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The role of nicotinic receptor beta-2 subunits in nicotine discrimination and conditioned taste aversion
Neuropharmacology 42 (2002) 530–539 www.elsevier.com/locate/neuropharm
The role of nicotinic receptor beta-2 subunits in nicotine discrimination and conditioned taste aversion M. Shoaib a, J. Gommans a, A. Morley a, I.P. Stolerman a,∗, R. Grailhe b, J.-P. Changeux b a
Section of Behavioural Pharmacology, Institute of Psychiatry, King’s College London, De Crespigny Park, London, UK b Neurobiologie Mole´culaire, Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France Received 29 August 2001; received in revised form 13 November 2001; accepted 4 December 2001
Abstract The subtypes of nicotinic receptors at which the behavioural effects of nicotine originate are not fully understood. These experiments use mice lacking the β2 subunit of nicotinic receptors to investigate its role in nicotine discrimination and conditioned taste aversion (CTA). Wild-type and mutant mice were trained either in a two-lever nicotine discrimination procedure using a tandem schedule of food reinforcement, or in a counterbalanced two-flavour CTA procedure. Rates of lever-pressing of wild-type and mutant mice did not differ. Wild-type mice acquired discrimination of nicotine (0.4 or 0.8 mg/kg) rapidly and exhibited steep dose– response curves. Mutant mice failed to acquire these nicotine discriminations and exhibited flat dose–response curves. Both wildtype and mutant mice acquired discrimination of nicotine (1.6 mg/kg) although discrimination performance was weak in the mutants. Nicotine initially reduced response rates in wild-type and mutant mice, and tolerance developed to this effect in each genotype. Both genotypes acquired discrimination of morphine (3 mg/kg) with similar degrees of accuracy, and dose–response curves for morphine discrimination in the two genotypes were indistinguishable. Nicotine produced dose-related CTA in both genotypes, but the magnitude of the effect was less in the mutants than in the wild-type controls. It is concluded that nicotinic receptors containing the β2 subunit play a major role in the discriminative stimulus and taste aversion effects of nicotine that may reflect psychological aspects of tobacco dependence. Such receptors appear to have a less crucial role in the response-rate, reducing effects of nicotine and in nicotine tolerance. 2002 Elsevier Science Ltd. All rights reserved. Keywords: Nicotine; Morphine; Drug discrimination; Conditioned taste aversion; Mice; Genetic modification
1. Introduction Nicotine produces a wide range of behavioural effects that may reflect the diversity of neuronal nicotinic receptors. The 12 known subunits of neuronal nicotinic receptors form pentameric structures associated with a wide spectrum of physiological and pharmacological functions (Corringer et al., 2000). Studies such as those described below have attempted to relate specific subtypes of these receptors to the behavioural effects of nicotine and to nicotine dependence as studied in animal models. This study uses mice with targeted deletions of
Corresponding author. Tel.: +44-20-7848-0370; fax: +44-207848-0579. E-mail address: [email protected] (I.P. Stolerman). ∗
the gene for the β2 subunit of nicotine receptors to investigate the receptor subtypes mediating the discriminative and aversive stimulus properties of nicotine. The positive reinforcing stimulus property of nicotine and its associated subjective effects for which drug discrimination experiments serve as a model play a central role in tobacco dependence. This postulated relationship between the discriminative stimulus effects of drugs and their effects on subjective states (mood) has been discussed many times (e.g. Schuster et al., 1981). Several nicotinic receptor subunits can be identified in mesocortico-limbic regions that are implicated in drug dependence (Le Nove`re et al., 1996; Picciotto et al., 1998; Klink et al., 2001). Experiments with nicotinic agonists and antagonists have supported the view that the stimulus properties of nicotine are mediated by heteromeric high-affinity receptors such as those of the α4β2 sub-
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type, rather than by α7 receptors. For example, in early studies, the potency of nicotinic agonists in nicotine discrimination experiments in rats was correlated with their potency as inhibitors of high-affinity tritiated nicotine binding (Romano et al., 1981; Reavill et al., 1988) that is now considered to mark mainly α4β2 receptors (Flores et al., 1992). Dihydro-β-erythroidine (DHβE) is a potent antagonist at most heteromeric nicotinic receptor subtypes, but it has low potency at homomeric α7-containing receptors; it can therefore be used to distinguish between heteromeric and α7-containing receptors but it cannot distinguish between, for example, α4β2 and α3β4 subtypes. DHβE antagonised the positive reinforcing effect of nicotine in rats (Watkins et al., 1999) and the discriminative stimulus effect of nicotine in both rats and mice (Stolerman et al., 1997; Gommans et al., 2000; Shoaib et al., 2000). In contrast, the potent α7 antagonist methyllycaconitine (MLA) appears unable to block the stimulus properties of nicotine in drug discrimination experiments in rats or mice (Brioni et al., 1996; Gommans et al., 2000). Conditioned taste aversions produced by abused drugs provide a potential model for their aversive effects that may set an upper limit to the amounts consumed (Cappell and Le Blanc, 1975; Kumar and Stolerman, 1977). DHβE antagonised the conditioned taste aversions produced by nicotine in rats and mice, whereas MLA did not block the effect in mice but was not tested in rats (Gommans et al., 2000; Shoaib et al., 2000). These studies, like those considered above on nicotine self-administration and discrimination, all supported the view that heteromeric receptors such as those of the α4β2 subtype play a critical role in the stimulus properties of nicotine. However, the interpretation of conditioned taste aversions produced by psychoactive drugs is controversial because such effects may reflect positive conditioned suppression and may be more closely related to the rewarding than to the aversive properties of the substances (Stolerman and D’Mello, 1981a; Grigson, 1997). While the pharmacological analyses of the stimulus properties of nicotine considered above were largely in agreement with each other, they were limited by reliance upon a small number of nicotinic agonists and antagonists that have limited selectivity for receptor subtypes. More recently, the construction of mice with targeted deletion of genes for β2 and other nicotinic receptor subunits has led to another avenue of investigation that complements the classical approach. In the β2 knockout mouse, the binding of tritiated nicotine was almost eliminated whereas there were normal levels of messenger RNA encoding the α2–α7 and β3–β4 subunits (Picciotto et al., 1995; Cordero-Erausquin et al., 2000). Nicotineinduced release of dopamine in the striatum was eliminated in β2 knockout mice and such mice also ceased responding when intravenous infusions of nicotine were
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substituted for cocaine in a self-administration experiment (Picciotto et al., 1998; Grady et al., 2002). The present studies extended the investigations in genetically modified mice to the discriminative stimulus effect of nicotine and to its ability to produce conditioned taste aversions. The procedures for nicotine discrimination also provided an opportunity to examine the effects of the β2 knockout on other parameters such as the acquisition of the lever-pressing task used to establish the behavioural baseline for drug discrimination, the response-rate reducing effect of nicotine on this baseline, and the development of tolerance to the latter effect. To test whether differences between wild-type and mutant mice were specific to nicotine, some animals were retrained to discriminate morphine from saline. Other animals were retrained to explore the effect of training with a larger dose of nicotine.
2. Materials and methods 2.1. Animals Picciotto et al. (1995) have described the construction of the genetically modified mice. Iffa-Credo (France) supplied male C57BL/6J wild-type control mice and male ACNβ2 mutant siblings from parents backcrossed for 12 generations to C57BL/6J inbred mice. For drug discrimination, 20 of the β2 knockout mice and 20 wildtype control mice were used. Conditioned taste aversion experiments were carried out in 32 β2 knockout mice and 32 wild-type control mice. Mice were about 8 weeks old at the start of experiments, which had a duration of 1 year (drug discrimination) or 6 weeks (taste aversion). All mice were housed individually in rooms at a controlled temperature (21°C) with a 12 h light–dark cycle (light from 0730 to 1930 h). The studies complied with local ethical requirements and were carried out in accordance with the Animals (Experimental Procedures) Act, 1986, under licence from the UK Home Office. 2.2. Apparatus For drug discrimination, six standard operant chambers for mice (MED Associates, Georgia, VT) contained in ventilated, sound-attenuating housings were used. Each chamber was equipped with two ultra-sensitive levers, one on each side of a recess in which a dispenser could deliver 25 mg pellets of food. System control, data acquisition and storage were accomplished with Arachnid software (Paul Fray Ltd, Cambridge) running under RISC OS. Taste aversion conditioning was carried out in the home cages to which drinking cylinders calibrated to ±0.1 ml were attached.
