Each chamber contained two levers separated by
a recessed tray into which a pellet dispenser could deliver 45-mg food
pellets (Bio-Serv, Inc., Frenchtown, NJ), a house light that was
centrally mounted on the front wall below the ceiling, and a device
producing white noise to mask extraneous sounds. Each press of a
lever with a force of 0.4 N through 1 mm was recorded as a response
and was accompanied by an audible click. The chamber was controlled by a computer using MED-PC software (Med Associates, Inc.,
East Fairfield, VT).
Training Procedure. Twenty-four rats were trained to press the
lever for food on a fixed ratio (FR) schedule of reinforcement 5 days
a week (Monday through Friday), always between 9:30 AM and 12:00
PM. At the start of each session, the white house light was turned on
and in its presence 10 consecutive responses on the active lever
delivered a food pellet (a fixed-ratio 10 schedule; FR 10) and initiated
a 3-s timeout during which lever presses had no programmed consequences and the chamber was dark. After each timeout, the house
light was turned on and food was again available. Each session
lasted 15 min. The location of the active lever (left versus right) was
randomly changed for each session to reduce position preferences
during the training period. Once all rats responded reliably under
the FR 10 schedule, they were divided into two groups: water-drinking rats and caffeine-drinking rats. Twelve animals received free
access in their home cages to caffeine in tap water (3 mg/ml caffeine
anhydrate base), whereas the other 12 control rats continued to
drink tap water. Daily caffeine intake was estimated once every
week throughout the remainder of the study based on the rat’s body
weight and 24-h fluid consumption of the established caffeine concentration (mg/kg per individual rat). Daily water intake in waterdrinking rats was monitored for comparison. Training was continued
for 2 weeks to eliminate temporal effects of caffeine on behavior in
rats.
Acquisition of Nicotine Discrimination. Water- and caffeinedrinking rats were then divided into four groups of six rats each and
trained, as described previously (Shoaib and Stolerman, 1996), under the FR10 schedule of food delivery to respond on one lever after
s.c. injection of nicotine and on the other lever after s.c. injection of
an equivalent volume of saline vehicle. Two groups of rats (waterand caffeine-drinking) were trained to discriminate 0.1 mg/kg of
nicotine from saline and two groups of rats (water- and caffeinedrinking) were trained to discriminate 0.4 mg/kg of nicotine from
saline. For half the rats in each group, the right lever was the drug
lever and for the other half, the left lever was the drug lever. This
remained constant throughout the study. Injections of nicotine or
saline were given s.c. 10 min before the session.
During discrimination training, 10 consecutive responses on the
stimulus-appropriate (correct) lever resulted in delivery of a food
pellet. Responses on the stimulus-inappropriate (incorrect) lever
were recorded but had no programmed consequences other than to
reset the FR requirement on the active lever. There were an equal
number of nicotine and saline sessions during each 2-week period of
training, and neither nicotine nor saline sessions prevailed for more
than three consecutive sessions. Discrimination training continued
until an animal was considered to be under stimulus control, that is,
Nicotine Discrimination and Chronic Caffeine
1056
Gasior et al.
Data were considered statistically significant at p ,.05. Two ED50
values were considered statistically different if their 95% confidence
limits did not overlap.
Results
Acquisition of Nicotine Discrimination. All 24 rats
met the criteria for stimulus control (Fig. 1). There were,
however, significant differences among the groups in the
number of training sessions necessary before rats met the
criteria for stimulus control (H35 11.655; p 5 .009). Waterand caffeine-drinking rats trained to discriminate 0.4 mg/kg
nicotine from saline acquired the task within a comparable
number of training sessions (p ..05) ranging from 28 to 43
(mean 6 S.E.M.: 37.2 6 2.3) and from 29 to 56 (mean 6
S.E.M.: 43.0 6 4.3), respectively. A significantly longer period of training was required for rats that were trained with
the lower 0.1 mg/kg dose of nicotine (p ,.05 versus rats
trained with the 0.4 mg/kg nicotine dose). At the 0.1 mg/kg
training dose of nicotine, the number of training sessions
required for water- and caffeine-drinking rats to meet the
criteria of stimulus control ranged from 38 to 94 (mean 6
S.E.M.: 60.3 6 8.0) and from 39 to 115 (mean 6 S.E.M.:
85.5 6 12.0), respectively (Fig. 1). There was a trend for
caffeine-drinking rats to show a slower rate of acquisition of
discriminative-stimulus control with the 0.1 mg/kg training
dose of nicotine (Fig. 1), but this did not reach statistical
significance (p ..05). Caffeine exposure did not effect rates of
responding. At the end of acquisition training, response rates
after 0.4 mg/kg of nicotine or saline in water-drinking rats
(mean 6 S.E.M.: 2.01 6 0.22 or 1.57 6 0.10 responses/s,
respectively) were not significantly different (p . .05) from
those after 0.4 mg/kg of nicotine or saline in caffeine-drinking
rats, (1.89 6 0.28 or 1.48 6 0.09 responses/s, respectively).
Similarly, response rates after 0.1 mg/kg of nicotine or saline
in water-drinking rats (mean 6 S.E.M.: 2.19 6 0.28 or 1.88 6
0.20 responses/s, respectively) were not significantly different (p ..05) from those after 0.1 mg/kg of nicotine or saline in
caffeine-drinking rats, (1.82 6 0.28 or 1.67 6 0.14 responses/s, respectively).
Dose-Response Tests with Nicotine and Blockade of
the Nicotine Cue by Mecamylamine in Water- and Caffeine-Drinking Rats. The percentage of nicotine-appropriate lever-press responses increased as the dose of nicotine
increased in both groups, regardless of the training dose of
nicotine (Fig. 2; Table 1). In both water- and caffeine-drinking rats trained with the 0.4-mg/kg dose of nicotine, the 4-fold
lower dose of nicotine (0.1 mg/kg) engendered nicotine-appropriate responding in all rats, whereas lower 0.05- and 0.025mg/kg doses of nicotine yielded either partial or no generalization. In contrast to the rats trained with 0.4 mg/kg of
nicotine, rats trained with 0.1 mg/kg of nicotine showed only
partial or no generalization when nicotine dose was reduced
2- or 4-fold below the training dose. There were no statistical
differences in the potency of nicotine as a discriminative
stimulus between water- and caffeine-drinking rats regardless of training dose (Tables 1 and 2; Fig. 2). A nicotine dose
of 0.8 mg/kg markedly and significantly (p ,.05) decreased
response rates but 0.4 mg/kg and lower doses of nicotine had
little effect on response rates of water- or caffeine-drinking
rats in rats trained with either 0.4 or 0.1 mg/kg of nicotine
(p ..05). There were no significant differences in effects on
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After 3 weeks, the rats were sacrificed at the time when the nicotinediscrimination study would typically be performed and blood samples were collected into 10-ml sterile tubes containing EDTA as an
anticoagulant. Tubes were centrifuged at 3500 rpm/min for 15 min to
separate plasma from blood cells. Plasma samples were then transferred to transport tubes. Measurements of plasma caffeine concentration were commercially performed at Labstat Incorporated
(Kitchener, Ontario, Canada) according to the HPLC method described in detail by Muir et al. (1980).
Data Presentation and Statistical Analysis. The percentage of
nicotine-appropriate responses during each training or test session
was obtained by dividing the number of responses on the nicotineappropriate lever by the total number of responses on both levers
during a session. The response rate (expressed as responses per
second) during each session was calculated by dividing the total
number of responses on both levers during a session by total session
length (in seconds). Average values (6 S.E.M.) for individual rats in
each of the four groups were calculated. The percentage of nicotineappropriate responses was considered as a measure of discrimination performance. The average response rate provided a second measure of the behavioral effects of treatment, a measure that was
independent of the distribution of responses between the two levers.
Additionally, daily fluid intake in milliliters per kilogram was calculated in water- and caffeine-drinking rats based on measured
individual body weights and intakes of fluid. The daily fluid intakes
of known caffeine concentration were then used to estimate intakes
of caffeine in individual rats (in milligrams per kilogram per day)
throughout the study. Daily caffeine intake and plasma caffeine
concentration were expressed as mean 6 S.E.M.
For the purpose of comparative presentation of experimental data,
dose-response functions for each testing condition were plotted in
paired graphs. The top graphs in Figs. 2 through 9 show absolute
percentages (6 S.E.M.) of nicotine-appropriate lever selections,
whereas the bottom graphs show mean percentage changes (6
S.E.M.) from baseline rates of responding after corresponding treatments in water- and caffeine-drinking rats. For each tested drug, the
mean response rate during treatments with drug vehicle served as
an individual baseline rate of responding (untransformed values
shown in Fig. 10)
An arc-sin transformation was used to normalize distributions of
percentages of nicotine-appropriate lever selections in generalization
and antagonism tests (Shoaib and Stolerman, 1996). Nicotine-appropriate lever selection data were excluded from analysis if a rat
emitted fewer than 10 responses during the test session; the response rate was denoted as zero in such a case and was included for
analysis of changes in rates of responding. Full generalization to the
nicotine cue was considered to exist if the percentage of responses on
the nicotine-appropriate lever was 80% or greater. Partial generalization to the nicotine cue was defined as nicotine-appropriate lever
responding ranging from 20 to 79%. No generalization to the nicotine
cue was considered to exist if nicotine-appropriate responding was
19% or less. Dose-dependent effects on discrimination and changes in
daily caffeine intake were analyzed using one-way repeated-measures ANOVA (within-group comparisons) or two-way repeated measures ANOVA on one repeated factor (between-group comparisons).
