Brain Research Bulletin, Vol. 37, No. 4, pp. 359-362, 1995 Copyright 0 1995 Ekvier Science Ltd Printed in the USA. All rightsreserved 0361-9230/95 $9.50 + .OO
Nicotine-induced Acute Tolerance: Studies Involving Schedule-controlled Behavior JOHN A. ROSECRANS,’ JENNY L. WILEY, CAROLINE E. BASS AND LORI D. KARAN Department of Pharmacology and Toxicology, Division of Substance Abuse Medicine, Virginia Commonwealth University, Richmond, VA 23298 [Received 19 July 1994; Accepted 20 December 19941 ABSTRACT: The major goal of the present study was to examine acute tolerance to nicotine-induced disruption of operant behavior following a single, noncontingent injection. Rats were trained to lever press for food reinforcement under a fixed ratio30 schedule. Once trained, rats were injected with either saline or nicotine (0.8 mg/kg) in their home cages. After either a 90- or 180-min delay, each rat was injected with nicotine (0.4 mg) and placed in the operant chamber for a 30-mln behavioral evaluation session. This experiment was replicated wlth sligM modifications 1 week later. The results of the present study suggest that 0.8 mg/kg of nicotine produces acute tolerance to the response rate decreasing effects of 0.4 mg/kg of nicotine. Decause the tolerance-producing dose of nicotine was injected while rats were not in the test environment, they did not have an opportunity to pracbce the target behavior while under the influence of the drug. Hence, the acute tolerance observed in this study appears to be, at least partly, pharmacological (vs. behavioral) in nature, and may be related to a desenslttxatlon of central nicotinic acetylcholinergic receptors (nAChRs). KEY WORDS: Nicotine, Nicotinic-acetylcholinerg~ receptors (nAChRs), Acute tolerance, Fixed ratio responding, Desensltixatton.
Nicotine, a nicotinic acetylcholinergic receptor (nAChR) agonist, has been shown to induce a rapid and well-defined tolerance to many of its acute behavioral and pharmacological effects [ 1,4,13]. Stolerman et al. [ 14), for example, provided evidence that tolerance to nicotine’s effects on spontaneous activity was rapid and long lasting. Tolerance to the behaviorally disruptive effects of nicotine in operant procedures also occurs rapidly. In a two-lever drug discrimination paradigm, rats trained to discriminate nicotine (0.4 mg/kg, SC) from vehicle became tolerant to the response rate suppression produced by the training dose within 4-5 days (Rosecrans, unpublished observations). Hendry and Rosecrans et al. [lo] have also shown that nicotine-induced (1.2 mg/kg SC) disruption of operant behavior in mice working under a fixed ratio-30 (RR-30) schedule of reinforcement dissipated within 30 days. Interestingly, mice administered nicotine developed tolerance to the disruptive effects of nicotine over the same time period, regardless of whether nicotine was adminis-
tered before or after the mice were tested in the operant chambers; that is, the dose-response curves were shifted equally and significantly to the right in both groups. These results suggested that the observed tolerance to nicotine’s response-rate decreasing effects was pharmacological in nature and was not contingent upon learning or other behavioral factors. This line of research has been continued in the rat with similar results. Villanueva et al.  trained rats to respond for food reward under a variable interval 15 s (VI-15 s) schedule. They reported that rats administered nicotine (0.8 mg/kg, SC) either immediately prior to (pregroup) or 1 h after (postgroup) the operant session developed tolerance to nicotine-induced decreases in responding at equal rates over a 14-28-day period. Thus, nicotine appeared to induce a rapid tolerance that appeared more dependent on pharmacological than behavioral mechanisms. The present investigation was conducted to evaluate whether acute tolerance developed to nicotine-induced disruption of operant behavior following a single noncontingent injection of nicotine. This design was similar to the earlier work of Stolerman et al. [ 141. Similar to the postgroup in chronic nicotine studies, rats in the present study received one dose of nicotine in their home cage either 90 or 180 min before being challenged with a behaviorally disruptive dose of nicotine. The design was similar to that used to demonstrate nicotine-induced acute tolerance to its discriminative stimulus . Thus, the first, or tolerance-inducing dose of nicotine was noncontingently related to the rat’s behavior. METHOD Subjects Thirty male Sprague-Dawley rats (225-250 g), purchased from Harlan Laboratories, were housed singly in stainless steel cages in a temperature- and humidity-controlled animal facility. They were maintained at 85% of their growing body weights throughout the study. Rats received ad lib water in their home cages. Apparatus
Operant sessions were conducted in standard two-lever operant chambers (BRQLVE, Laurel, MD, and Lafayette Instruments Co., Lafayette, IN). During each session, a house light was
’ To whom requests for reprints should be addressed. 359
ROSECRANS ET AL.