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2.3. Drug discrimination: training procedure The procedure was based on Stolerman et al. (1999). Mice were placed on a restricted diet to maintain them at 85% of their free-feeding weights. Water was available ad libitum. Initially, mice were trained to respond for food pellets delivered automatically under a variabletime 30 s schedule; during this stage of the procedure no response lever was present in the chamber. One lever was then inserted into the chambers and training to leverpress began, initially on a continuous reinforcement schedule, and then under fixed ratio schedules that increased progressively to FR-10. The duration of each session was 15 min. After this response was acquired, the original lever was removed and a single lever was inserted on the opposite side of the chamber. The mice were again trained to perform under the FR-10 schedule of food presentation. A variable interval (VI) component was introduced into the schedule (Stolerman et al., 1999). Under this tandem schedule of reinforcement, the tenth bar press after a randomly determined, variable interval of time was reinforced (tandem VI FR schedule). Initially the mean value of the VI component was 15 s; after five sessions, this was increased to the final value of 30 s (range 7–53 s). Responses during the intervening periods were recorded but not reinforced. Once a baseline of tandem VI-30 FR-10 responding on either lever was obtained, discrimination training commenced. At this point both levers were present during training. The 20 mutant and the 20 wild-type mice were each divided into two groups that were trained to discriminate 0.4 and 0.8 mg/kg doses of nicotine (s.c.) from saline (n ⫽ 10). These doses were based on previous work (Stolerman et al., 1999). Nicotine was administered 10 min prior to training sessions of 15 min duration. Pressing the left lever was reinforced after drug injections and pressing the right lever was reinforced after saline for half of the mice in each group; these arrangements were reversed in the remaining animals. Each mouse had a unique order of nicotine and saline sessions and the sequence of injections was random except that there were never more than three nicotine or saline sessions in succession. 2.4. Drug discrimination: extinction tests Discrimination performance appeared to have stabilised after 60 training sessions and dose–response (generalisation) tests began with nicotine. Nicotine was administered in doses of 0.0, 0.1, 0.2, 0.4, 0.8, 1.2 or 1.6 mg/kg. Each dose was tested once in each mouse, in unique randomised sequences. Ten minutes after injection, mice were placed in the chambers for 5 min with both levers present but no reinforcers available (extinction tests). Mice were tested twice a week and each test day was preceded by a saline training session
to minimise residual drug action on test days. A small proportion of mice died before completion of this work, reducing sizes of some groups to seven or eight animals; the reasons for the deaths are not known and they did not seem to be related to the experimental procedures. 2.5. Drug discrimination: retraining After completion of the extinction tests detailed above, mice originally trained to discriminate 0.8 mg/kg of nicotine were retrained with a larger dose of nicotine whereas mice originally trained with 0.4 mg/kg of nicotine were retrained with morphine. The new training dose of nicotine was 1.2 mg/kg for 10 sessions; this dose was then increased to 1.6 mg/kg for the duration of the study. Drug-lever assignments for these animals were unchanged from those during the original training. After 30 training sessions under these conditions, the dose–response curve for nicotine was determined again using extinction test procedures as above. The doses tested were 0.0, 0.4, 0.8 and 1.6 mg/kg. For mice trained to discriminate morphine from saline, the training dose was 4 mg/kg (s.c.) for 10 sessions, administered 30 min prior to sessions. The dose was then reduced to 3 mg/kg to minimise response rate-suppression. Drug-lever assignments were reversed from those during original training. After 30 training sessions under these conditions, the dose–response curve for morphine was determined. The doses tested were 0.0, 0.3, 1.0, 3.0 and 5.6 mg/kg. 2.6. Conditioned taste aversion The procedures were based on those described previously (Gommans et al., 2000). Mice were accustomed to drinking tap water in two daily 30 min sessions. In the first session, they were allowed to drink 0.9% solutions of either sodium saccharin or sodium chloride in distilled water on every second day. Immediately after drinking a solution, mice were injected with either nicotine or vehicle (“drug-flavour pairing”). These pairings of nicotine with the two flavours were counterbalanced in each group. Such conditioning with drug and vehicle alternated for a total of four sessions. For the second daily drinking session and on intervening days, tap water was offered. Two days after the last conditioning session, a test session took place in which the mice were presented with both flavoured solutions simultaneously. On the next day, this test was repeated with the positions of the two flavours reversed. Different groups of mice were used for each dose of nicotine. 2.7. Data analyses Two- or three-factor analysis of variance were carried out, using repeated measure designs where appropriate.
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For drug discrimination, the percentage of mice that first pressed the correct lever 10 times was used to assess performance during acquisition; drug stimulus generalisation in dose–response studies was assessed from the number of presses on the drug-appropriate lever expressed as a percentage of total presses (a minimum of 10 responses in total was required for calculation of this index). The total number of responses served as an index of response rate; response rates after administration of nicotine were expressed as percentages of rates after saline. For conditioned taste aversion (CTA), the mean amounts consumed of the drug-paired flavour were calculated as percentages of the total fluid intake. Percentage data for nicotine discrimination and CTA were subjected to arcsine transformation to normalise distributions. All statistical tests were performed with Unistat 5 (Unistat Ltd, London). 2.8. Drugs Nicotine bitartrate (BDH, Poole, UK), was dissolved in isotonic saline and the pH was adjusted to 7 with NaOH. Morphine HCl (BDH) was also dissolved in saline. All injections were given subcutaneously in a volume of 1 ml/100 g and all doses were calculated as those of the base.
3. Results 3.1. Acquisition of schedule-controlled behaviour There were no appreciable differences in rates of responding between the wild-type and β2-knockout mice under the continuous reinforcement, fixed ratio (FR-10), or tandem VI-FR schedules. Figs. 1(a) and (b) show that response rates increased progressively during the periods of exposure to continuous reinforcement and FR-10 schedules (F(7, 260) ⫽ 26.9, and F(6, 228) ⫽
Fig. 1. Acquisition of lever-pressing for food in groups of 20 wildtype (쎲) and β2-knockout mice (䊊). Results are shown for three stages of training during which food was presented on continuous reinforcement (FR-1), fixed-ratio 10 (FR-10) and tandem variable interval 30 s FR-10 schedules. Line graphs show results as means±SEM for successive sessions; insets show means for all sessions under each schedule.
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45.3; p ⬍ 0.001 in each case) but there was no increase in rate under the tandem schedule (Fig. 1(c), F(3, 106) ⫽ 2.38). The overall differences between wild-type and mutant mice did not approach significance at any stage (maximum F(1, 38) ⫽ 1.6). The genotype × session interaction was negligible under continuous reinforcement (F(7, 260) ⫽ 1.04), but it approached significance under the FR-10 schedule (F(6, 228) ⫽ 2.09; p ⫽ 0.055). This interaction may be attributed to faster responding by the knockout mice than the wildtype controls during the later sessions on the FR-10 schedule (Fig. 1(b)). However, no similar effect was seen during testing under the tandem VI 30 FR-10 schedule (Fig. 1(c)). 3.2. Acquisition of nicotine discrimination Significant differences between mouse genotypes were detected when nicotine was introduced as a discriminative stimulus. Initially performance of both genotypes was close to the chance level of 50%. Wildtype mice learned to discriminate nicotine readily and after 50 sessions, lever-selection accuracy reached 68 and 87% with training doses of 0.4 and 0.8 mg/kg respectively (Figs. 2(a) and (b)). The β2-knockout mice failed to acquire the discrimination at either training dose and accuracy was not markedly different from 50% at any stage. Analysis of variance confirmed the effects of genotype (F(1, 34) ⫽ 42.3, p ⬍ 0.001), nicotine dose (F(1, 34) ⫽ 27.8, p ⬍ 0.001) and training session (F(5, 169) ⫽ 12.0, p ⬍ 0.001). The significant genotype × training session interaction confirmed the development of a profound difference between performance of wildtype and mutant mice (F(5, 169) ⫽ 6.03, p ⬍ 0.001). During the initial 10 sessions of training, the smaller 0.4 mg/kg training dose of nicotine had little effect on response rates whereas the larger dose (0.8 mg/kg) decreased rates markedly in both wild-type and mutant mice (Fig. 2(d)). Analysis of variance confirmed the significance of the dose-related effect of nicotine (F(1, 34) ⫽ 23.5, p ⬍ 0.001). Rates of responding after saline injections were similar for the two genotypes (wild-type, 370±40, mutant 336±40 lever presses in 15 min). In both genotypes the response to nicotine dissipated over successive blocks of training sessions (nicotine dose×session interaction F(5, 169) ⫽ 8.75, p ⬍ 0.001); there was no significant main effect of genotype (F(1, 34) ⫽ 1.01) and none of the interactions involving genotype were significant. Thus, both the initial response rate-reducing effect of nicotine and the development of tolerance to this effect could be seen in wild-type and mutant mice (Fig. 2(d)). 3.3. Dose–response (generalisation) tests with nicotine of
In wild-type mice trained to discriminate 0.8 mg/kg nicotine, the percentage of drug-appropriate
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larger doses of nicotine prevented assessment of discriminative effects at the largest dose tested (1.6 mg/kg) and reduced the numbers of animals for which data were available at other doses. Thus, results presented for 1.2 mg/kg of nicotine in Figs. 3(a) and (b) are for six to eight wild type and five to six mutant animals only. Nicotine reduced the rate of responding in a doserelated manner in both wild-type and mutant mice (Figs. 3(c) and (d)). Three-factor analysis of variance confirmed the rate-depressant effect of nicotine (F(6, 161) ⫽ 11.0, p ⬍ 0.001) but there was no significant overall difference between genotypes (F(1, 32) ⬍ 1) and no interaction involving the genotype factor (F ⬍ 1 in all cases). There was no significant effect of training dose on the response rate-reducing effect of nicotine, as shown by the absence of either a main effect of training dose (F(1, 32) ⬍ 1) or a training dose×test dose interaction (F(5, 161) ⫽ 1.66). Rates of responding after saline administration were similar for wild-type and mutant mice (142±17 and 143±16 responses in 15 min, respectively).