Two-way repeated-measures ANOVA on two repeated factors was
used for “before versus after” comparisons of dose-response functions. One-way ANOVA for repeated measures was used to analyze
changes in fluid and caffeine intakes. Post hoc analysis was performed, when appropriate, using Dunnett’s test (multiple comparisons versus control performance within the same group). When possible, doses required to evoke 50% nicotine-appropriate responses or
to decrease response rate by 50% (ED50 values with 95% confidence
limits) were calculated by linear regression analysis over the ascending or descending portion of the log dose-response curve, respectively
(Internal Bioassay software, National Institutes of Health, National
Institute on Drug Abuse, Intramural Research Program). Finally,
when appropriate, Student’s t test for unpaired groups was used.
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1999
Nicotine Discrimination and Chronic Caffeine
1057
response rates of graded doses of nicotine between water- and
caffeine-drinking rats trained with either 0.4 mg/kg or 0.1
mg/kg of nicotine (p ..05; Table 1).
The nicotinic-receptor antagonist mecamylamine dose-dependently blocked the discriminative effects of nicotine in
rats trained with both 0.4 mg/kg and 0.1 mg/kg of nicotine
(Fig. 3; Table 1). A 1.0-mg/kg dose of mecamylamine was
needed to fully block the discriminative effects of 0.4 mg/kg of
nicotine in both water- and caffeine-drinking rats, whereas a
lower dose of 0.3 mg/kg of mecamylamine fully blocked the
discriminative effects of 0.1 mg/kg of nicotine in all of the
water-drinking rats and in five of six of the caffeine-drinking
rats. In both water-and caffeine-drinking rats trained with
the 0.4-mg/kg dose of nicotine, mecamylamine also dosedependently antagonized the increases in rates of responding
produced by 0.4 mg/kg of nicotine (p ,.05 versus vehicle); at
doses of 0.56 and 1.0 mg/kg of mecamylamine, rates of responding reached the baseline levels of responding (p ..05
versus vehicle). This effect of mecamylamine could not be
assessed in rats trained with 0.1 mg/kg of nicotine, because
this dose of nicotine did not produce clear increases in rates
of responding (Fig. 3). When administered alone, 1.0 mg/kg or
0.3 mg/kg of mecamylamine engendered only saline-like responses and had no effect on rates of responding in either
water- or caffeine-drinking rats regardless of nicotine training dose (Fig. 3). There were no significant differences in
either potency or efficacy of the blocking effects of
mecamylamine in water- and caffeine-drinking rats, regardless of the training dose of nicotine (Tables 1 and 2). Likewise, the effects of mecamylamine on rates of responding in
water-drinking rats did not differ from those in caffeinedrinking rats (Table 1).
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 8, 2017
Fig. 1. Acquisition of nicotine discrimination. Open symbols represent water-drinking rats, solid symbols represent caffeinedrinking rats. Circles represent groups of
water- and caffeine-drinking rats trained to
discriminate 0.4 mg/kg of nicotine from saline. Triangles represent groups trained to
discriminate 0.1 mg/kg of nicotine from saline. Each group consisted of six rats. Top,
each symbol represents cumulative percentage of rats that met criteria for stimulus
control (y-axis) over successive training sessions with nicotine or saline (x-axis) in water- and caffeine-drinking groups. Bottom,
each symbol represents the number of sessions necessary for an individual rat to meet
the criteria for stimulus control in waterand caffeine-drinking rats trained to discriminate 0.1 or 0.4 mg/kg of nicotine from
saline. Symbols with error bars represent
mean number of sessions (6S.E.M.) in each
group. See Materials and Methods for the
criteria for stimulus control.
1058
Gasior et al.
Vol. 288
Tests for Generalization to Caffeine, Amphetamine
Cocaine, and GBR-12909. In rats trained to discriminate
0.4 mg/kg of nicotine from saline, caffeine (1.0 –56 mg/kg)
failed to engender nicotine-appropriate responding in waterdrinking rats or in caffeine-drinking rats (Fig. 2; Table 3).
The maximum percentage of nicotine-appropriate responses
was 35.1% (S.E.M., 6 20.5) after 56 mg/kg of caffeine in
water-drinking rats and it did not reach the assigned level of
statistical significance (p ..05 versus vehicle). In waterdrinking rats, caffeine produced dose-dependent and biphasic changes in rates of responding (Table 3). Rates of responding increased after lower doses of caffeine (1.0 and 3.0 mg/kg)
and decreased significantly after higher doses of caffeine (30
and 56 mg/kg). In contrast, in caffeine-drinking rats, there
were no increases in rates of responding after lower doses of
caffeine, but 30 to 56 mg/kg of caffeine did decrease response
rates. There was a statistically significant interaction between two factors (water/caffeine drinking and dose of caffeine; F4,405 2.643, p 5 .048), indicative of a trend in caffeine-drinking rats to show tolerance to the rate increasing
effects of lower doses of caffeine (1.0 and 3.0 mg/kg) (Fig. 2).
Higher doses of caffeine (10 –56 mg/kg) produced dose-dependent decreases in rates of responding with comparable
(p ..05) potency and efficacy in water- and caffeine-drinking
rats (Fig. 2, Tables 3 and 4), indicative of the lack of tolerance
to the rate-decreasing effects of caffeine in rats chronically
exposed to caffeine.
Amphetamine generalized in a dose-dependent manner to
both the 0.4-mg/kg and 0.1-mg/kg nicotine cue in waterdrinking rats (Fig. 4; Table 3). The maximum percentage of
nicotine-appropriate responses was 79.6 (S.E.M., 6 18.1) and
80.0% (S.E.M., 6 18.3) after 1.0 and 1.7 mg/kg doses of
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 8, 2017
Fig. 2. Dose-response functions for the discriminative stimulus effects of nicotine and caffeine. Circles represent water (E)- and caffeine (F)-drinking
rats trained to discriminate 0.4 mg/kg of nicotine from saline. Triangles represent water (ƒ)- and caffeine ()-drinking rats trained to discriminate
0.1 mg/kg of nicotine from saline. Top, mean percentage of nicotine-appropriate responses (6S.E.M.; n 5 5– 6 rats) after injections with increasing
doses of nicotine (s.c.; 10 min before test), caffeine (i.p.; 15 min before test), or control vehicle. Control points (vehicle instead of drug) were included
to show the degree of stimulus control produced by vehicle given under test conditions (these points often overlap). Caffeine was tested only in the
groups of rats trained to discriminate 0.4 mg/kg of nicotine from saline. Horizontal lines at 20% and 80% separate arrays of nicotine-appropriate
responses, considered as no generalization (0 –19%), partial generalization (20 –79%) and full generalization (80 –100%). Doses shown on the abscissa
are in mg/kg, log scale. Bottom, mean percentage of change (6S.E.M.) from the individual baseline rate of responding during vehicle control after
different doses of nicotine or caffeine. The dashed line at 0% denotes no change from vehicle-control response rates. Asterisks represent performance
significantly (p ,.05) different from vehicle (Dunnett’s test after one-way repeated measures ANOVA). See Tables 1 to 4 for the outcome of
between-group comparisons and for ED50 values calculated, where appropriate, from these dose-response functions.
1999
1059
Nicotine Discrimination and Chronic Caffeine
TABLE 1
ANOVA table for the effects of nicotine and mecamylamine in water- and caffeine-drinking rats trained to discriminate either 0.4 mg/kg of
nicotine from saline or 0.1 mg/kg of nicotine from saline
% Generalization
Drug
Training dose: 0.4 mg/kg nicotine
Nicotine
Nicotine (2nd time)
Mecamylamine
1 Nicotine (0.4 mg/kg)
Training dose: 0.1 mg/kg nicotine
Nicotine
Nicotine (2nd time)
Mecamylamine
1 Nicotine (0.1 mg/kg)
Response rate
Water-dr
Caff-dr
Water-dr versus
Caff-dr
Water-dr
Caff-dr
Water-dr versus
Caff-dr
F6,30 5 44.77
p , .001
F6,30 5 11.826
p , .001
F4,20 5 10.63
p , .001
F6,30 5 77.14
p , .001
F6,24 5 20.83
p , .001
F4,16 5 14.44
p , .001
F1,50 5 4.194
p 5 .068
F1,45 5 0.075
p 5 .791
F1,27 5 0.023
p 5 .883
F6,30 5 5.550
p , .001
F6,30 5 29.75
p , .001
F4,20 5 3.287
p 5 .032
F6,30 5 4.978
p 5 .001
F6,24 5 8.248
p , .001
F4,16 5 3.893
p 5 .022
F1,50 5 0.117
p 5 .739
F1,45 5 0.026
p 5 .876
F1,27 5 0.098
p 5 .762
F4,20 5 20.48
p , .001
F5,20 5 38.99
p , .001
F4,16 5 9.095
p , .001
F4,20 5 24.90
p , .001
F5,25 5 19.38
p , .001
F4,20 5 9.950
p , .001
F1,30 5 0.087
p 5 .774
F1,36 5 0.049
p 5 .831
F1,27 5 0.063
p 5 .807
F5,25 5 38.66
p , .001
F5,20 5 16.19
p , .001
F4,16 5 0.545
p 5 .705
F5,25 5 22.53
p , .001
F5,25 5 11.80
p , .001
F4,20 5 0.445
p 5 .774
F1,40 5 3.273
p 5 .101
F1,36 5 3.748
p 5 .085
F1,27 5 0.010
p 5 .922
TABLE 2
ED50 values (with 95% CL) of drugs tested
Training dose: 0.4 mg/kg nicotine
Drug
Nicotine
Nicotine
(2nd time)
Mecamylamine
ED50 (% generalization)
Training dose: 0.1 mg/kg nicotine
ED50 (response rate)
ED50 (% generalization)
ED50 (response rate)
Water-dr
Caff-dr
Water-dr
Caff-dr
Water-dr
Caff-dr
Water-dr
Caff-dr
0.050
(0.039–0.062)
0.085
(0.058–0.126)
0.28
(0.18–0.44)
0.058
(0.049–0.068)
0.088
(0.064–0.121)
0.28
(0.19–0.42)
n.c.
n.c.
n.c.
n.c.
n.c.