illuminated and a pellet dispenser delivered 45 mg Bio Serv (Frenchtown, NJ) food pellets to a food cup located between the two levers. Behavioral contingencies and data collection were programmed using a MED-PC system (MED Associates, Georgia, VT). Drugs
(-)-Nicotine-bitartrate (Supplied by Dr. E. May, V.C.U.) was dissolved in saline. Doses refer to the free base. Procedure
Upon arrival, rats were given a 2-week adaptation period prior to being shaped to lever press for food. Subjects were initially trained to press one of the two levers under a fixed ratio 1 (FR1) schedule of food reinforcement, which was slowly increased to FR-30. The location of the training lever (left vs. right) was counterbalanced across subjects to reduce position preferences. The 30-min sessions were conducted daily Monday-Friday. Rats were fed, but were not trained, on the weekends. After responding on the FR-30 schedule had stabilized (10 sessions), subjects were divided into three experimental groups with approximately equivalent response rates. Each group was evaluated for acute tolerance to the response-disruptive effects of nicotine (0.4 mg/kg, SC). During the test session, three groups of 10 rats each were administered nicotine or saline as follows: Group A. Ten rats each received saline (1 ml/kg, SC) at either 90 (n = 5) or 180 (n = 5) min prior to receiving a dose of nicotine (0.4 mgikg, SC). Rats were immediately placed in the operant chamber following this second injection and a 30-min session was started 2-3 min later. Group B. Ten rats were administered nicotine (0.8 mg/kg, SC) in their home cage 180 min prior to receiving a dose of nicotine (0.4 mg/kg, SC). Rats were immediately placed in the operant chamber following this second injection and a 30-min session was started 2-3 min later. Group C. Ten rats were administered nicotine (0.8 mg/kg, SC) in their home cage 90 min prior to receiving a dose of nicotine (0.4 mg/kg, SC). Again, rats were placed in the operant chamber and, following a 2-3 min delay, a 30-min session began. For each rat, the number of responses was recorded separately in six S-min bins during the 30-min session. One week after the completion of this first study, a second experiment was conducted with these rats. In the second study, the interinjection interval between administration of the 0.8 and 0.4 mg/kg doses of nicotine was reversed for groups B and C. The interinjection interval (90 vs. 180 min) was also reversed for each control rat. Statistical Analysis
Mean response rates (responses/s) were calculated for each group separately in 5-min bins for each test session and were expressed as a percentage of baseline. The baseline was defined as the number of responses at each bin on the day before nicotine dosing. A three-way analysis of variance (ANOVA) was performed with one between-subject factor (group: A, B, or C) and two within-subject factors (bin: l-6 and Experiment: 1 or 2). Newman-Keuls’ tests (a = 0.05) were used for post hoc analysis of significance differences revealed by the overall ANOVA. RESULTS The results of the three-way ANOVA reveal significant main effects for group, F(2, 24) = 5.4, p = 0.01, bin, F(5, 120) = 16.6, p < 0.0001, and experiment, F(1, 24) = 12.7, p =0.002. Because a significant three-way interaction, F(10, 120) = 2.1,
p = 0.03, was also found, post hoc analysis focused on deter-
mining differences between the means of each group in each bin in each experiment. The results of the first experiment are illustrated in Fig. 1; the results of the second experiment are shown in Fig. 2. There were few differences within each group across the two experiments; hence, the results for each experiment are similar and will be described simultaneously. In both experiments, the greatest degree of response rate suppression for all three groups occurred during the first 5 min of testing with the control group (saline + nicotine) exhibiting a significantly larger decrease in percentage of baseline responding than did either of the two groups that had received a noncontingent injection of 0.8 mgikg nicotine. Response rates were significantly increased during later sessions for all three groups in both experiments compared to responding during the first 5 mitt; however, responding in the control group remained significantly lower than in either of the other two groups until 20 (group B in Experiment 1 and groups B and C in Experiment 2) or 25 min (group C in Experiment 1) following the start of the test session. There were no significant differences in responding between the two groups that had received two nicotine injections. DISCUSSION The results of the first experiment conducted in this study show that the administration of nicotine (0.8 mg/kg, SC) 90 or 180 min prior to a challenge dose of nicotine (0.4 mg/kg, SC) produced a significant degree of acute tolerance to the response rate suppression normally induced by this dose of nicotine (Fig. 1). This effect was replicated in a second experiment conducted 1 week later (Fig. 2). Because the tolerance-producing dose of nicotine was injected while rats were in their home cages and not in the test environment, they did not have an opportunity to practice the target behavior while under the influence of the drug in the first study. Thus, tolerance in this situation appeared to be less contingent on behavior and more on some pharmacological mechanism. In contrast, rats did have the opportunity to practice the target behavior during the first exposure in the operant chamber and, thus, a behavioral as