Fig. 2. Results of nicotine discrimination training at the doses shown in two groups of wild-type (쎲) and two groups of β2-knockout mice (䊊). Upper sections show discrimination performance as accuracy of lever selection for successive blocks of 10 training sessions each; horizontal dashed line represents chance level of responding. Lower sections show total responses on both levers in nicotine sessions as percentages of total responses in saline sessions; horizontal dashed line represents saline control responding. All data shown as means±SEM. Nine mice of each genotype were trained with 0.4 mg/kg, of nicotine and 10 mice of each genotype were trained with 0.8 mg/kg of nicotine.
responding was 7.4% in tests with saline and increased in proportion to the dose of nicotine to reach a maximum of 90.8%. Fig. 3 (upper section) shows that there was little difference between such dose–response curves for wild-type mice trained with 0.4 and 0.8 mg/kg of nicotine. In marked contrast, the dose–response curves for nicotine in the β2 mutant mice were flat and did not deviate appreciably from the chance level in any instance. These data were examined by means of three-factor repeated measure analysis of variance, the factors being mouse genotype, training dose of nicotine and test dose of nicotine. The main effects of genotype (F(1, 32) ⫽ 6.58, p ⬍ 0.02) and test dose of nicotine (F(5, 145) ⫽ 26.4, p ⬍ 0.001) were significant; importantly, the significant interaction of nicotine test dose with genotype (F(5, 145) ⫽ 13.7, p ⬍ 0.001) reflected the different discriminative responses of wildtype and mutant mice as a function of dose. Neither the main effect of training dose nor any other interaction was significant. The response-rate reducing effect of the
Fig. 3. Dose–response curves for discriminative stimulus and response rate-reducing effects of nicotine in wild-type and β2-knockout mice trained with nicotine in doses of 0.4 mg/kg (䊊, n ⫽ 8) and 0.8 mg/kg (쎲, n ⫽ 10). Upper sections show discriminative responding on the drug-appropriate lever expressed as a percentage of the total numbers of responses on both levers (means±SEM). These data are shown for doses up to 1.2 mg/kg of nicotine; in tests at 1.6 mg/kg, mice did not respond sufficiently for assessment of discriminative effects. Lower sections show total numbers of responses after nicotine as percentages of responses after saline. All data shown as means±SEM from 5-min extinction tests.
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3.4. Retraining drug discriminations with large doses of nicotine or with morphine When the training dose of nicotine was increased first to 1.2 mg/kg and then to 1.6 mg/kg, stimulus control by drug states was retained in the wild-type mice although discrimination accuracy seemed rather unstable (Fig. 4(a)). Mutant mice were inaccurate at first but they did acquire the discrimination and exhibited final accuracy close to that of the wild-type animals. Two-factor analysis of variance confirmed the differences between genotypes (F(1, 18) ⫽ 36.6, p ⬍ 0.001), training sessions (F(3, 54) ⫽ 14.5, p ⬍ 0.001) and the genotype×session interaction (F(3, 54) ⫽ 6.45, p ⬍ 0.001). Similarly, Fig. 4(b) shows that mice retrained to discriminate morphine gradually acquired the discrimination and exhibited accuracy in the range of 80–90%, as supported by a significant effect of training sessions (F(3, 39) ⫽ 8.54, p ⬍ 0.001). With respect to morphine discrimi-
Fig. 4. Results of retraining wild-type (쎲) and β2-knockout (䊊) mice to discriminate nicotine or morphine in the doses shown. Upper sections show results for accuracy of lever selection over successive blocks of 10 training sessions (mean±SEM). Lower sections show total responses on both levers in nicotine or morphine sessions as percentages of total responses in saline sessions. Each group of nicotinetrained animals consisted of 10 mice whereas there were seven mutant and eight wild-type morphine-trained animals.
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nation, there was no clear difference between wild-type and mutant animals (between genotypes F(1, 13) ⫽ 2.75; genotype×session interaction F(3, 39) ⬍ 1). Nicotine reduced response rates throughout retraining (Fig. 6(c)), an effect that varied in magnitude across training sessions (F(3, 54) ⫽ 52.6, p ⬍ 0.001), and was rather more pronounced with the 1.6 mg/kg than with the 1.2 mg/kg dose of nicotine. There was no difference between the genotypes (F(1, 18) ⬍ 1). Rates of responding after saline injections were also not significantly different between genotypes (wild-type, 512±47, mutant 404±60 lever presses in 15 min). Morphine also reduced response rates in the mutant mice during the first block of ten sessions where the dose of morphine was 4.0 mg/kg (Fig. 6(d); session × geno-
Fig. 5. Dose–response curves for discriminative stimulus and response rate-reducing effects of nicotine in wild-type (쎲) and β2knockout mice (䊊) trained with nicotine or morphine in the doses shown. Test doses of nicotine or morphine were administered as indicated on abscissae. Upper sections show responding on the drug-appropriate lever expressed as a percentage of the total numbers of responses on both levers (means±SEM). The response-rate reducing effect of the 1.6 mg/kg dose of nicotine prevented assessment of drug-appropriate responding in some mice, reducing the numbers of animals for whom discrimination data can be shown to nine and five animals for wildtype and mutant animals, respectively, at this dose. For the same reason, data for discriminative effects of 5.6 mg/kg of morphine can be shown for only seven of the eight wild-type mice tested. Lower sections show total numbers of responses after nicotine as percentages of responses after saline for all mice tested (groups of 10 nicotine-trained and seven to eight morphine-trained mice).
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type interaction F(3, 39) ⫽ 3.11, p ⬍ 0.05). The effect was less obvious after the dose of morphine was reduced to 3.0 mg/kg to avoid compromising discrimination training and thus, the difference between the overall effect of morphine in the two strains was not significant (F(1, 13) ⫽ 2.71). Rates of responding after saline injections were also not significantly different between genotypes (wild-type, 374±53, mutant 425±62 lever presses in 15 min).
yielded steep dose–response curves for its discriminative effect (F(4, 51) ⫽ 40.1, p ⬍ 0.001) and for effects on rates of lever-pressing (F(4, 52) ⫽ 6.3, p ⬍ 0.001). These effects were very similar in the two genotypes (interaction F(4, 51) ⬍ 1 and F(4,52) ⫽ 1.87, for drugappropriate responding and response rate, respectively). Figs. 5(b) and (d) illustrate these results.
3.5. Dose–response tests after retraining drug discriminations
When the flavoured solutions previously associated with nicotine and saline were presented simultaneously, wild-type mice consumed less of the drug-paired flavoured solutions than of the control solution. Significant CTA was present for nicotine doses of 0.8, 1.2 and 2.0 mg/kg (Fig. 6(a)). Mutant mice also exhibited CTA, but the effects were smaller and reached significance only at doses of 1.2 and 2.0 mg/kg. Two-factor analysis of variance confirmed that CTA was stronger in wild-type than in mutant mice (F(1, 56) ⫽ 4.94, p ⬍ 0.05), although the effect of nicotine dose was only marginally significant (F(3, 56) ⫽ 2.70, p ⬍ 0.055). The genotype × dose interaction was not significant (F(3, 56) ⬍ 1) indicating that the difference between genotypes was similar at all doses of nicotine.
Wild-type mice retrained to discriminate 1.6 mg/kg of nicotine exhibited a steep dose–response curve indicative of a very high level of discrimination accuracy (Fig. 5(a)). Results for nicotine in doses of 0.4 mg/kg and above were clearly different from those for saline. In the mutant mice, the dose–response curve was much shallower, due to poor discrimination accuracy under the saline control condition. Furthermore, it was only at the 1.6 mg/kg training dose that nicotine increased drugappropriate responding above the saline level. Two-factor analysis of variance revealed an overall effect of test dose (F(3, 48) ⫽ 44.4, p ⬍ 0.001) and a significant dose×genotype interaction (F(3, 48) ⫽ 10.3, p ⬍ 0.001). Fig. 5(c) shows that nicotine reduced response rates in a dose-related manner (F(4, 72) ⫽ 36.4, p ⬍ 0.001) and that wild-type and mutant mice were equally sensitive to this effect (interaction F(4, 72) ⬍ 1). Mice retrained to discriminate morphine (3.0 mg/kg)
Fig. 6. Conditioned taste aversion (CTA) produced by nicotine at the doses shown in wild-type (䊏) and β2-knockout mice (䊐) shown as percentage intakes of drug-associated flavoured solutions in two-stimulus tests where both drug- and saline-associated solutions were available simultaneously. Horizontal dashed line represents response level in absence of either preference or aversion (50%). Each group consisted of eight mice All data are from 30-min tests (means±SEM).