0.050
(0.038–0.066)
0.037
(0.020–0.068)
0.073
(0.038–0.138)
n.c.
n.c.
0.049
(0.036–0.067)
0.044
(0.036–0.054)
0.055
(0.029–0.105)
0.34
(0.25–0.46)
0.47
(0.27–0.81)
n.c.
n.c.
n.c.
n.c., not calculated. Data represent ED50 values of drugs in mg/kg (with 95% CL in parentheses) calculated, where appropriate, from dose-response functions shown in
Figs. 2, 3, and 8. Data from dose-response functions were treated quantitatively. Each ED50 value reflects a dose of a drug in mg/kg predicted to produce 50%
nicotine-appropriate responses (% generalization) or reduce rates of responding to 50% of the individual baseline level of responding. In the case of mecamylamine, the ED50
value reflects a dose of mecamylamine predicted to block nicotine-appropriate responses by 50% in rats treated with a training dose of nicotine that produced 100%
nicotine-appropriate responses. For abbreviation details, see the footnote to Table 1.
amphetamine in rats trained to discriminate 0.4-mg/kg and
0.1-mg/kg doses of nicotine, respectively. In contrast to water-drinking rats, caffeine-drinking rats responded only on
the saline-appropriate lever after receiving the same range of
doses of amphetamine, regardless of the training dose of
nicotine (Fig. 4; Table 3). Amphetamine, in a dose-dependent
manner, disrupted responding as indicated by statistically
significant decreases in rates of responding after higher
doses of amphetamine (Fig. 4; Table 3). The 3.0-mg/kg dose of
amphetamine completely suppressed responding in both water- and caffeine-drinking rats. The effects of amphetamine
on rates of responding were both qualitatively and quantitatively comparable in water- and caffeine-drinking rats. This
was confirmed by nonsignificant (p ..05) differences in both
potency and efficacy of amphetamine to suppress responding
in water- and caffeine-drinking rats, regardless of the training dose of nicotine (Tables 3 and 4).
Like amphetamine, cocaine engendered nicotine-appropriate responses in water- but not caffeine-drinking rats (Fig. 5).
There was a clear significant difference (p ,.05) in the percentage of nicotine-appropriate responses between waterand caffeine-drinking rats trained to discriminate the lower
dose of nicotine (Fig. 5). Cocaine dose-dependently increased
the percentage of nicotine-appropriate responses, reaching a
maximum of 83.2% (S.E.M., 6 16.6) nicotine-lever selection
after a 10-mg/kg dose. In caffeine-drinking rats, however,
cocaine failed to generalize to 0.1 mg/kg nicotine (p ..05;
Table 3). In water-drinking rats trained with the higher
0.4-mg/kg dose of nicotine, there was no clear dose-response
relation with cocaine. The maximum percentage of nicotineappropriate responses was 50.7% (S.E.M., 6 22.04; p ,.05)
after a 10-mg/kg dose of cocaine, but with higher 13- and
17-mg/kg doses of cocaine the percentage of nicotine-appropriate responses was only 33.3% (S.E.M., 6 21.1). In contrast, cocaine engendered between 0.0 and 20.7% 6 19.8
nicotine-appropriate responses in caffeine-drinking rats
(p ..05 versus vehicle). However, the percentage of nicotineappropriate responses in water- and caffeine-drinking rats
were not significantly different (p ..05) after treatment with
cocaine (Table 3). Cocaine produced dose-dependent decreases in rates of responding, and statistical comparisons
revealed that cocaine was equipotent (Table 4) and equieffective (Table 3; Fig. 5) in both water-and caffeine-drinking rats,
regardless of the nicotine training dose.
The selective dopamine-uptake inhibitor, GBR-12909, produced dose-dependent nicotine-appropriate responding only
in water-drinking rats, with maximal effects of 83.3%
(S.E.M., 6 16.7) at 10 mg/kg and 13 mg/kg in groups trained
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 8, 2017
Shown are the F values, degrees of freedom, and significance levels of difference (p values) revealed by ANOVA of the dose-response functions (percentage of generalization
to the training dose of nicotine and changes in rates of responding) for each treatment. One-way repeated measures ANOVA was used to ascertain a statistically significant
effect of drug treatment versus vehicle treatment (first, second, fourth, and fifth data columns). Two-way repeated measures ANOVA on one repeated factor was used to
compare dose-response effects of the same treatment in water-drinking (Water-dr) versus caffeine-drinking rats (Caff-dr) (third and sixth data columns). Dose-response
effects of nicotine (s.c., 10 min before test session) were first evaluated after all rats met the criteria for stimulus control in the respective groups and once again at the end
of study. See Figs. 2, 3, and 8 for drugs’ dose-response functions and other details.
1060
Gasior et al.
Vol. 288
to discriminate 0.4 mg/kg of nicotine and 0.1 mg/kg of nicotine from saline, respectively (Fig. 5; Table 3). In contrast,
GBR-12909 produced only saline-appropriate responses in
caffeine-drinking rats, regardless of the training dose of nicotine. GBR-12909 significantly and dose-dependently reduced rates of responding in both water- and caffeine-drinking rats with comparable potency and efficacy, regardless of
the training dose of nicotine (Tables 3 and 4).
Tests for Generalization to Nonselective and Selective Dopaminergic Agents. The nonselective D1/D2 dopamine receptor agonist, apomorphine, significantly and dosedependently generalized to the nicotine cue in water-
drinking groups, with a maximal effect of 66.6% (S.E.M.,
6 21.1) at 0.17 mg/kg and 63.9% (S.E.M., 6 18.0) at 0.3
mg/kg in rats trained to discriminate saline from 0.4 mg/kg
and 0.1 mg/kg of nicotine, respectively (Fig. 6; Tables 4 and
5). Apomorphine, in contrast, failed to generalize to the nicotine cue in caffeine-drinking rats, regardless of the training
dose of nicotine (p ..05 versus vehicle). Apomorphine also
significantly and dose-dependently decreased rates of responding (Fig. 6). The rate-decreasing potency and efficacy of
apomorphine were comparable (p ..05) in water- and caffeine-drinking rats, regardless of the training dose of nicotine
(Tables 4 and 5; Fig. 6).
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 8, 2017
Fig. 3. Results of antagonism tests with mecamylamine and CGS 10746B in water- and caffeine- drinking rats trained to discriminate nicotine from
saline. Circles represent water (E)- and caffeine (F)-drinking rats trained to discriminate 0.4 mg/kg of nicotine from saline. Triangles represent water
(ƒ)- and caffeine ()-drinking rats trained to discriminate 0.1 mg/kg of nicotine from saline. Mecamylamine was tested in water- and caffeine-drinking
rats trained to discriminate 0.1 or 0.4 mg/kg of nicotine from saline. CGS 10746B was tested only in the groups of rats trained to discriminate 0.4 mg/kg
of nicotine from saline. Top, mean percentage of nicotine-appropriate responses (6S.E.M.; n 5 5– 6 rats) after injections with increasing doses of
mecamylamine (s.c., 10 min before nicotine or saline) or CGS 10746B (i.p., 20 min before nicotine or saline). Doses shown on the abscissa are in mg/kg,
log scale. Mecamylamine and CGS 10746B engendered only vehicle-appropriate responses when given before saline instead of nicotine (points labeled
mec-1.0 1 sal, mec-0.3 1 sal, and CGS-30 1 sal on abscissa; these points often overlap). Nicotine, when administered with a vehicle instead of
mecamylamine or CGS 10746B, engendered nicotine-appropriate responses in all rats (points labeled with 0). Bottom, mean percentage of change
(6S.E.M.) from the individual baseline rates of responding after different doses of mecamylamine or CGS 10746B. The individual baseline level of
responding was recorded during a test session with appropriate vehicles administered instead of either nicotine or an antagonist. The dashed line at
0% denotes no change from the individual baseline rate of responding. Asterisks represent performance significantly (p ,.05) different from vehicle
(Dunnett’s test after one-way repeated measures ANOVA). X symbols represent performance after different doses of mecamylamine or CGS 10746B
1 nicotine that was significantly (p ,.05) different from performance after mecamylamine vehicle or CGS 10746B vehicle 1 nicotine.