well as a pharmacological component of tolerance may have contributed to the results obtained in the replication study (Fig. 2). These results are consistent with previous operant research using a similar methodology in which the postinjection of nicotine also produced tolerance to the effects of a challenge dose of nicotine, which strongly suggests that the mechanisms involved were more pharmacological than behavioral [3,10,15]. This experiment also provided results quite similar to that observed in drug discrimination studies . In these latter studies, a select population (52%) of the rats trained to discriminate nicotine (0.4 mg/kg, SC) exhibited acute tolerance when nicotine (0.8 mg/kg, SC) was administered to them in their home cage 30- 180 min before a discrimination test session with the training dose. In the discrimination study, the ability to detect the training dose of nicotine was antagonized by the previous injection with 0.8 mg/kg of nicotine, i.e., acute tolerance. The time interval for maximal discriminative stimulus attenuation was 90 min, which is analogous to that observed in this experiment. One plausible mechanism that may help explain the acute pharmacological tolerance to nicotine’s behavioral effects in this and the drug discrimination study  has emerged from the work of several investigators who postulate that tolerance to nicotine’s effects may be related to its ability to induce a desensitization of central nAChRs [6-81. Recent research has also indicated that the nicotine-induced acute tolerance effects on brain dopamine release may be subject to a similar desensitization mechanism
NICOTINE AND BEHAVIOR
Percent of Baseline
FIG. I. Effects of nicotine (0.4 mgkg, SC) on fixed ratio (FR-30) responding. Control rats were administered saline (n = 10) 90 or 180 min prior to the administration of nicotine and behavioral evaluation. The other two groups were administered nicotine (0.8 mg!kg, SC) 90 or 180 min prior to the second nicotine dose. Rats were administered tolerance inducing nicotine (0.8 mgkg, SC) doses in their home cages and were placed in the operant chamber within 2 min after receiving the second nicotine dose (0.4 mgkg, SC).
[2,12]. Although the exact mechanism(s) of acute in vivo desensitization (acute tolerance) has yet to be completely elucidated, we can propose a multistage model that can provide at least one explanation of nicotine-induced acute tolerance in these behavioral paradigms. In the first stage of this model, nicotine is viewed as eliciting its effects on behavior by an activation of a channellinked nAChR. In this process the receptor opens an ion channel allowing the entrance of one or more cations (Na+ and/or Ca”) into select pre- and postsynaptic neuron sites. The second proposed stage involves a change in the affinity state of the nAChR (to a higher state) leading to a desensitization of the receptor causing cation channel closure. This would prevent additional nicotine-induced activation of the nAChR (acute tolerance). The last stage of this model appears to involve a restoration of the nAChR to its initial activated state. Drug discrimination research indicates that the desensitization period averages about 40-50 min . These latter studies also indicate that the level and duration of desensitization is variable between subjects, suggesting specific differences in nAChR function. Nicotine, therefore., appears to have the potential to act as a receptor antagonist at its nAChR in addition to its agonist properties. The length and interval between doses necessary to induce desensitization and acute tolerance may also be related to the half-life of nicotine levels at the nAChR. Previous work conducted in the male rat indicates that the nicotine brain area halflife was about 45-60 min following a dose of 0.4 mg/kg, SC . Extrapolating to this experiment is difficult, but we can speculate
that there is probably a measurable nicotine level at least at the 90-min interval following a dose of 0.8 mg/kg, SC; a minimal level would be. expected at the 180 min. Thus, whether nicotine needs to be present to induce a desensitization of the receptor is difficult to say. The major point, however, is the fact that regardless of whether nicotine is present or not at the receptor, tolerance rather than a potentiated effect was observed, which is best explained by a desensitization of the nAChR. To sum, this research appears to present us with a unique depiction as to how nicotine is producing its behavioral effects, which appears to involve the activation and/or desensitization of specific brain nAChRs. In relation to the tobacco user, the ability of nicotine to induce an activation and/or a desensitization of specific nAChRs may be especially important [l 11. The rate of smoking, for example, may be best explained on the basis of the ability of brain nAChRs to respond to a specific nicotine level. Thus, the duration of nAChR desensitization may be a major biological factor that determines the rate of smoking. In addition, the paradoxical effects of smoking (nicotine can either stimulate or depress behavior and/or arousal levels), may also be partially explainable on the basis of this unique desensitization process, that is, whether nicotine is activating or desensitizing a specific neuronal system. Thus, contingent upon dose (or rate of use) of nicotine obtained via tobacco, a smoker or tobacco user may be able to regulate their behavior and/or arousal levels within a narrow margin. Lastly, whether a subject is able to
ROSECRANS ET AL.