3.6. Conditioned taste aversion (CTA)
4. Discussion The major findings of the studies were that the β2knockout mice were severely deficient in their sensitivity to the stimulus properties of nicotine in both drug discrimination and conditioned taste aversion experiments. These mice were not affected with respect to rates of lever-pressing in the undrugged state and they were also unimpaired in a discrimination procedure involving a different drug (morphine). It also appeared that there was some selectivity with respect to the effects of nicotine that were attenuated in the mutant mice; the response rate-reducing effect of nicotine, and the development of tolerance to that effect, seemed to be less critically dependent upon β2-containing receptors. The findings therefore extend, and to some extent, contrast with previous studies that had found these mutant mice to be rather similarly insensitive to nicotine across a range of behavioural procedures. The experiments presented several opportunities for comparing the effect of the β2 knockout on rates of lever-pressing in undrugged mice. This comparison was possible for the continuous reinforcement, FR-10 and tandem schedule performance prior to nicotine discrimination training, during acquisition of the nicotine discrimination, and during dose–response testing both before and after retraining of different drug discriminations. The mutation does not affect these baseline response rates, showing that functional β2-containing
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receptors are not required for performance of a simple operant response. In relation to these observations and other data considered below, it is thought that artefacts due to genetic background may be minimal because the animals had been back-crossed for 12 generations with C57BL/6J mice. The possibility that adaptive responses may have masked an effect on the baseline cannot be excluded, although, for comparing drug effects, the lack of baseline differences is a major advantage. The marked impairment of nicotine discrimination was the main finding of the experiments (Figs. 2(a), 2(b), 3(a) and 3(b)). Indeed, at the 0.4–0.8 mg/kg training doses of nicotine that are the largest doses normally used in such studies, the ability to discriminate nicotine from the non-drug state seemed to be completely lost. The dose–response studies failed to show any discriminative response to nicotine, even when the dose was increased above that used in training to a 1.6 mg/kg dose that was so large that few animals performed the lever-pressing response. These findings support the results of preliminary discrimination experiments (Oglesby et al., 1998) and indicate that β2-containing receptors play a major role in the discriminative stimulus effects of nicotine, as had been suspected from previous studies with nicotinic agonists and antagonists (Reavill et al., 1988; Stolerman et al., 1997; Gommans et al., 2000). The results appeared robust since very similar data were obtained regardless of the use of 0.4 or 0.8 mg/kg doses of nicotine for training. The similarity of the dose–response curves at these two training doses, even in wild-type animals, may be surprising since training drug discriminations with smaller doses often increases sensitivity and shifts dose– response curves to the left (Colpaert et al., 1980; Stolerman and D’Mello, 1981b). However, when the training dose of nicotine was varied (Stolerman et al., 1984, 1999), shifts in dose–response curves were less apparent than in earlier studies with psychomotor stimulants or opioids. When animals were retrained with 1.6 mg/kg of nicotine, it was possible to demonstrate relatively weak discriminative performance in the mutants (Figs. 4(a) and 5(a)). Marubio et al. (1999) have proposed that α3β4 receptors may mediate a peripheral component of the antinociceptive response to nicotine in mice; these receptors may also be considered as a candidate for mediating the discrimination of large doses of nicotine. An impairment of nicotine discrimination might be interpreted as either a diminished response to those effects of nicotine that normally support stimulus control or to an impairment in the ability to learn a conditional discrimination of the type required for performance of the two-lever task. The failure to find any impairment of the acquisition of morphine discrimination (Fig. 4(b)), and the lack of any difference between the dose– response curves for morphine in wild-type and mutant mice (Fig. 5(b)), suggest strongly that the explanation is weakened sensitivity to nicotine rather than a general
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deficit in learning ability. Preliminary observations showing intact cocaine discrimination in β2-knockout mice also support this argument (Oglesby et al., 1998). The observation that the response rate-reducing effect of nicotine was largely intact in the β2-knockout mice was made during the early stages of acquiring nicotine discrimination (Fig. 2(d)). It was confirmed during dose– response tests where relatively large doses of nicotine were needed to produce the effect in both wild-type and mutant mice, and during retraining with a larger dose of nicotine (Figs. 3(a), 3(b), 4(a) and 5(a)). The response rate-reducing effect of morphine was also present and actually slightly enhanced during retraining of mutant mice (Fig. 4(d)); this may indicate a possible interaction between the nicotinic and opioid systems that should be investigated further. However, a similar effect was not detected during dose–response tests with morphine (Fig. 5(d)). The development of tolerance to the rate-reducing effect of nicotine, as observed during the acquisition phase (Fig. 3(d)), explains adequately the need to administer large doses of nicotine to produce response ratereductions during testing (Figs. 4(c) and (d)). Earlier studies have also described tolerance to the rate-reducing effect of nicotine on operant responses in mice and rats (Hendry and Rosecrans, 1982; Villanueva et al., 1992). However, some caution should be observed in interpreting these results because the experiments were not designed primarily to study effects on response rates or tolerance; dose–response data for the acute effects of nicotine on response rates were not obtained and comparisons between animals chronically treated with nicotine and saline were not made. These caveats notwithstanding, the data suggest that β2-containing receptors may play a less critical role in the depressant effects of nicotine on rates of operant behaviour, and on tolerance to such effects, than they do in its discriminative stimulus effects. This conclusion is also compatible with evidence from studies with the antagonist DHβE, suggesting that the receptor mechanism mediating the response rate-reducing effect of nicotine in rats differed from that for the discriminative stimulus effect (Stolerman et al., 1997). The experiments on conditioned taste aversion indicate that the effect was attenuated in β2-knockout mice across the range of doses of nicotine that were studied (Fig. 6). The β2-containing receptors therefore appear to play a role in this effect. However, these studies did not control for the possibility that mutation weakened the ability of mice to learn any taste aversion, as contrasted with a selective attenuation of the response to nicotine. Previous studies on the mechanisms of nicotine-produced conditioned taste aversion have not differentiated the receptors from those involved in nicotine discrimination (Kumar et al., 1983; Reavill et al., 1986) and in this sense the present findings are not surprising.
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In summary, deletion of the gene that encodes the β2 subunits of nicotinic receptors dramatically impaired discrimination of nicotine and weakened nicotine-induced CTA. This compromised behaviour was observed in the absence of deficits in a simple lever-pressing task or in sensitivity to the unconditioned depressant action of nicotine on lever-pressing. The impairment of discrimination may be specific to nicotine since the mutant mice showed no deficits in acquisition or performance of morphine discrimination. Thus, nicotinic receptors containing β2 subunits mediate the stimulus properties of nicotine that constitute important psychological elements in nicotine dependence; β2-containing receptors appear to be of lesser significance for nicotine tolerance, a physiological adaptation that is present in the nicotine-dependent state. The involvement of β2 subunits in the stimulus properties of nicotine and their relative insignificance in nicotine tolerance is suggestive of a possible dissociation between the mechanisms of the psychological and the physiological aspects of nicotine dependence that deserves further investigation.
Acknowledgements This work was supported by a European Union TMR grant, the Medical Research Council (UK), the Colle`ge de France, the Centre National de la Recherche Scientifique, the Council for Tobacco Research, the Association pour la Recherche sur le Cancer and the Commission of the European Communities.