1999
1061
Nicotine Discrimination and Chronic Caffeine
TABLE 3
ANOVA table for the effects of caffeine, amphetamine, cocaine, GBR-12909, and CGS 10746B in water- and caffeine-drinking rats trained to
discriminate either 0.4 mg/kg of nicotine from saline or 0.1 mg/kg of nicotine from saline
% Generalization
Drug
Water-dr
Caff-dr
Water-dr versus
Caff-dr
Water-dr
Caff-dr
Water-dr versus
Caff-dr
F5,25 5 0.999
p 5 .438
F4,16 5 0.994
p 5 .423
F5,20 5 0.977
p 5 .455
F4,15 5 1.318
p 5 .308
F3,12 5 1.001
p 5 .426
F1,40 5 3.032
p 5 .112
F1,20 5 6.010
p 5 .034
F1,36 5 1.017
p 5 .340
F1,24 5 13.55
p 5 .005
F1,16 5 5.801
p 5 .043
F5,25 5 12.67
p , .001
F5,25 5 11.98
p , .001
F5,25 5 3.869
p 5 .010
F5,25 5 11.19
p , .001
F4,16 5 21.12
p , .001
F5,25 5 26.05
p , .001
F5,25 5 9.792
p , .001
F5,20 5 2.898
p 5 .040
F5,20 5 4.737
p 5 .005
F4,16 5 28.99
p , .001
F1,40 5 0.106
p 5 .752
F1,40 5 0.261
p 5 .621
F1,36 5 0.623
p 5 .450
F1,36 5 0.166
p 5 .693
F1,24 5 3.427
p 5 .101
F4,20 5 1.566
p 5 .222
F5,25 5 1.073
p 5 .396
F3,15 5 1.014
p 5 .414
F1,29 5 10.12
p 5 .010
F1,30 5 11.03
p 5 .008
F1,20 5 6.698
p 5 .027
F4,20 5 8.591
p , .001
F5,25 5 5.181
p 5 .002
F3,15 5 7.056
p 5 .004
F4,20 5 20.26
p , .001
F4,20 5 3.213
p 5 .034
F3,15 5 7.265
p 5 .003
F1,30 5 1.255
p 5 .289
F1,30 5 0.002
p 5 .964
F1,20 5 0.003
p 5 .958
Shown are the F values, degrees of freedom, and significance levels of difference (p values) revealed by ANOVA of the dose-response functions (percentage of generalization
to the training dose of nicotine and changes in rates of responding) for each treatment as listed in the first column. See Figs. 2 to 5 for dose-response functions of the drugs
and the footnote to Table 1 for other details.
TABLE 4
ED50 values (with 95% CL) of drugs tested
Training dose: 0.4 mg/kg nicotine
Drug
Caffeine
Amphetamine
Cocaine
GBR-12909
CGS 10746B
Apomorphine
PD 128,907
SKF-82958
NPA
ED50 (% generalization)
Training dose: 0.1 mg/kg nicotine
ED50 (response rate)
ED50 (% generalization)
ED50 (response rate)
Water-dr
Caff-dr
Water-dr
Caff-dr
Water-dr
Caff-dr
Water-dr
Caff-dr
n.c.
n.c.
n.e.
n.e.
n.e.
n.c.
42.9
(32.1–57.3)
1.00
(0.61–1.45)
12.6
(8.1–19.5)
15.2
(9.87–23.5)
9.43
(6.89–12.9)
0.21
(0.15–0.32)
0.07
(0.05–0.09)
0.07
(0.04–0.12)
0.0039
(1.8–8.5 z 1023)
n.e.
0.51
(0.33–0.79)
n.c.
36.3
(23.5–56.2)
0.82
(0.61–0.92)
17.8
(9.03–35.2)
12.9
(10.1–16.7)
13.8
(10.5–18.2)
n.c.
0.65
(0.31–0.73)
1.50
(0.51–4.42)
7.31
(5.21–10.3)
n.e.
n.c.
2.11
(0.98–4.76)
11.2
(6.98–17.9)
20.7
(8.72–49.3)
n.e.
1.14
(0.82–1.59)
19.9
(4.48–58.3)
14.5
(7.23–30.0)
n.e.
0.11
(0.06–0.21)
0.24
(0.12–0.48)
0.20
(0.09–0.47)
0.0036
(3.1–4.3 z 1023)
0.08
(0.05–0.15)
0.19
(0.12–0.29)
0.09
(0.06–0.14)
0.0045
(2.9–7.2 z 1023)
5.85
(3.68–9.28)
10.1
(2.36–42.6)
0.21
(0.09–0.49)
n.c.
0.05
(0.01–0.20)
n.c.
n.c.
n.c.
n.c.
n.c.
n.c.
n.c.
n.c.
0.08
(0.06–0.10)
0.05
(0.04–0.08)
0.0030
(2.3–4.0 z 1023)
n.c.
n.c.
n.e.
0.17
(0.07–0.41)
n.c.
n.c.
0.03
(0.01–0.08)
n.c.
n.c.
n.c.
n.c.
n.c., not calculated; n.e., not evaluated. Data represent ED50 values of drugs in mg/kg (with 95% CL in parentheses) calculated, where appropriate, from dose-response
functions shown in Figs. 2 to 8. Data from dose-response functions were treated quantitatively. Each ED50 value reflects a dose of a drug in mg/kg predicted to produce 50%
nicotine-appropriate responses (% generalization) or reduce rates of responding to 50% of the individual baseline level of responding. In the case of CGS 10746B, the ED50
value reflects a dose of CGS 10746B predicted to block nicotine-appropriate responses by 50% in rats treated with a training dose of nicotine that produced 100%
nicotine-appropriate responses. See the footnote to Table 1 for details.
The selective D1 receptor agonist, SKF-82958, engendered
nicotine-appropriate responding in a dose-dependent manner
in water-drinking rats, regardless of the training dose of
nicotine. In contrast to water-drinking rats, SKF-82958
failed to generalize to either the 0.4-mg/kg or 0.1-mg/kg nicotine cues in caffeine-drinking rats. SKF-82958, however,
significantly and dose-dependently decreased rates of responding with a comparable potency and efficacy in waterdrinking rats in comparison with caffeine-drinking rats, regardless of the training dose of nicotine (Tables 4 and 5).
Generalization tests with selective D2 (NPA) and D3 (PD
128,907) dopamine-receptor agonists produced only salineappropriate responding in both water- and caffeine-drinking
rats (p ..05 versus vehicle), regardless of the training dose of
nicotine (Figs. 6 and 7). Both compounds however, dosedependently decreased rates of responding. Regardless of the
training dose of nicotine, there were no differences in the
rate-decreasing potency or efficacy of the compounds in water-drinking rats in comparison with those in caffeine-drinking rats (Tables 4 and 5; Figs. 6 and 7).
Antagonism of the Nicotine Cue with the DopamineRelease Inhibitor CGS 10746B. The dopamine-release inhibitor, CGS10746B, when administered 10 min before the
training dose of 0.4 mg/kg of nicotine, dose-dependently, but
not completely, reduced the discriminative effects of nicotine
in water-drinking rats but not in caffeine-drinking rats (Fig.
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 8, 2017
Training dose: 0.4 mg/kg nicotine
Caffeine
F5,25 5 1.924
p 5 .126
Amphetamine
F3,15 5 6.458
p 5 .005
Cocaine
F5,25 5 2.824
p 5 .037
GBR-12909
F4,18 5 4.113
p 5 .014
CGS 10746B
F3,12 5 4.656
p 5 .022
Training dose: 0.1 mg/kg nicotine
Amphetamine
F4,20 5 3.657
p 5 .022
Cocaine
F5,25 5 4.637
p 5 .004
GBR-12909
F3,15 5 8.580
p 5 .001
Response rate
1062
Gasior et al.
Vol. 288
3; Table 4). The most effective doses of CGS10746B appeared
to be 10 and 17 mg/kg (reduction of the percentage of nicotine-appropriate responses from 100% to 38.8% (S.E.M.,
6 21.2) and 47.7% (6 18.3), respectively, p ,.05 versus 0.4
mg/kg of nicotine alone. These doses of CGS 10746B also
significantly decreased the rate of responding. A higher dose
of CGS 10746B (30 mg/kg) almost completely suppressed
responding. In contrast to the different effects of CGS1076B
upon the discriminative effects of nicotine in water- and caffeine-drinking rats, CGS 10746B dose-dependently and with a
comparable (p ..05) efficacy and potency reduced rates of responding in water- and caffeine-drinking rats (Table 4).
Re-evaluation of the Dose-Response Curves of the
Discriminative-Stimulus Effects of Nicotine. Dose-response functions for the discriminative-stimulus effects of
nicotine were re-evaluated in each group after completion of
the above-mentioned tests. In each group, nicotine again
produced dose-dependent increases in the percentage of nicotine-appropriate responding with a comparable (p ..05)
potency in water- and caffeine-drinking rats, regardless of
the training dose of nicotine (Fig. 8; Tables 1 and 2). Likewise, the discriminative-stimulus effects of nicotine did not
change significantly over time (Fig. 8). This was further
confirmed by comparable ED50 values of nicotine (Table 2).
Similarly, the effects of nicotine on rates of responding did
not differ between water-and caffeine-drinking rats, and the
effects of nicotine on rates of responding did not change over
the time (p ..05) (Fig. 8; Table 1 and 2).
Generalization of Amphetamine to the Nicotine Cue
in Rats When the Water- and Caffeine-Drinking Conditions Were Reversed. In rats trained to discriminate 0.4
mg/kg of nicotine from saline, caffeine solution (3 mg/ml) was
substituted for water solution in water-drinking rats and
caffeine solution was replaced with water solution in caffeine-drinking rats. Rats were then allowed 3 weeks to habituate to the new drinking solutions, during which time they
were kept in their home cages with no training. After 3
weeks, the ability of amphetamine (10 min before testing,
i.p.) to generalize to the 0.4-mg/kg nicotine cue was re-evaluated (Fig. 9; Table 6). The water-drinking group of rats, in
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 8, 2017
Fig. 4. Dose-response functions for the
discriminative stimulus generalization
of amphetamine to the nicotine cue in
water- and caffeine-drinking rats. Circles represent water (E)- and caffeine
(F)-drinking rats trained to discriminate
0.4 mg/kg of nicotine from saline. Triangles represent water (ƒ)- and caffeine
()-drinking rats trained to discriminate
0.1 mg/kg of nicotine from saline. Top,
mean percentage of nicotine-appropriate
responses (6S.E.M.; n 5 5– 6 rats) after
injections with increasing doses of amphetamine or control vehicle (i.p.; 10 min
before test). Bottom, mean percentage of
change (6S.E.M.) from the individual
baseline rates of responding after different doses of amphetamine. The individual baseline level of responding was recorded during a test session with an
appropriate vehicle administered instead of amphetamine. The dashed line
at 0% denotes no change from the individual baseline rate of responding. Doses
shown on the abscissa are in mg/kg, log
scale. Asterisks represent performance
significantly (p ,.05) different from vehicle (Dunnett’s test after one-way repeated measures ANOVA). See Fig. 2 for
other details and Tables 3 and 4 for the
outcome of between-group comparisons
and for ED50 values calculated, where
appropriate, from these dose-response
functions.