Percent of Baseline
FIG. 2. Effects of a second nicotine dose (0.4 mg/kg, SC) on fixed ratio (FR-30) schedule of responding; this exoetiment was conducted 7 davs after the first experiment. Groups were treated similarly as in Fig. 1. The same rat groups were evaluated a second time.
exhibit nicotine-induced desensitization (acute tolerance) or not may be central to whether an individual takes up the tobacco habit [ill. ACKNOWLEDGEMENTS J.L.W. was supported by a USPHS Training Grant, DA-07027. This research was supported by a grant to Dr. Lori Karan, USPHS, X2ODAOO183-02.
REFERENCES 1. Dong. L.; Houdi, A. A.; Van Loom, G. R. Desensitization of central nicotinic isomers and a quatemary analogue. Pharmacol. Biochem. Behav. 38843-852; 1991. 2. Grady, S. R.; Marks, M. J.; Collins, A. C. Desensitization of nicotine-stimulated [‘Hldopamine release form mouse strialtal synaptasomes. J. Neurochem. 621390-1398; 1994. 3. Hendry, J. S.; Rosecrans, J. A. The development of pharmacological tolerance to the effect of nicotine on schedule-controlled responding in mice. Psychopharmacology (Berlin) 77:339-343; 1982. 4. Hulihan-Giblin, B. A.; Lumpkin, M. D.; Kellar, K. J. Acute effects of nicotine on prolactin release in the rat: Agonist and antagonist effects of a single injection of nicotine. J. Pharmacol. Exp. Ther. 252:15-20; 1990. 5. James, J. R.; Villanueva, H. F.; Johnson, J. H.; Arezo, S.; Rosecrans, J. A. Evidence that nicotine can acutely desensitize central nicotinic acetylcholinergic receptors. Psychopharmacology (Berlin) 114:456462; 1994.
6. Marks, M. J.; Burch, J. B.; Collins, A. C. Effects of chronic nicotine infusion on tolerance development and cholinergic receptors. J. Pharmacol. Exp. Ther. 224:806-816; 1983. 7. No&erg, A.; Wahlstrom, G.; Amelo, G.; Larrson, C. Effects of long-term nicotine treatment on [3H]Nicotine binding sites in rat brain. Drug Alcohol Depend. 16:9-17; 1985. 8. Ochoa, E. L. M.; Chattopadhyay, A.; McNamee. M. G. Desensitization of the nicotinic acetylcholinergic receptor: Molecuhu mechanisms and effects of modulators. Cell. Mol. Neurobiol. 9: 141- 178; 1989. 9. Rosecrans, J. A. Brain area nicotine levels in male and female rats with different levels of spontaneous activity. Neuropharmacology 11:863-870; 1972. 10. Rosecrans, J. A.; Stimlar, C. A.; Hendry. J. S.; Melker, L. T. Nicotineinduced tolerance and dependence in tats and mice: Studies involving schedulecontrolled behavior. In: Non&erg, A.; Fuxe, K.; Holmstedt, B.; Sundwall, A., eds. Nicotinic teceptors in the CNS: Theii role in synaptic transmission. New York: Blsevier Science; 1989239-247. 11. Rosecrans, J. A.; Karan. L. D. Neurobehavioral mechanisms of nicotine action: Role in the initiation and maintenance of tobacco dependence. J. Subst. AbuseTreat. 10:161-170; 1993. 12. Rowell, P. P.; Hillebrand, J. A. Characterization of nicotine-induced desensitization of evoked dopamine release from rat striatal synaptosomes. J. Neurochem. 63:561-569; 1994. 13 Sharp, B. M.; Beyer, H. S. Rapid desensitization of the acute. stimulatory effects of nicotine on rat plasma adrenocorticotrophin and prolactin. J. Pharmacol. Exp. Ther. 238:486-491; 1986. 14. Stolerman, 1. P.; Bunker, P.; Jarvik. M. E. Nicotine tolerance m rats; Role of dose and dose interval. Psychopharmacology (Berlin) 34317-324; 1974. 15. Villanueva, H. F.; Arezo, S.; James, J. R.; Rosecrans, J. A. A characterization of nicotine-induced tolerance: Evidence of pharmacological tolerance in the rat. Behav. Pharmacol. 3:255-260; 1992.