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interpeduncular nucleus: a function mediated by a different nAChR than dopamine release from striatum. Journal of Neurochemistry 76, 258–268. Grigson, P.S., 1997. Conditioned taste aversions and drugs of abuse: a reinterpretation. Behavioral Neuroscience 111, 129–136. Hendry, J.S., Rosecrans, J.A., 1982. The development of pharmacological tolerance to the effect of nicotine on schedule-controlled responding in mice. Psychopharmacology 77, 339–343. Klink, R., d’Exaerde, A.D., Zoli, M., Changeux, J.-P., 2001. Molecular and physiological diversity of nicotinic acetylcholine receptors in the midbrain dopaminergic nuclei. Journal of Neuroscience 21, 1452–1463. Kumar, R., Stolerman, I.P., 1977. Experimental and clinical aspects of drug dependence. In: Iversen, L.L., Iversen, S.D., Snyder, S.H. (Eds.), Handbook of Psychopharmacology, vol. 7. Plenum, New York, pp. 321–367. Kumar, R., Pratt, J.A., Stolerman, I.P., 1983. Characteristics of conditioned taste aversion produced by nicotine in rats. British Journal of Pharmacology 79, 245–253. Le Nove`re, N., Zoli, M., Changeux, J.-P., 1996. Neuronal nicotinic receptor alpha 6 subunit mRNA is selectively concentrated in catecholaminergic nuclei of the rat brain. European Journal of Neuroscience 8, 2428–2439. Marubio, L.M., del Mar Arroyo-Jimenez, M., Cordero-Erausquin, M., Le´ na, C., Le Nove`re, N., d’Exaerde, A.D., Huchet, M., Damaj, M.I., Changeux, J.-P., 1999. Reduced antinociception in mice lacking neuronal nicotinic receptor subunits. Nature 398, 805–810. Oglesby, M.W., Epping-Jordan, M.P., Pich, E.M., Picciotto, M.R., Changeux, J.-P., 1998. Attenuated nicotine discrimination in mice lacking high affinity nicotine receptors. Society for Neuroscience Abstracts 24, 1196. Picciotto, M.R., Zoli, M., Le´ na, C., Bessis, A., Lallemand, Y., Le Nove`re, N., Vincent, P., Pich, E.M., Brulet, P., Changeux, J.-P., 1995. Abnormal avoidance learning in mice lacking functional highaffinity nicotine receptor in the brain. Nature 374, 65–67. Picciotto, M.R., Zoli, M., Rimondini, R., Le´ na, C., Marubio, L.M., Pich, E.M., Fuxe, K., Changeux, J.-P., 1998. Acetylcholine receptors containing the β2 subunit are involved in the reinforcing properties of nicotine. Nature 391, 173–177. Reavill, C., Stolerman, I.P., Kumar, R., Garcha, H.S., 1986. Chlorisondamine blocks acquisition of the conditioned taste aversion produced by (⫺)-nicotine. Neuropharmacology 25, 1067. Reavill, C., Jenner, P., Kumar, R., Stolerman, I.P., 1988. High-affinity binding of [3H](⫺)-nicotine to rat brain membranes and its inhibition by analogues of nicotine. Neuropharmacology 27, 235. Romano, C., Goldstein, A., Jewell, N.P., 1981. Characterization of the receptor mediating the nicotine discriminative stimulus. Psychopharmacology 74, 310–315. Schuster, C.R., Fischman, M.W., Johanson, C.-E., 1981. Internal stimulus control and the subjective effects of drugs. In: Thompson, T., Johanson, C.-E. (Eds.), Behavioral Pharmacology of Human Drug Dependence, NIDA Research Monographs, vol. 37. US Government Printing Office, Washington, DC, pp. 116–129. Shoaib, M., Zubaran, C., Stolerman, I.P., 2000. Antagonism of stimulus properties of nicotine by dihydro-β-erythroidine (DHβE) in rats. Psychopharmacology 149, 140–146. Stolerman, I.P., D’Mello, G,D., 1981a. Oral self-administration and the relevance of conditioned taste aversions. In: Thompson, T., Dews, P.B., McKim, W.A. (Eds.), Advances in Behavioural Pharmacology, vol. 3. Academic Press, New York, pp. 169–214. Stolerman, I.P., D’Mello, G.D., 1981b. Role of training conditions in discrimination of central nervous system stimulants by rats. Psychopharmacology 73, 295–303. Stolerman, I.P., Garcha, H.S., Pratt, J.A., Kumar, R., 1984. Role of training dose in discrimination of nicotine and related compounds by rats. Psychopharmacology 84, 413–419. Stolerman, I.P., Chandler, C.J., Garcha, H.S., Newton, J.M., 1997.
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The role of nicotinic receptor beta-2 subunits in nicotine discrimination and conditioned taste aversion M. Shoaib a, J. Gommans a, A. Morley a, I.P. Stolerman a,∗, R. Grailhe b, J.-P. Changeux b a
Section of Behavioural Pharmacology, Institute of Psychiatry, King’s College London, De Crespigny Park, London, UK b Neurobiologie Mole´culaire, Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France Received 29 August 2001; received in revised form 13 November 2001; accepted 4 December 2001
Abstract The subtypes of nicotinic receptors at which the behavioural effects of nicotine originate are not fully understood. These experiments use mice lacking the β2 subunit of nicotinic receptors to investigate its role in nicotine discrimination and conditioned taste aversion (CTA). Wild-type and mutant mice were trained either in a two-lever nicotine discrimination procedure using a tandem schedule of food reinforcement, or in a counterbalanced two-flavour CTA procedure. Rates of lever-pressing of wild-type and mutant mice did not differ. Wild-type mice acquired discrimination of nicotine (0.4 or 0.8 mg/kg) rapidly and exhibited steep dose– response curves. Mutant mice failed to acquire these nicotine discriminations and exhibited flat dose–response curves. Both wildtype and mutant mice acquired discrimination of nicotine (1.6 mg/kg) although discrimination performance was weak in the mutants. Nicotine initially reduced response rates in wild-type and mutant mice, and tolerance developed to this effect in each genotype. Both genotypes acquired discrimination of morphine (3 mg/kg) with similar degrees of accuracy, and dose–response curves for morphine discrimination in the two genotypes were indistinguishable. Nicotine produced dose-related CTA in both genotypes, but the magnitude of the effect was less in the mutants than in the wild-type controls. It is concluded that nicotinic receptors containing the β2 subunit play a major role in the discriminative stimulus and taste aversion effects of nicotine that may reflect psychological aspects of tobacco dependence. Such receptors appear to have a less crucial role in the response-rate, reducing effects of nicotine and in nicotine tolerance. 2002 Elsevier Science Ltd. All rights reserved. Keywords: Nicotine; Morphine; Drug discrimination; Conditioned taste aversion; Mice; Genetic modification
1. Introduction Nicotine produces a wide range of behavioural effects that may reflect the diversity of neuronal nicotinic receptors. The 12 known subunits of neuronal nicotinic receptors form pentameric structures associated with a wide spectrum of physiological and pharmacological functions (Corringer et al., 2000). Studies such as those described below have attempted to relate specific subtypes of these receptors to the behavioural effects of nicotine and to nicotine dependence as studied in animal models. This study uses mice with targeted deletions of
Corresponding author. Tel.: +44-20-7848-0370; fax: +44-207848-0579. E-mail address: [email protected] (I.P. Stolerman). ∗
the gene for the β2 subunit of nicotine receptors to investigate the receptor subtypes mediating the discriminative and aversive stimulus properties of nicotine. The positive reinforcing stimulus property of nicotine and its associated subjective effects for which drug discrimination experiments serve as a model play a central role in tobacco dependence. This postulated relationship between the discriminative stimulus effects of drugs and their effects on subjective states (mood) has been discussed many times (e.g. Schuster et al., 1981). Several nicotinic receptor subunits can be identified in mesocortico-limbic regions that are implicated in drug dependence (Le Nove`re et al., 1996; Picciotto et al., 1998; Klink et al., 2001). Experiments with nicotinic agonists and antagonists have supported the view that the stimulus properties of nicotine are mediated by heteromeric high-affinity receptors such as those of the α4β2 sub-
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type, rather than by α7 receptors. For example, in early studies, the potency of nicotinic agonists in nicotine discrimination experiments in rats was correlated with their potency as inhibitors of high-affinity tritiated nicotine binding (Romano et al., 1981; Reavill et al., 1988) that is now considered to mark mainly α4β2 receptors (Flores et al., 1992). Dihydro-β-erythroidine (DHβE) is a potent antagonist at most heteromeric nicotinic receptor subtypes, but it has low potency at homomeric α7-containing receptors; it can therefore be used to distinguish between heteromeric and α7-containing receptors but it cannot distinguish between, for example, α4β2 and α3β4 subtypes. DHβE antagonised the positive reinforcing effect of nicotine in rats (Watkins et al., 1999) and the discriminative stimulus effect of nicotine in both rats and mice (Stolerman et al., 1997; Gommans et al., 2000; Shoaib et al., 2000). In contrast, the potent α7 antagonist methyllycaconitine (MLA) appears unable to block the stimulus properties of nicotine in drug discrimination experiments in rats or mice (Brioni et al., 1996; Gommans et al., 2000). Conditioned taste aversions produced by abused drugs provide a potential model for their aversive effects that may set an upper limit to the amounts consumed (Cappell and Le Blanc, 1975; Kumar and Stolerman, 1977). DHβE antagonised the conditioned taste aversions produced by nicotine in rats and mice, whereas MLA did not block the effect in mice but was not tested in rats (Gommans et al., 2000; Shoaib et al., 2000). These studies, like those considered above on nicotine self-administration and discrimination, all supported the view that heteromeric receptors such as those of the α4β2 subtype play a critical role in the stimulus properties of nicotine. However, the interpretation of conditioned taste aversions produced by psychoactive drugs is controversial because such effects may reflect positive conditioned suppression and may be more closely related to the rewarding than to the aversive properties of the substances (Stolerman and D’Mello, 1981a; Grigson, 1997). While the pharmacological analyses of the stimulus properties of nicotine considered above were largely in agreement with each other, they were limited by reliance upon a small number of nicotinic agonists and antagonists that have limited selectivity for receptor subtypes. More recently, the construction of mice with targeted deletion of genes for β2 and other nicotinic receptor subunits has led to another avenue of investigation that complements the classical approach. In the β2 knockout mouse, the binding of tritiated nicotine was almost eliminated whereas there were normal levels of messenger RNA encoding the α2–α7 and β3–β4 subunits (Picciotto et al., 1995; Cordero-Erausquin et al., 2000). Nicotineinduced release of dopamine in the striatum was eliminated in β2 knockout mice and such mice also ceased responding when intravenous infusions of nicotine were
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substituted for cocaine in a self-administration experiment (Picciotto et al., 1998; Grady et al., 2002). The present studies extended the investigations in genetically modified mice to the discriminative stimulus effect of nicotine and to its ability to produce conditioned taste aversions. The procedures for nicotine discrimination also provided an opportunity to examine the effects of the β2 knockout on other parameters such as the acquisition of the lever-pressing task used to establish the behavioural baseline for drug discrimination, the response-rate reducing effect of nicotine on this baseline, and the development of tolerance to the latter effect. To test whether differences between wild-type and mutant mice were specific to nicotine, some animals were retrained to discriminate morphine from saline. Other animals were retrained to explore the effect of training with a larger dose of nicotine.