1999
Nicotine Discrimination and Chronic Caffeine
1063
which amphetamine had previously fully generalized to the
nicotine cue, no longer showed generalization of amphetamine to the nicotine cue after 3 or more weeks of caffeine
exposure. Surprisingly, the caffeine-drinking group of rats, in
which amphetamine had previously failed to generalize to
the nicotine cue, continued to show no generalization to the
nicotine cue with amphetamine after 3 or more weeks maintenance on water.
There continued to be no differences between water- and
caffeine-drinking rats in the effects of amphetamine on rates
of responding after the exchange of solutions. Amphetamine
decreased rates of responding with a comparable (p ..05)
efficacy and potency in both groups (Fig. 9; Table 6). Adding
caffeine to the drinking water did result in a significant
decrease (1.99-fold; p ,.05) in sensitivity to the rate-decreasing effects of amphetamine (Table 6), which was most evident
with doses of 1.0 and 1.7 mg/kg of amphetamine (Fig. 9). In
contrast, sensitivity to the rate-decreasing effect of amphet-
amine did not change as a result of removal of caffeine from
the drinking water in caffeine-drinking rats (Fig. 9; Table 6).
Absolute Values of Rates of Responding during the
Study. Absolute values of rates of responding after administration of the appropriate vehicle under the testing condition were grouped (Fig. 10) and analyzed for changes in the
baseline levels that might result from repeated exposure to
different compounds or chronic caffeine exposure. One-way
repeated measures ANOVA revealed a stable baseline level
of responding throughout the study in water- (F12,58 5 1.649,
p 5 .103) and caffeine- (F12,58 5 0.525, p 5 .888) drinking
rats trained to discriminate 0.4 mg/kg of nicotine from saline
and in water- (F9,45 5 1.008, p 5 .449) and caffeine- (F9,45 5
1.750, p 5 .105) drinking rats trained to discriminate 0.1
mg/kg of nicotine from saline. There were also no significant
differences between water- and caffeine-drinking groups
trained with 0.4 mg/kg (F1,101 5 0.259, p 5 .622) or 0.1 mg/kg
of nicotine (F1,87 5 4.677, p 5 .056) according to two-way
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Fig. 5. Dose-response functions for the stimulus generalization of cocaine and GBR-12909 in water- and caffeine-drinking rats. Circles represent water
(E)- and caffeine (F)-drinking rats trained to discriminate 0.4 mg/kg of nicotine from saline. Triangles represent water (ƒ)- and caffeine ()-drinking
rats trained to discriminate 0.1 mg/kg of nicotine from saline. Top, mean percentage of nicotine-appropriate responses (6S.E.M.; n 5 5– 6 rats) after
injections with increasing doses of cocaine, GBR-12909, or control vehicle (i.p.; 10 min and 15 min before test). Bottom, mean percentage of change
(6S.E.M.) from the individual baseline rates of responding (responses per second) after different doses of the drugs. The individual baseline level of
responding was recorded during a test session with appropriate vehicles administered instead of either cocaine or GBR-12909. The dashed line at 0%
denotes no change from the individual baseline rate of responding. Doses shown on the abscissa are in mg/kg, log scale. Asterisks represent
performance significantly (p ,.05) different from vehicle (Dunnett’s test after one-way repeated measures ANOVA). See Fig. 2 for other details and
Tables 3 and 4 for the outcome of between-group comparisons and for ED50 values calculated, where appropriate, from these dose-response functions.
1064
Gasior et al.
Vol. 288
repeated measures ANOVA. In the groups trained with 0.1
mg/kg of nicotine, however, there was a tendency for waterdrinking rats to show higher levels of baseline responding
than caffeine-drinking rats.
Daily Intake and Plasma Concentration of Caffeine
in Caffeine-Drinking Rats. The bottom plot in Fig. 10
shows the daily intake of caffeine The daily caffeine intake
ranged from 93.9 6 15.2 to 199.2 6 17.1 mg/kg in the group
of rats trained to discriminate 0.4 mg/kg of nicotine from
saline, and from 86.1 6 9.1 to 187.9 6 8.0 mg/kg in the group
of rats trained to discriminate 0.1 mg/kg of nicotine from
saline, with average values from 76 measurements being
138.9 6 24.5 mg/kg/day and 131.0 6 23.3 mg/kg/day of caffeine in these groups. Overall, throughout the study (over 11⁄2
years), daily caffeine intakes in these two groups were indistinguishable during acquisition training and drug testing.
Consumption of caffeinated water, however, remained from
10 to 15% below that of tap water throughout the study (data
not shown). The latter had no effect on food consumption,
body weights, or baseline performance (Fig. 10) of rats
throughout the study.
Relative to the above mentioned groups of caffeine-drinking rats, the rats used for determination of plasma levels of
caffeine showed a comparable daily caffeine intake (ranged
from 131.6 6 8.1 to 167.3 6 18.2 mg/kg/day) that resulted in
a mean plasma caffeine concentration of 29.11 6 6.61 mg/ml.
Discussion
In the present study, chronic caffeine exposure during acquisition of a nicotine discrimination by rats changed the
qualitative nature of the nicotine discrimination without af-
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Fig. 6. Dose-response functions for the stimulus generalization of apomorphine and PD 128,907 in water- and caffeine-drinking rats. Circles represent
water (E)- and caffeine (F)-drinking rats trained to discriminate 0.4 mg/kg of nicotine from saline. Triangles represent water (ƒ)- and caffeine
()-drinking rats trained to discriminate 0.1 mg/kg of nicotine from saline. Top, mean percentage of nicotine-appropriate responses (6S.E.M.; n 5 5– 6
rats) after injections with increasing doses of apomorphine, PD 128,907, or control vehicle (i.p.; 10 min and 15 min before test). Bottom, mean
percentage of change (6S.E.M.) from the individual baseline rates of responding (responses per second) after different doses of apomorphine or PD
128,907. The individual baseline rate of responding was recorded during a test session with appropriate vehicles administered instead of either
apomorphine or PD 128,907. The dashed line at 0% denotes no change from the individual baseline rate of responding. Doses shown on the abscissa
are in mg/kg, log scale. Asterisks represent performance significantly (p ,.05) different from vehicle (Dunnett’s test after one-way repeated measures
ANOVA). See Fig. 2 for other details and Tables 4 and 5 for the outcome of between-group comparisons and for ED50 values calculated, where
appropriate, from these dose-response functions.
1999
Nicotine Discrimination and Chronic Caffeine
1065
TABLE 5
ANOVA table for the effects of apomorphine, PD 128,907, SKF-82958, and NPA in water- and caffeine-drinking rats trained to discriminate either
0.4 mg/kg of nicotine from saline or 0.1 mg/kg of nicotine from saline
% Generalization
Drug
Water-dr
Caff-dr
Water-dr versus
Caff-dr
Water-dr
Caff-dr
Water-dr versus
Caff-dr
F3,12 5 0.995
p 5 .428
F3,12 5 1.625
p 5 .236
F3,12 5 1.000
p 5 .426
F4,15 5 0.776
p 5 .588
F1,18 5 5.481
p 5 .044
F1,18 5 0.905
p 5 .366
F1,18 5 5.279
p 5 .047
F1,25 5 1.382
p 5 .269
F3,15 5 13.72
p , .001
F4,20 5 23.63
p , .001
F4,20 5 15.29
p , .001
F4,20 5 30.89
p , .001
F3,12 5 8.035
p 5 .003
F4,16 5 20.55
p , .001
F4,16 5 11.65
p , .001
F4,16 5 5.970
p 5 .004
F1,18 5 3.951
p 5 .078
F1,27 5 0.581
p 5 .465
F1,27 5 1.174
p 5 .307
F1,27 5 0.331
p 5 .579
F3,15 5 1.437
p 5 .271
F3,15 5 1.893
p 5 .174
F4,17 5 1.858
p 5 .164
F3,12 5 1.007
p 5 .423
F1,20 5 4.736
p 5 .055
F1,20 5 2.706
p 5 .131
F1,27 5 29.18
p , .001
F1,27 5 0.380
p 5 .553
F3,15 5 11.80
p , .001
F3,15 5 7.389
p 5 .003
F4,20 5 5.580
p 5 .003
F4,16 5 22.82
p , .001
F3,15 5 14.653
p , .001
F3,15 5 11.05
p , .001
F4,20 5 13.93
p , .001
F4,20 5 9.353
p , .001
F1,20 5 2.244
p 5 .165
F1,20 5 0.401
p 5 .541
F1,30 5 3.688
p 5 .084
F1,18 5 0.182
p 5 .680
Shown are the F values, degrees of freedom, and significance levels of difference (p values) revealed by ANOVA of the dose-response functions (percentage of generalization
to the training dose of nicotine and changes in rates of responding) for each treatment as listed in the first column. One-way repeated measures ANOVA was used to ascertain
a statistically significant effect of drug treatment versus vehicle treatment (first, second, fourth, and fifth data columns). Two-way repeated measures ANOVA on one factor
was used to compare dose-response effects of the same treatment in water-drinking versus caffeine-drinking rats (third and sixth data columns). See Figs. 6 and 7 and the
footnote to Table 1 for dose-response functions and other details.
fecting the rate at which the discrimination was acquired.