2. Materials and methods 2.1. Animals Picciotto et al. (1995) have described the construction of the genetically modified mice. Iffa-Credo (France) supplied male C57BL/6J wild-type control mice and male ACNβ2 mutant siblings from parents backcrossed for 12 generations to C57BL/6J inbred mice. For drug discrimination, 20 of the β2 knockout mice and 20 wildtype control mice were used. Conditioned taste aversion experiments were carried out in 32 β2 knockout mice and 32 wild-type control mice. Mice were about 8 weeks old at the start of experiments, which had a duration of 1 year (drug discrimination) or 6 weeks (taste aversion). All mice were housed individually in rooms at a controlled temperature (21°C) with a 12 h light–dark cycle (light from 0730 to 1930 h). The studies complied with local ethical requirements and were carried out in accordance with the Animals (Experimental Procedures) Act, 1986, under licence from the UK Home Office. 2.2. Apparatus For drug discrimination, six standard operant chambers for mice (MED Associates, Georgia, VT) contained in ventilated, sound-attenuating housings were used. Each chamber was equipped with two ultra-sensitive levers, one on each side of a recess in which a dispenser could deliver 25 mg pellets of food. System control, data acquisition and storage were accomplished with Arachnid software (Paul Fray Ltd, Cambridge) running under RISC OS. Taste aversion conditioning was carried out in the home cages to which drinking cylinders calibrated to ±0.1 ml were attached.
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2.3. Drug discrimination: training procedure The procedure was based on Stolerman et al. (1999). Mice were placed on a restricted diet to maintain them at 85% of their free-feeding weights. Water was available ad libitum. Initially, mice were trained to respond for food pellets delivered automatically under a variabletime 30 s schedule; during this stage of the procedure no response lever was present in the chamber. One lever was then inserted into the chambers and training to leverpress began, initially on a continuous reinforcement schedule, and then under fixed ratio schedules that increased progressively to FR-10. The duration of each session was 15 min. After this response was acquired, the original lever was removed and a single lever was inserted on the opposite side of the chamber. The mice were again trained to perform under the FR-10 schedule of food presentation. A variable interval (VI) component was introduced into the schedule (Stolerman et al., 1999). Under this tandem schedule of reinforcement, the tenth bar press after a randomly determined, variable interval of time was reinforced (tandem VI FR schedule). Initially the mean value of the VI component was 15 s; after five sessions, this was increased to the final value of 30 s (range 7–53 s). Responses during the intervening periods were recorded but not reinforced. Once a baseline of tandem VI-30 FR-10 responding on either lever was obtained, discrimination training commenced. At this point both levers were present during training. The 20 mutant and the 20 wild-type mice were each divided into two groups that were trained to discriminate 0.4 and 0.8 mg/kg doses of nicotine (s.c.) from saline (n ⫽ 10). These doses were based on previous work (Stolerman et al., 1999). Nicotine was administered 10 min prior to training sessions of 15 min duration. Pressing the left lever was reinforced after drug injections and pressing the right lever was reinforced after saline for half of the mice in each group; these arrangements were reversed in the remaining animals. Each mouse had a unique order of nicotine and saline sessions and the sequence of injections was random except that there were never more than three nicotine or saline sessions in succession. 2.4. Drug discrimination: extinction tests Discrimination performance appeared to have stabilised after 60 training sessions and dose–response (generalisation) tests began with nicotine. Nicotine was administered in doses of 0.0, 0.1, 0.2, 0.4, 0.8, 1.2 or 1.6 mg/kg. Each dose was tested once in each mouse, in unique randomised sequences. Ten minutes after injection, mice were placed in the chambers for 5 min with both levers present but no reinforcers available (extinction tests). Mice were tested twice a week and each test day was preceded by a saline training session
to minimise residual drug action on test days. A small proportion of mice died before completion of this work, reducing sizes of some groups to seven or eight animals; the reasons for the deaths are not known and they did not seem to be related to the experimental procedures. 2.5. Drug discrimination: retraining After completion of the extinction tests detailed above, mice originally trained to discriminate 0.8 mg/kg of nicotine were retrained with a larger dose of nicotine whereas mice originally trained with 0.4 mg/kg of nicotine were retrained with morphine. The new training dose of nicotine was 1.2 mg/kg for 10 sessions; this dose was then increased to 1.6 mg/kg for the duration of the study. Drug-lever assignments for these animals were unchanged from those during the original training. After 30 training sessions under these conditions, the dose–response curve for nicotine was determined again using extinction test procedures as above. The doses tested were 0.0, 0.4, 0.8 and 1.6 mg/kg. For mice trained to discriminate morphine from saline, the training dose was 4 mg/kg (s.c.) for 10 sessions, administered 30 min prior to sessions. The dose was then reduced to 3 mg/kg to minimise response rate-suppression. Drug-lever assignments were reversed from those during original training. After 30 training sessions under these conditions, the dose–response curve for morphine was determined. The doses tested were 0.0, 0.3, 1.0, 3.0 and 5.6 mg/kg. 2.6. Conditioned taste aversion The procedures were based on those described previously (Gommans et al., 2000). Mice were accustomed to drinking tap water in two daily 30 min sessions. In the first session, they were allowed to drink 0.9% solutions of either sodium saccharin or sodium chloride in distilled water on every second day. Immediately after drinking a solution, mice were injected with either nicotine or vehicle (“drug-flavour pairing”). These pairings of nicotine with the two flavours were counterbalanced in each group. Such conditioning with drug and vehicle alternated for a total of four sessions. For the second daily drinking session and on intervening days, tap water was offered. Two days after the last conditioning session, a test session took place in which the mice were presented with both flavoured solutions simultaneously. On the next day, this test was repeated with the positions of the two flavours reversed. Different groups of mice were used for each dose of nicotine. 2.7. Data analyses Two- or three-factor analysis of variance were carried out, using repeated measure designs where appropriate.
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For drug discrimination, the percentage of mice that first pressed the correct lever 10 times was used to assess performance during acquisition; drug stimulus generalisation in dose–response studies was assessed from the number of presses on the drug-appropriate lever expressed as a percentage of total presses (a minimum of 10 responses in total was required for calculation of this index). The total number of responses served as an index of response rate; response rates after administration of nicotine were expressed as percentages of rates after saline. For conditioned taste aversion (CTA), the mean amounts consumed of the drug-paired flavour were calculated as percentages of the total fluid intake. Percentage data for nicotine discrimination and CTA were subjected to arcsine transformation to normalise distributions. All statistical tests were performed with Unistat 5 (Unistat Ltd, London). 2.8. Drugs Nicotine bitartrate (BDH, Poole, UK), was dissolved in isotonic saline and the pH was adjusted to 7 with NaOH. Morphine HCl (BDH) was also dissolved in saline. All injections were given subcutaneously in a volume of 1 ml/100 g and all doses were calculated as those of the base.
3. Results 3.1. Acquisition of schedule-controlled behaviour There were no appreciable differences in rates of responding between the wild-type and β2-knockout mice under the continuous reinforcement, fixed ratio (FR-10), or tandem VI-FR schedules. Figs. 1(a) and (b) show that response rates increased progressively during the periods of exposure to continuous reinforcement and FR-10 schedules (F(7, 260) ⫽ 26.9, and F(6, 228) ⫽
Fig. 1. Acquisition of lever-pressing for food in groups of 20 wildtype (쎲) and β2-knockout mice (䊊). Results are shown for three stages of training during which food was presented on continuous reinforcement (FR-1), fixed-ratio 10 (FR-10) and tandem variable interval 30 s FR-10 schedules. Line graphs show results as means±SEM for successive sessions; insets show means for all sessions under each schedule.
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45.3; p ⬍ 0.001 in each case) but there was no increase in rate under the tandem schedule (Fig. 1(c), F(3, 106) ⫽ 2.38). The overall differences between wild-type and mutant mice did not approach significance at any stage (maximum F(1, 38) ⫽ 1.6). The genotype × session interaction was negligible under continuous reinforcement (F(7, 260) ⫽ 1.04), but it approached significance under the FR-10 schedule (F(6, 228) ⫽ 2.09; p ⫽ 0.055). This interaction may be attributed to faster responding by the knockout mice than the wildtype controls during the later sessions on the FR-10 schedule (Fig. 1(b)). However, no similar effect was seen during testing under the tandem VI 30 FR-10 schedule (Fig. 1(c)). 3.2. Acquisition of nicotine discrimination Significant differences between mouse genotypes were detected when nicotine was introduced as a discriminative stimulus. Initially performance of both genotypes was close to the chance level of 50%. Wildtype mice learned to discriminate nicotine readily and after 50 sessions, lever-selection accuracy reached 68 and 87% with training doses of 0.4 and 0.8 mg/kg respectively (Figs. 2(a) and (b)). The β2-knockout mice failed to acquire the discrimination at either training dose and accuracy was not markedly different from 50% at any stage. Analysis of variance confirmed the effects of genotype (F(1, 34) ⫽ 42.3, p ⬍ 0.001), nicotine dose (F(1, 34) ⫽ 27.8, p ⬍ 0.001) and training session (F(5, 169) ⫽ 12.0, p ⬍ 0.001). The significant genotype × training session interaction confirmed the development of a profound difference between performance of wildtype and mutant mice (F(5, 169) ⫽ 6.03, p ⬍ 0.001). During the initial 10 sessions of training, the smaller 0.4 mg/kg training dose of nicotine had little effect on response rates whereas the larger dose (0.8 mg/kg) decreased rates markedly in both wild-type and mutant mice (Fig. 2(d)). Analysis of variance confirmed the significance of the dose-related effect of nicotine (F(1, 34) ⫽ 23.5, p ⬍ 0.001). Rates of responding after saline injections were similar for the two genotypes (wild-type, 370±40, mutant 336±40 lever presses in 15 min). In both genotypes the response to nicotine dissipated over successive blocks of training sessions (nicotine dose×session interaction F(5, 169) ⫽ 8.75, p ⬍ 0.001); there was no significant main effect of genotype (F(1, 34) ⫽ 1.01) and none of the interactions involving genotype were significant. Thus, both the initial response rate-reducing effect of nicotine and the development of tolerance to this effect could be seen in wild-type and mutant mice (Fig. 2(d)). 3.3. Dose–response (generalisation) tests with nicotine of
In wild-type mice trained to discriminate 0.8 mg/kg nicotine, the percentage of drug-appropriate
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larger doses of nicotine prevented assessment of discriminative effects at the largest dose tested (1.6 mg/kg) and reduced the numbers of animals for which data were available at other doses. Thus, results presented for 1.2 mg/kg of nicotine in Figs. 3(a) and (b) are for six to eight wild type and five to six mutant animals only. Nicotine reduced the rate of responding in a doserelated manner in both wild-type and mutant mice (Figs. 3(c) and (d)). Three-factor analysis of variance confirmed the rate-depressant effect of nicotine (F(6, 161) ⫽ 11.0, p ⬍ 0.001) but there was no significant overall difference between genotypes (F(1, 32) ⬍ 1) and no interaction involving the genotype factor (F ⬍ 1 in all cases). There was no significant effect of training dose on the response rate-reducing effect of nicotine, as shown by the absence of either a main effect of training dose (F(1, 32) ⬍ 1) or a training dose×test dose interaction (F(5, 161) ⫽ 1.66). Rates of responding after saline administration were similar for wild-type and mutant mice (142±17 and 143±16 responses in 15 min, respectively).
Fig. 2. Results of nicotine discrimination training at the doses shown in two groups of wild-type (쎲) and two groups of β2-knockout mice (䊊). Upper sections show discrimination performance as accuracy of lever selection for successive blocks of 10 training sessions each; horizontal dashed line represents chance level of responding. Lower sections show total responses on both levers in nicotine sessions as percentages of total responses in saline sessions; horizontal dashed line represents saline control responding. All data shown as means±SEM. Nine mice of each genotype were trained with 0.4 mg/kg, of nicotine and 10 mice of each genotype were trained with 0.8 mg/kg of nicotine.
responding was 7.4% in tests with saline and increased in proportion to the dose of nicotine to reach a maximum of 90.8%. Fig. 3 (upper section) shows that there was little difference between such dose–response curves for wild-type mice trained with 0.4 and 0.8 mg/kg of nicotine. In marked contrast, the dose–response curves for nicotine in the β2 mutant mice were flat and did not deviate appreciably from the chance level in any instance. These data were examined by means of three-factor repeated measure analysis of variance, the factors being mouse genotype, training dose of nicotine and test dose of nicotine. The main effects of genotype (F(1, 32) ⫽ 6.58, p ⬍ 0.02) and test dose of nicotine (F(5, 145) ⫽ 26.4, p ⬍ 0.001) were significant; importantly, the significant interaction of nicotine test dose with genotype (F(5, 145) ⫽ 13.7, p ⬍ 0.001) reflected the different discriminative responses of wildtype and mutant mice as a function of dose. Neither the main effect of training dose nor any other interaction was significant. The response-rate reducing effect of the
Fig. 3. Dose–response curves for discriminative stimulus and response rate-reducing effects of nicotine in wild-type and β2-knockout mice trained with nicotine in doses of 0.4 mg/kg (䊊, n ⫽ 8) and 0.8 mg/kg (쎲, n ⫽ 10). Upper sections show discriminative responding on the drug-appropriate lever expressed as a percentage of the total numbers of responses on both levers (means±SEM). These data are shown for doses up to 1.2 mg/kg of nicotine; in tests at 1.6 mg/kg, mice did not respond sufficiently for assessment of discriminative effects. Lower sections show total numbers of responses after nicotine as percentages of responses after saline. All data shown as means±SEM from 5-min extinction tests.
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3.4. Retraining drug discriminations with large doses of nicotine or with morphine When the training dose of nicotine was increased first to 1.2 mg/kg and then to 1.6 mg/kg, stimulus control by drug states was retained in the wild-type mice although discrimination accuracy seemed rather unstable (Fig. 4(a)). Mutant mice were inaccurate at first but they did acquire the discrimination and exhibited final accuracy close to that of the wild-type animals. Two-factor analysis of variance confirmed the differences between genotypes (F(1, 18) ⫽ 36.6, p ⬍ 0.001), training sessions (F(3, 54) ⫽ 14.5, p ⬍ 0.001) and the genotype×session interaction (F(3, 54) ⫽ 6.45, p ⬍ 0.001). Similarly, Fig. 4(b) shows that mice retrained to discriminate morphine gradually acquired the discrimination and exhibited accuracy in the range of 80–90%, as supported by a significant effect of training sessions (F(3, 39) ⫽ 8.54, p ⬍ 0.001). With respect to morphine discrimi-
Fig. 4. Results of retraining wild-type (쎲) and β2-knockout (䊊) mice to discriminate nicotine or morphine in the doses shown. Upper sections show results for accuracy of lever selection over successive blocks of 10 training sessions (mean±SEM). Lower sections show total responses on both levers in nicotine or morphine sessions as percentages of total responses in saline sessions. Each group of nicotinetrained animals consisted of 10 mice whereas there were seven mutant and eight wild-type morphine-trained animals.
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nation, there was no clear difference between wild-type and mutant animals (between genotypes F(1, 13) ⫽ 2.75; genotype×session interaction F(3, 39) ⬍ 1). Nicotine reduced response rates throughout retraining (Fig. 6(c)), an effect that varied in magnitude across training sessions (F(3, 54) ⫽ 52.6, p ⬍ 0.001), and was rather more pronounced with the 1.6 mg/kg than with the 1.2 mg/kg dose of nicotine. There was no difference between the genotypes (F(1, 18) ⬍ 1). Rates of responding after saline injections were also not significantly different between genotypes (wild-type, 512±47, mutant 404±60 lever presses in 15 min). Morphine also reduced response rates in the mutant mice during the first block of ten sessions where the dose of morphine was 4.0 mg/kg (Fig. 6(d); session × geno-
Fig. 5. Dose–response curves for discriminative stimulus and response rate-reducing effects of nicotine in wild-type (쎲) and β2knockout mice (䊊) trained with nicotine or morphine in the doses shown. Test doses of nicotine or morphine were administered as indicated on abscissae. Upper sections show responding on the drug-appropriate lever expressed as a percentage of the total numbers of responses on both levers (means±SEM). The response-rate reducing effect of the 1.6 mg/kg dose of nicotine prevented assessment of drug-appropriate responding in some mice, reducing the numbers of animals for whom discrimination data can be shown to nine and five animals for wildtype and mutant animals, respectively, at this dose. For the same reason, data for discriminative effects of 5.6 mg/kg of morphine can be shown for only seven of the eight wild-type mice tested. Lower sections show total numbers of responses after nicotine as percentages of responses after saline for all mice tested (groups of 10 nicotine-trained and seven to eight morphine-trained mice).
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type interaction F(3, 39) ⫽ 3.11, p ⬍ 0.05). The effect was less obvious after the dose of morphine was reduced to 3.0 mg/kg to avoid compromising discrimination training and thus, the difference between the overall effect of morphine in the two strains was not significant (F(1, 13) ⫽ 2.71). Rates of responding after saline injections were also not significantly different between genotypes (wild-type, 374±53, mutant 425±62 lever presses in 15 min).
yielded steep dose–response curves for its discriminative effect (F(4, 51) ⫽ 40.1, p ⬍ 0.001) and for effects on rates of lever-pressing (F(4, 52) ⫽ 6.3, p ⬍ 0.001). These effects were very similar in the two genotypes (interaction F(4, 51) ⬍ 1 and F(4,52) ⫽ 1.87, for drugappropriate responding and response rate, respectively). Figs. 5(b) and (d) illustrate these results.