The effect of chronic exposure to caffeine on the acquired
nicotine cue in the present study appeared to be selective to
its dopaminergic component, since a number of dopaminergic
compounds, including amphetamine, cocaine, GBR-12909,
apomorphine, and SKF-82958, failed to generalize to the
nicotine cue in caffeine-drinking rats, whereas there was a
complete or partial generalization with these compounds in
water-drinking rats. However, chronic caffeine exposure did
not appear to change the nicotinic component of the nicotine
cue, as there were no differences between caffeine- and water-drinking rats in the nicotine dose-response functions nor
in the ability of the nicotinic-receptor blocker mecamylamine
to block the nicotine discriminative cue.
Rates of acquisition of nicotine discrimination for both
lower and higher doses of nicotine in the present study with
rats were similar to those reported in the literature (Chance
et al., 1977; Stolerman et al., 1984; Rosecrans, 1989). As with
Shoaib et al. (1997), there was no evidence for the development of either tolerance or sensitization to the discriminative
stimulus effects of nicotine over time as a result of repeated
treatment with nicotine and with various other compounds in
the generalization and antagonism tests (Fig. 8). For example, the effects of graded doses of nicotine on rates of responding at the end of the study were comparable to those at the
beginning of the study. Furthermore, the present findings
from the generalization and antagonism tests generally resembled previous findings. Specifically, nicotine discrimination has been reported to be dose-related, with ED50 values
typically ranging from 0.04 to 0.1 mg/kg depending on the
training dose and conditions (Stolerman, 1988; Rosecrans,
1989; Schechter and Meehan, 1992; Shoaib et al., 1997). In
the present study, the ED50 values of nicotine ranged from
0.044 to 0.088 mg/kg. The noncompetitive nicotinic-receptor
antagonist mecamylamine dose-dependently blocked the discriminative stimulus effects of nicotine in the present study
(Fig. 3) with comparable potency and efficacy to those found
in the earlier studies (Romano et al., 1981; Stolerman et al.,
1983, 1984). The present findings with partial generalization
of amphetamine, cocaine, and the nonselective dopamine receptor agonist apomorphine to the nicotine cue, as well as the
failure of caffeine to generalize to the nicotine cue (Figs. 2, 4,
5, and 6), have also been previously reported (Chance et al.,
1977; Stolerman et al., 1984; Rosecrans, 1989). Finally, the
dopamine-release inhibitor CGS 10746B has previously been
shown to attenuate a nicotine discrimination in rats
(Schechter and Meehan, 1992). In the present study, the
blocking effect of CGS 10746B was also incomplete and its
behavioral effects were characterized by a small separation
between doses attenuating the discriminative-stimulus effect
of nicotine and those producing a marked reduction in rates
of responding.
In a previous study with Long-Evans rats trained to discriminate nicotine from saline, 5.0- and 30-mg/kg doses of
GBR-12909 failed to generalize to the nicotine cue (Corrigall
and Coen, 1994); the higher dose produced complete suppression of responding in four of six rats. In the present study,
however, GBR-12909 produced full generalization in waterdrinking Sprague-Dawley rats to both the 0.1- and 0.4-mg/kg
nicotine cues at doses of 10 and 13 mg/kg (intermediate doses
of GBR-12909 not studied by Corrigall and Coen, 1994) and
partial generalization at 5.6 mg/kg. Higher doses of GBR12909, as with Corrigall and Coen (1994), suppressed responding. Given that there is a symmetrical generalization
between GBR-12909 and cocaine in rats and monkeys (Melia
and Spealman, 1991; Witkin et al., 1991; Spealman, 1993),
likely due to overlapping pharmacological mechanisms of
action (Feldman et al., 1997), and that cocaine partially generalized to the nicotine cue (present study and Stolerman et
al., 1984), partial generalization of GBR-12909 to the nicotine cue would have been expected.
The role of D1 and D2 dopamine receptors in the mediation
of nicotine’s discriminative stimulus effects has been studied
by Stolerman and coworkers. The selective D1 dopamine-
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Training dose: 0.4 mg/kg nicotine
Apomorphine
F3,15 5 5.174
p 5 .012
PD 128,907
F4,20 5 1.297
p 5 .305
SKF-82958
F3,15 5 4.334
p 5 .022
NPA
F4,19 5 1.493
p 5 .244
Training dose: 0.1 mg/kg nicotine
Apomorphine
F3,15 5 3.316
p 5 .049
PD 128,907
F3,15 5 0.746
p 5 .541
SKF-82958
F4,20 5 4.285
p 5 .012
NPA
F3,15 5 2.150
p 5 .137
Response rate
1066
Gasior et al.
Vol. 288
receptor agonist SKF 38393 partially generalized to the nicotine cue (Stolerman and Reavil, 1989). Moreover, the selective D1 dopamine antagonist SCH 23390 significantly
attenuated nicotine discrimination, whereas two neuroleptics with selectivity for D2 dopamine receptors had no effect
(Reavil and Stolerman, 1987). In the present study, the selective D1 dopamine-receptor agonist SKF-82958 generalized
to the nicotine cue in water-drinking rats, whereas the selective D2 dopamine-receptor agonist NPA produced only saline-appropriate responses (Fig. 7), supporting these earlier
findings indicating involvement of D1 but not D2 dopamine
receptors in mediation of the discriminative-stimulus effects
of nicotine in rodents.
It has recently been suggested that D3 dopamine autoreceptors may be involved in the pathogenesis of neuropsychiatric disorders such as schizophrenia and drug addiction, and
the potential clinical use of selective D3 dopamine receptor
agonists is now being explored (Caine and Koob, 1993; Acri et
al., 1995; Lamas et al., 1996; Levant, 1997; Sanger et al.,
1997; Witkin et al., 1998). Stimulation of D3 dopamine autoreceptors by selective compounds results in a dose-dependent
inhibition of dopamine release in vivo and in vitro, and also
has been implicated in blocking the reinforcing effects of
amphetamine and cocaine (for review see Levant, 1997). Of
importance for the present study, the discriminative stimulus effects of D3 dopamine-receptor agonists (e.g., 7-hydroxydipropylaminotetralin hydrobromide, PD 128,907) are similar to those of cocaine and the nonselective dopamine
receptor agonist apomorphine in both rats and monkeys (Acri
et al., 1995; Lamas et al., 1996; Sanger et al., 1997). Thus, the
selective D3 dopamine receptor agonist PD 128,907 was
tested in the present study to verify the extent to which it
was similar to the other dopaminergic compounds that partially generalized to the nicotine cue. PD 128,907 engendered
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Fig. 7. Dose-response functions for the stimulus generalization of SKF-82958 and NPA in water- and caffeine-drinking rats. Circles represent water
(E)- and caffeine (F)-drinking rats trained to discriminate 0.4 mg/kg of nicotine from saline. Triangles represent water (ƒ)- and caffeine ()-drinking
rats trained to discriminate 0.1 mg/kg of nicotine from saline. Top, mean percentage of nicotine-appropriate responses (6S.E.M.; n 5 5– 6 rats) after
injections with increasing doses of SKF-82958, NPA, or control vehicle (i.p.; 10 min before test). Bottom, mean percentage of change (6S.E.M.) from
the individual baseline rate of responding (responses per second) after different doses of SKF-82958 or NPA. The individual baseline rate of responding
was recorded during a test session with appropriate vehicles administered instead of SKF-82958 or NPA. The dashed line at 0% denotes no change
from the individual baseline rate of responding. Doses shown on the abscissa are in mg/kg, log scale. Asterisks represent performance significantly
(p ,.05) different from vehicle (Dunnett’s test after one-way repeated measures ANOVA). See Fig. 2 for other details and Tables 4 and 5 for the
outcome of between-group comparisons and for ED50 values calculated, where appropriate, from these dose-response functions.
1999
Nicotine Discrimination and Chronic Caffeine
1067
only saline-appropriate responses in water-drinking rats up
to doses that markedly suppressed responding. Moreover,
chronic caffeine exposure in caffeine-drinking rats did not
change the discriminative or response rate-decreasing effects
of PD 128,907. Although more studies will be needed to
assess the role of D3 dopamine autoreceptors in mediating
the discriminative stimulus effects of nicotine, the present
study provides the first evidence suggesting their minimal
role and adds to accumulating evidence differentiating the
nicotine cue from those of other psychomotor stimulants such
as amphetamine and cocaine.
It has been observed for a number of pharmacologically
diverse drugs, including nicotine, that the results of generalization tests can vary depending on the training dose of
drug (Stolerman et al., 1984; Mumford and Holtzman, 1991;
Schechter, 1997). Typically, pharmacological specificity in
drug discrimination studies decreases with lower and in-
creases with higher doses of a training drug. In the present
study, a 4-fold difference in the training dose of nicotine was
insufficient to produce marked differences in the pharmacological specificity of the nicotine cue. Patterns of generalization to dopaminergic agents were qualitatively identical for
the lower and higher training doses of nicotine in waterdrinking rats. Likewise, ED50 values calculated from doseresponse functions were statistically comparable (Table 4).
Only in the case of cocaine did different nicotine-training
doses quantitatively affect the outcome. Cocaine appeared
more efficacious in producing nicotine-appropriate responses
in water-drinking rats trained with 0.1 mg/kg of nicotine
relative to water-drinking rats trained with 0.4 mg/kg of
nicotine (83.2% versus 50.7%).