3.5. Dose–response tests after retraining drug discriminations
When the flavoured solutions previously associated with nicotine and saline were presented simultaneously, wild-type mice consumed less of the drug-paired flavoured solutions than of the control solution. Significant CTA was present for nicotine doses of 0.8, 1.2 and 2.0 mg/kg (Fig. 6(a)). Mutant mice also exhibited CTA, but the effects were smaller and reached significance only at doses of 1.2 and 2.0 mg/kg. Two-factor analysis of variance confirmed that CTA was stronger in wild-type than in mutant mice (F(1, 56) ⫽ 4.94, p ⬍ 0.05), although the effect of nicotine dose was only marginally significant (F(3, 56) ⫽ 2.70, p ⬍ 0.055). The genotype × dose interaction was not significant (F(3, 56) ⬍ 1) indicating that the difference between genotypes was similar at all doses of nicotine.
Wild-type mice retrained to discriminate 1.6 mg/kg of nicotine exhibited a steep dose–response curve indicative of a very high level of discrimination accuracy (Fig. 5(a)). Results for nicotine in doses of 0.4 mg/kg and above were clearly different from those for saline. In the mutant mice, the dose–response curve was much shallower, due to poor discrimination accuracy under the saline control condition. Furthermore, it was only at the 1.6 mg/kg training dose that nicotine increased drugappropriate responding above the saline level. Two-factor analysis of variance revealed an overall effect of test dose (F(3, 48) ⫽ 44.4, p ⬍ 0.001) and a significant dose×genotype interaction (F(3, 48) ⫽ 10.3, p ⬍ 0.001). Fig. 5(c) shows that nicotine reduced response rates in a dose-related manner (F(4, 72) ⫽ 36.4, p ⬍ 0.001) and that wild-type and mutant mice were equally sensitive to this effect (interaction F(4, 72) ⬍ 1). Mice retrained to discriminate morphine (3.0 mg/kg)
Fig. 6. Conditioned taste aversion (CTA) produced by nicotine at the doses shown in wild-type (䊏) and β2-knockout mice (䊐) shown as percentage intakes of drug-associated flavoured solutions in two-stimulus tests where both drug- and saline-associated solutions were available simultaneously. Horizontal dashed line represents response level in absence of either preference or aversion (50%). Each group consisted of eight mice All data are from 30-min tests (means±SEM).
3.6. Conditioned taste aversion (CTA)
4. Discussion The major findings of the studies were that the β2knockout mice were severely deficient in their sensitivity to the stimulus properties of nicotine in both drug discrimination and conditioned taste aversion experiments. These mice were not affected with respect to rates of lever-pressing in the undrugged state and they were also unimpaired in a discrimination procedure involving a different drug (morphine). It also appeared that there was some selectivity with respect to the effects of nicotine that were attenuated in the mutant mice; the response rate-reducing effect of nicotine, and the development of tolerance to that effect, seemed to be less critically dependent upon β2-containing receptors. The findings therefore extend, and to some extent, contrast with previous studies that had found these mutant mice to be rather similarly insensitive to nicotine across a range of behavioural procedures. The experiments presented several opportunities for comparing the effect of the β2 knockout on rates of lever-pressing in undrugged mice. This comparison was possible for the continuous reinforcement, FR-10 and tandem schedule performance prior to nicotine discrimination training, during acquisition of the nicotine discrimination, and during dose–response testing both before and after retraining of different drug discriminations. The mutation does not affect these baseline response rates, showing that functional β2-containing
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receptors are not required for performance of a simple operant response. In relation to these observations and other data considered below, it is thought that artefacts due to genetic background may be minimal because the animals had been back-crossed for 12 generations with C57BL/6J mice. The possibility that adaptive responses may have masked an effect on the baseline cannot be excluded, although, for comparing drug effects, the lack of baseline differences is a major advantage. The marked impairment of nicotine discrimination was the main finding of the experiments (Figs. 2(a), 2(b), 3(a) and 3(b)). Indeed, at the 0.4–0.8 mg/kg training doses of nicotine that are the largest doses normally used in such studies, the ability to discriminate nicotine from the non-drug state seemed to be completely lost. The dose–response studies failed to show any discriminative response to nicotine, even when the dose was increased above that used in training to a 1.6 mg/kg dose that was so large that few animals performed the lever-pressing response. These findings support the results of preliminary discrimination experiments (Oglesby et al., 1998) and indicate that β2-containing receptors play a major role in the discriminative stimulus effects of nicotine, as had been suspected from previous studies with nicotinic agonists and antagonists (Reavill et al., 1988; Stolerman et al., 1997; Gommans et al., 2000). The results appeared robust since very similar data were obtained regardless of the use of 0.4 or 0.8 mg/kg doses of nicotine for training. The similarity of the dose–response curves at these two training doses, even in wild-type animals, may be surprising since training drug discriminations with smaller doses often increases sensitivity and shifts dose– response curves to the left (Colpaert et al., 1980; Stolerman and D’Mello, 1981b). However, when the training dose of nicotine was varied (Stolerman et al., 1984, 1999), shifts in dose–response curves were less apparent than in earlier studies with psychomotor stimulants or opioids. When animals were retrained with 1.6 mg/kg of nicotine, it was possible to demonstrate relatively weak discriminative performance in the mutants (Figs. 4(a) and 5(a)). Marubio et al. (1999) have proposed that α3β4 receptors may mediate a peripheral component of the antinociceptive response to nicotine in mice; these receptors may also be considered as a candidate for mediating the discrimination of large doses of nicotine. An impairment of nicotine discrimination might be interpreted as either a diminished response to those effects of nicotine that normally support stimulus control or to an impairment in the ability to learn a conditional discrimination of the type required for performance of the two-lever task. The failure to find any impairment of the acquisition of morphine discrimination (Fig. 4(b)), and the lack of any difference between the dose– response curves for morphine in wild-type and mutant mice (Fig. 5(b)), suggest strongly that the explanation is weakened sensitivity to nicotine rather than a general
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deficit in learning ability. Preliminary observations showing intact cocaine discrimination in β2-knockout mice also support this argument (Oglesby et al., 1998). The observation that the response rate-reducing effect of nicotine was largely intact in the β2-knockout mice was made during the early stages of acquiring nicotine discrimination (Fig. 2(d)). It was confirmed during dose– response tests where relatively large doses of nicotine were needed to produce the effect in both wild-type and mutant mice, and during retraining with a larger dose of nicotine (Figs. 3(a), 3(b), 4(a) and 5(a)). The response rate-reducing effect of morphine was also present and actually slightly enhanced during retraining of mutant mice (Fig. 4(d)); this may indicate a possible interaction between the nicotinic and opioid systems that should be investigated further. However, a similar effect was not detected during dose–response tests with morphine (Fig. 5(d)). The development of tolerance to the rate-reducing effect of nicotine, as observed during the acquisition phase (Fig. 3(d)), explains adequately the need to administer large doses of nicotine to produce response ratereductions during testing (Figs. 4(c) and (d)). Earlier studies have also described tolerance to the rate-reducing effect of nicotine on operant responses in mice and rats (Hendry and Rosecrans, 1982; Villanueva et al., 1992). However, some caution should be observed in interpreting these results because the experiments were not designed primarily to study effects on response rates or tolerance; dose–response data for the acute effects of nicotine on response rates were not obtained and comparisons between animals chronically treated with nicotine and saline were not made. These caveats notwithstanding, the data suggest that β2-containing receptors may play a less critical role in the depressant effects of nicotine on rates of operant behaviour, and on tolerance to such effects, than they do in its discriminative stimulus effects. This conclusion is also compatible with evidence from studies with the antagonist DHβE, suggesting that the receptor mechanism mediating the response rate-reducing effect of nicotine in rats differed from that for the discriminative stimulus effect (Stolerman et al., 1997). The experiments on conditioned taste aversion indicate that the effect was attenuated in β2-knockout mice across the range of doses of nicotine that were studied (Fig. 6). The β2-containing receptors therefore appear to play a role in this effect. However, these studies did not control for the possibility that mutation weakened the ability of mice to learn any taste aversion, as contrasted with a selective attenuation of the response to nicotine. Previous studies on the mechanisms of nicotine-produced conditioned taste aversion have not differentiated the receptors from those involved in nicotine discrimination (Kumar et al., 1983; Reavill et al., 1986) and in this sense the present findings are not surprising.
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In summary, deletion of the gene that encodes the β2 subunits of nicotinic receptors dramatically impaired discrimination of nicotine and weakened nicotine-induced CTA. This compromised behaviour was observed in the absence of deficits in a simple lever-pressing task or in sensitivity to the unconditioned depressant action of nicotine on lever-pressing. The impairment of discrimination may be specific to nicotine since the mutant mice showed no deficits in acquisition or performance of morphine discrimination. Thus, nicotinic receptors containing β2 subunits mediate the stimulus properties of nicotine that constitute important psychological elements in nicotine dependence; β2-containing receptors appear to be of lesser significance for nicotine tolerance, a physiological adaptation that is present in the nicotine-dependent state. The involvement of β2 subunits in the stimulus properties of nicotine and their relative insignificance in nicotine tolerance is suggestive of a possible dissociation between the mechanisms of the psychological and the physiological aspects of nicotine dependence that deserves further investigation.
Acknowledgements This work was supported by a European Union TMR grant, the Medical Research Council (UK), the Colle`ge de France, the Centre National de la Recherche Scientifique, the Council for Tobacco Research, the Association pour la Recherche sur le Cancer and the Commission of the European Communities.
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