Different training doses of nicotine also had little effect on
its final potency as a discriminative stimulus after acquisition. When dose-response functions of nicotine’s discrimina-
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Fig. 8. Re-evaluation of the dose-response functions for the discriminative-stimulus effects of nicotine in water- and caffeine-drinking rats. Open and
solid symbols represent water- and caffeine-drinking rats, respectively. Circles and triangles represent dose-response curves evaluated at the
beginning of the study (re-plotted from Fig. 2 for comparison); squares represent dose-response curves re-evaluated in the same groups. Top, mean
percentage of nicotine-appropriate responses (6 S.E.M.; n 5 5– 6 rats) after injections with increasing doses of nicotine or saline (s.c.; 10 min before
test). Bottom, mean percentage of change (6S.E.M.) from the individual baseline rate of responding (responses per second) after different doses of
nicotine. The individual baseline rate of responding was recorded during a test session with saline administered instead of nicotine. The dashed line
at 0% denotes no change from the individual baseline rate of responding. Doses shown on the abscissa are in mg/kg, log scale. Asterisks represent
performance significantly (p ,.05) different from vehicle (Dunnett’s test after one-way repeated measures ANOVA). See Fig. 2 for other details and
Tables 1 and 2 for the outcome of between-group comparisons and for ED50 values calculated, where appropriate, from these dose-response functions.
1068
Gasior et al.
Vol. 288
tive stimulus effects were evaluated, immediately after all
rats met the criteria of stimulus control, the ED50 values for
nicotine were similar in water- and caffeine-drinking rats
trained with 0.1 mg/kg relative to 0.4 mg/kg of nicotine (Table 2). However, when dose-response functions were re-evaluated after generalization and antagonism tests were completed, the ED50 values for nicotine were 1.9-fold lower in
water-drinking rats and 2.4-fold lower in caffeine-drinking
rats trained with 0.1 mg/kg of nicotine relative to rats trained
with 0.4 mg/kg of nicotine. Furthermore, the ED50 values for
mecamylamine needed to block the discriminative effects of
the 0.1-mg/kg training dose of nicotine were 5.1-fold lower in
water-drinking rats and 3.8-fold lower in caffeine-drinking
rats relative to the doses needed to block the discriminative
effects of the 0.4 mg/kg training dose of nicotine. As expected
(Stolerman et al., 1984), a 4-fold decrease in the training dose
of nicotine appeared sufficient to significantly (1.6- and 2.0fold; p ,.05) decrease the rate of acquisition of the nicotine
discrimination in both water- and caffeine-drinking rats.
A large body of evidence suggests that nicotine exerts stim-
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Fig. 9. Dose-response functions for the stimulus generalization of amphetamine re-evaluated in water- and caffeine-drinking rats trained to
discriminate 0.4 mg/kg of nicotine from saline. Water and caffeine solutions were exchanged, so that water-drinking rats (E symbols) were exposed
to caffeine solution (f), whereas caffeine-drinking rats (F) were maintained on tap water since then (L). Open (E) and solid (F) symbols representing,
respectively, water- and caffeine-drinking rats are re-plotted from Fig. 4 for comparison. Squares and diamonds represent groups of animals after their
solutions were changed. Each plot shows pairs of dose-response function evaluated in the same subjects (before and after; n 5 5– 6 per data point).
Top, mean percentage of nicotine-appropriate responses (6S.E.M.) after injections with increasing doses of amphetamine or control vehicle (s.c.; 10
min before test). Doses shown on the abscissa are in mg/kg, log scale. Bottom, mean percentage of change (6S.E.M.) from the individual baseline rate
of responding (responses per second) after different doses of amphetamine. The individual rate of responding was recorded during a test session with
vehicle administered instead of amphetamine. The dashed line at 0% denotes no change from the individual baseline rate of responding. Asterisks
represent performance significantly (p ,.05) different from vehicle (Dunnett’s test after one-way repeated measures ANOVA). See Fig. 2 for other
details and Table 6 for the outcome of between-group comparisons and for ED50 values calculated, where appropriate, from these dose-response
functions.
1999
1069
Nicotine Discrimination and Chronic Caffeine
TABLE 6
ANOVA table and ED50 values for the effects of amphetamine evaluated in water- and caffeine-drinking rats
Groups
% Generalization
Amphetamine (training dose: 0.4 mg/kg nicotine)
Water-dr (W)a
F3,15 5
Water-dr now Caff-dr (W3C)
F4,16 5
a
Caff-dr (C)
F4,16 5
Caff-dr now Water-dr (C3W)
F4,16 5
W versus Ca
W3C versus C3W
W versus W3C
C versus C3W
F1,20
F1,20
F1,8
F1,8
6.458;
1.062;
0.994;
0.995;
p
p
p
p
5
5
5
5
.005
.407
.423
.439
5 6.010; p 5 .034
5 0.448; p 5 .522
5 12.96; p 5 .023
5 1.006; p 5 .373
ED50
0.51 (0.33–0.79)
n.c.
n.c.
n.c.
Response Rate
ED50
F5,25
F5,20
F5,25
F5,20
5
5
5
5
11.98;
12.86;
9.792;
7.557;
p
p
p
p
,
,
,
,
.001
.001
.001
.001
F1,40
F1,32
F1,16
F1,16
5
5
5
5
0.261;
0.091;
22.33;
4.457;
p
p
p
p
5
5
5
5
.621
.770
.009
.102
0.82 (0.61–0.92)
1.63 (1.18–2.25)
1.00 (0.61–1.45)
1.37 (1.04–1.88)
Shown are the F values, degrees of freedom, and significance levels of difference (p values) revealed by ANOVA of the dose-response functions (percentage of generalization
to 0.4 mg/kg of nicotine and changes in rates of responding) for amphetamine. W and C represent water- and caffeine-drinking rats, respectively. W3 C represents
water-drinking rats that had tap water replaced with caffeine solution in this study. C3 W represents caffeine-drinking rats that had caffeine solution replaced with tap
water. See caption of Fig. 9 and Results for more details on this study. One-way repeated measures ANOVA was used to ascertain a statistically significant effect of drug
treatment versus vehicle treatment (four upper rows). Two-way repeated measures ANOVA on one repeated factor was used to compare dose-response effects between groups
(fifth and sixth rows). Two-way repeated measures ANOVA on two repeated factors was used to compare dose-response effects within the same group (before versus after;
seventh and eighth rows). Where appropriate, ED50 values (with 95% CL) were calculated from the dose-response functions (Fig. 9). See footnote to Table 1 for additional
details.
a
Data from Table 3.
The failure of dopaminergic compounds to generalize to the
nicotine cue in caffeine-drinking rats in the present study is
unlikely due to an inability of rats chronically exposed to
caffeine to respond to dopaminergic drugs. Similar regimens
of caffeine exposure in rats failed to change amphetamineand cocaine-induced stimulation of ambulatory activity but
potentiated that of nicotine (Holtzman, 1983; Finn and Holtzman, 1987; Shoaib et al., 1996). More recently, we have
shown that a chronic regimen of caffeine exposure, identical
with that in the present study, markedly potentiated the
response-rate increasing effects of amphetamine and cocaine,
but not those of nicotine, in rats responding under a FI
schedule of food reinforcement (Jaszyna et al., 1998). In
contrast, caffeine-drinking rats appeared to be less sensitive
to the response-rate decreasing effect of the selective D1
dopamine receptor agonist SKF-82958 than water-drinking
rats, but sensitivity to the behavioral effects of the selective
D2 dopamine receptor agonist NPA remained unaffected by
chronic caffeine exposure. Chronic caffeine exposure also appears to have no effect on the overall behavior of rats, as
there were no differences in FR or FI rates of responding
between water- and caffeine-drinking rats throughout the
previous study by Jaszyna et al. (1998) and throughout the
present study (Fig. 10). Furthermore, the baseline level of
ambulatory activity in caffeine-drinking rats did not differ
from that of water-drinking rats (Shoaib et al., 1996). Thus,
it can be concluded that chronic caffeine exposure changed
the discriminative stimulus properties of nicotine and these
changes could be due to specific pharmacological effects of
caffeine on the dopaminergic component of nicotine’s discriminative cue.
In the present study, rats were exposed to caffeine for 2
weeks before acquisition training was initiated. Chronic caffeine exposure had little effect on rates at which rats learned
to discriminate nicotine from saline, as the number of sessions required for robust stimulus control was comparable in
water- and caffeine-drinking rats. There was, however, a
trend for acquisition of the 0.1-mg/kg nicotine discrimination
to be retarded in caffeine-drinking rats. It is possible that
chronic caffeine exposure weakened the discriminative stimulus cue of nicotine, and in turn, more training sessions were
needed to establish stimulus control. Given that there were
no changes in the nicotinic component but marked changes in
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ulus control of behavior primarily by activation of nicotinic
acetylcholine (ACh) receptors in the brain (Stolerman, 1990;
Wiley et al., 1996). Thus, drugs that show high affinity for
nicotinic ACh receptors in the central nervous system dosedependently and stereoselectively generalize to a nicotinediscriminative cue with potencies positively correlating with
their binding affinities for nicotinic ACh receptors (Wiley et
al., 1996). The discriminative stimulus effects of nicotine,
however, are not exclusively specific to nicotine-like drugs, as
some dopaminergic compounds have been shown to produce
partial generalization to the nicotine cue. Such a dopaminergic component of the discriminative stimulus properties of
nicotine might be explained by neuroanatomical studies
showing that many nicotinic ACh receptors are located on
presynaptic dopamine-containing neurons in brain regions
potentially involved in mediating the discriminative stimulus effects of nicotine (Koob, 1992; Dani and Heinemann,
1996; Shoaib and Stolerman, 1996; Wonnacott et al., 1996).
Such presynaptic nicotinic ACh receptors, when activated by
nicotine, can lead to release of dopamine into the synaptic
cleft in the striatum and cortex (Nisell et al., 1995; Wonnacott et al., 1996). In the present study, chronic caffeine exposure appeared to eliminate the dopaminergic component of
the discriminative stimulus effects of nicotine. All dopaminergic compounds that generalized to the nicotine cue in
water-drinking rats failed to generalize to the nicotine cue in
caffeine-drinking rats, regardless of their specific pharmacological mechanism of action upon dopaminergic transmission. This effect of chronic caffeine exposure appeared to be
independent of the training dose of nicotine. In contrast, the
primary component governing the discriminative stimulus
properties of nicotine remained unchanged by chronic caffeine exposure, suggesting that caffeine produced changes
downstream from activation of nicotinic ACh receptors. This
speculation is strengthened by the results of antagonism
tests with mecamylamine and CGS 10746B. Mecamylamine
equieffectively blocked the discriminative stimulus effects of
nicotine in both water- and caffeine-drinking rats. In contrast, CGS 10746B partially blocked the discriminative stimulus effects of nicotine in water-drinking rats but not in
caffeine-drinking rats. The behavioral effects of CGS 10746B
on rates of responding however, did not differ between waterand caffeine-drinking rats.
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Gasior et al.
Vol. 288
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Fig. 10. Top and middle plots show untransformed mean values (6S.E.M.) of the baseline rates of responding in water- (open symbols) and caffeine(solid symbols) drinking rats trained to discriminate 0.4 of mg/kg nicotine (top) or 0.1 mg/kg of nicotine (middle) from saline (n 5 5– 6 rats per group)
during all test sessions with the different drugs as indicated in the legend. The average response rate during a session (expressed as responses per
second) in an individual rat was calculated by dividing the total number of responses emitted by a rat on both levers by the total session length in
seconds (900 s). These rates of responding were used as baseline levels of responding to calculate mean percentage of changes from individual level
of responding produced by the respective drugs (Figs. 2–9). Bottom, calculated average intake (6S.E.M., n 5 5– 6 per data point) of caffeine (in
mg/kg/day) during successive weeks in rats trained to discriminate 0.4 mg/kg of nicotine (F) or 0.1 mg/kg of nicotine () from saline. The shaded
horizontal bars represent the number of weeks necessary for all rats from groups trained with either 0.4 mg/kg or 0.1 mg/kg of nicotine to meet the
criteria of reliable stimulus control (see Fig. 1), whereas generalization and antagonism tests were performed during the remaining weeks (see Figs.
2–9).
1999
1071
ported (Holtzman, 1983; Jaszyna et al., 1998). To minimize
these variations, each dose of a respective drug was tested in
a randomized order. Nevertheless, the present regimen of
chronic caffeine exposure had no effect on body weights,
baseline levels of FR responding (Fig. 10), FI responding
(Jaszyna et al., 1998), or on ambulatory activity (Shoaib et
al., 1996).
In the present study, the plasma level of caffeine in rats
drinking water containing 3 mg/ml caffeine and showing an
average 135 mg/kg/day caffeine intake was, on average, 29.11
mg/ml. Such a plasma concentration of caffeine is comparable
to those measured in rats after a single bolus injection of
behaviorally active 20- to 40-mg/kg doses of caffeine (e.g.,
Modrow et al., 1981; Hirsh, 1984; Nehlig et al., 1992; Lau and
Falk, 1994). In humans, an oral dose of 1 mg/kg of caffeine
(equivalent to the caffeine content in one cup of coffee) produces plasma concentrations of 1 to 2 mg/ml, whereas doses of
5 to 8 mg/kg (equivalent to the caffeine intake of a heavy
coffee drinker) would produce plasma concentrations of about
8 to 10 mg/ml (e.g., Benowitz, 1990; James, 1991; Sawynok,
1995). Any direct comparisons of doses and plasma levels of
caffeine in experimental animals relative to humans ought to
be interpreted with extreme caution, because there are large
between-species differences in metabolism and sensitivity to
the stimulatory effects of caffeine (James, 1991). In general,
a dosage correction based on differences in metabolic weight
predicts a 3- to 4-fold reduction of an equivalent caffeine dose
in humans relative to rats (James, 1991). Similarly, and
again very approximate, a dosage correction based on differences in sensitivity to the stimulatory effects of caffeine on
operant behavior predicts a 3- to 16-fold reduction of an
equivalent caffeine dose in nonhuman primates (e.g., Katz
and Goldberg, 1987; Howell and Landrum, 1997) relative to
rats (e.g., Logan et al., 1989; Horger et al., 1991; Jaszyna et
al., 1998).
A pharmacological explanation for the qualitative changes
in the discriminative stimulus properties of nicotine produced by chronic caffeine exposure in the present study can
only be speculative at this point, as there are no neurochemical data directly correlating changes in the discriminative
stimulus effects of nicotine or other psychomotor stimulant
drugs with changes produced by chronic caffeine exposure at
the receptor level. Caffeine acts as a competitive, nonselective A1/A2 adenosine receptor antagonist at “physiological
concentrations” and has been shown to change the amount
and function of central A1 and A2 adenosine receptors after
chronic treatment (for review, see Nehlig et al., 1992; Jacobson et al., 1996). The existence of an antagonistic interaction
between adenosine and dopamine receptors in the brain has
been confirmed in behavioral and biochemical studies (Ferre
et al., 1992). It appears that specific adenosine receptors are
colocalized with specific dopamine receptors (A1 with D1 and
A2 with D2, respectively) at postsynaptic membranes in the
striatum. Functionally, activation of A1 adenosine receptors
results in the inhibition of D1 dopamine receptor-mediated
increases in cAMP levels. Likewise, activation of A2 adenosine receptors inhibits D2 dopamine receptor-mediated decreases in cAMP levels. Therefore, caffeine, by blocking A1
and A2 adenosine receptors, can remove the inhibitory tone
of endogenous adenosine from D1 and D2 dopamine receptors, which in turn can lead to the stimulation of dopaminergic neurotransmission in the brain (Ferre et al., 1992; Fred-
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 8, 2017
the dopaminergic component of the discriminative stimulus
cue of nicotine, as revealed by later studies, it seems reasonable to speculate that chronic caffeine exposure reduced this
dopaminergic component and thus retarded the acquisition
of the 0.1 mg/kg of nicotine discrimination. Because 0.4
mg/kg of nicotine would produce stronger stimulation of nicotinic receptors than 0.1 mg/kg, this could overshadow the
effect of caffeine on the dopaminergic component of the nicotine cue during the acquisition phase. There was also a
trend for caffeine-drinking rats trained with 0.1 mg/kg of
nicotine to show lower response rates than water-drinking
rats trained with the same dose of nicotine (Fig. 10). This
could additionally affect the rate of acquisition of the nicotine
discrimination in this group. Nevertheless, the general lack
of effect of chronic caffeine exposure on rates of acquisition of
the nicotine discrimination were somewhat surprising based
on our previous findings that Sprague-Dawley rats chronically exposed to the same concentrations of caffeine in their
drinking water acquired self-administration of i.v. nicotine
significantly faster and reached higher rates of responding
than did water-drinking control animals (Shoaib et al., 1996).
This apparent difference may be attributed to different brain
regions mediating the discriminative and reinforcing effects
of nicotine (Stolerman and Shoaib, 1991; Nisell et al., 1995;
Shoaib and Stolerman, 1996) and/or to different dose thresholds for caffeine to produce facilitation and retardation of
these effects.
Caffeine appeared to have both acute and long-lasting effects on the discriminative stimulus properties of nicotine. In
contrast to water-drinking rats, amphetamine engendered
only saline-appropriate responses in rats chronically exposed
to caffeine (Fig. 4). When caffeine solution was replaced by
tap water and dose-response functions for amphetamine
were re-evaluated in the same subjects 3 weeks later, amphetamine still failed to generalize to the nicotine cue (Fig.
9). This suggests that chronic caffeine exposure during the
acquisition phase and subsequent testing and training
phases produced long-lasting changes in the discriminative
stimulus properties of nicotine, as no caffeine or its metabolites would be expected 21 days after termination of chronic
caffeine exposure in rodents (Bonati et al., 1984 –1985;
Gasior et al., 1996). On the other hand, although amphetamine was shown to generalize to the nicotine cue in waterdrinking rats (Fig. 4), when tap water was replaced by caffeine solution and amphetamine was re-evaluated 3 weeks
later, amphetamine failed to generalize to the nicotine cue.
This suggests that chronic caffeine exposure can also qualitatively change the properties of an established nicotine cue.
It is important to note that there was no training during this
3-week period to avoid retraining animals under new conditions in terms of water and caffeine exposure.
Chronic oral exposure to caffeine in the drinking water, as
in the present study, has previously been shown to produce
rapid, complete, and insurmountable tolerance to the stimulatory effects of caffeine on behavior in rats (Holtzman, 1983;
Finn and Holtzman, 1987; Newland and Brown, 1997; Jaszyna et al., 1998), and similar tolerance is seen after repeated
daily i.m. injections of caffeine in monkeys (Katz and Goldberg, 1987; Howell and Landrum, 1997). Although caffeine
intake in rats remained stable throughout the present study,
week-to-week variations were considerable (Fig. 10). Similar
degrees of variation in oral caffeine intake have been re-
Nicotine Discrimination and Chronic Caffeine
1072
Gasior et al.
Acknowledgments
We thank Dr. James Goldberg for helpful comments about the
manuscript, Eric Thorndike (Preclinical Pharmacology Laboratory,
National Institute on Drug Abuse) for his programming assistance,
Dr. H. Cooper Eckhardt (Novartis Pharmaceuticals Corporation) for
help with obtaining CGS 10746B, and J. Zavitsky (Labstat Incorporated) for measuring plasma caffeine concentrations.
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