Psychopharmacology (1997) 134 : 121–130

© Springer-Verlag 1997

O R I G I NA L I N V E S T I G AT I O N

Mohammed Shoaib · Charles W. Schindler Steven R. Goldberg · James R. Pauly

Behavioural and biochemical adaptations to nicotine in rats: influence of MK801, an NMDA receptor antagonist

Received: 23 January 1997 /Final version : 8 May 1997

Abstract Chronic exposure of rats to nicotine can result in sensitization to the stimulant effects of nicotine on locomotor activity. At a biochemical level, chronic exposure to nicotine increases the number of CNS nicotinic binding sites, and this has been suggested as the basis for sensitization to nicotine. The present experiment was conducted to examine the effects of MK801, an NMDA receptor antagonist, on sensitization to nicotine. In addition, the hypothesis that MK801 may block behavioural sensitization by preventing the up-regulation of nicotinic receptors was tested by measuring receptor numbers in the same individuals using quantitative autoradiography with [3H]-cytisine and [3H]-MK801. Male Sprague-Dawley rats were chronically treated with nicotine (0.4 mg /kg SC) or saline daily for 7 days. Over the next 2 days, in a counterbalanced order, rats were challenged with nicotine (0.4 mg/kg SC) or saline and locomotor activity was monitored. In saline-pretreated rats, nicotine produced a small increase in activity. Nicotine-pretreated rats exhibited higher levels of activity following a nicotine challenge. This sensitized response was attenuated in rats administered MK801 (0.3 mg /kg IP) 30 min before each daily nicotine injection. Rats pretreated with MK801 alone showed activity scores no different from saline pretreated control groups. Biochemical studies revealed increased [3H]-cytisine binding following

M. Shoaib1 (*) · C.W. Schindler · S.R. Goldberg Preclinical Pharmacology Branch, Addiction Research Center, National Institute on Drug Abuse, Baltimore MD 21224, USA J.R. Pauly Division of Pharmacology and Experimental Therapeutics, College of Pharmacy, University of Kentucky, Lexington, KY 40536, USA Present address : 1 Section of Behavioural Pharmacology, Institute of Psychiatry, London SE5 8AF, UK Fax (+44) 171/740-5305, e-mail: [email protected]

chronic nicotine treatment; however, receptor increases were significantly attenuated by MK801 pretreatment. Binding of [3H]-MK801 remained unchanged across the four groups. The results suggest that MK801 prevents behavioural sensitization to nicotine via the prevention of receptor up-regulation. Although the findings support the notion that receptor up-regulation may be the basis for the increased responsiveness to nicotine, other interpretations are possible. Key words Nicotine · Rats · MK801 · NMDA receptor

Introduction Sensitization and tolerance are phenomena commonly observed in subjects exposed repeatedly to psychoactive substances (Goudie and Emmett-Oglesby 1989). With regard to tobacco smoking, there has been considerable interest in the neural adaptations to chronic nicotine exposure. The majority of studies in rodents have used locomotor activity as the endpoint measure and have shown that nicotine can produce biphasic effects on ambulatory behaviour. Studies that have evaluated neuroadaptations to chronic nicotine treatment have demonstrated the development of both tolerance and sensitization. The interpretation and integration of these studies is difficult, since experimental variables such as the species of animal used (e.g. rat versus mouse) and chronic treatment regimen employed (infusions versus injections) are rarely consistent between studies. In general, large doses of nicotine can profoundly depress locomotor activity, an effect to which tolerance develops, characterized by a shift of the doseresponse curve to the right (Morrison and Stephenson 1972; Stolerman et al. 1974). In contrast, the administration of small doses of nicotine can produce moderate increases in locomotor activity of rats, and repeated

122

injections of nicotine result in an upward shift of the dose-response curve, that is commonly referred to as “sensitization” (Ksir et al. 1987). Several studies have examined the effects of repeated administration of nicotine on brain nicotinic receptors, particularly in attempts to determine whether alteration of these sites would play a role in the development of tolerance or sensitization to nicotine. Many investigators using [3H]-nicotine or [3H]-ACh as radioligands have found an increase in brain nicotinic cholinergic receptors (nAChR) following chronic nicotine exposure (Marks et al. 1983; Schwarz and Kellar 1985; Ksir et al. 1987). However, the magnitude of nicotineinduced increases in nicotinic binding sites varies widely across brain regions (Pauly et al. 1991). These changes represent an increase in the number of bindings sites with no corresponding change in binding affinity. Similar increases in nAChRs were observed in post-mortem brains from smokers in comparison to brains from non-smokers (Benwell et al. 1988). Thus studies from both rodent and human subjects indicate that chronic exposure to nicotine increases the number of neuronal nAChRs. However, the significance of nicotine-induced receptor up-regulation in behaviour maintained by tobacco smoking is still unknown. This issue was recently reviewed by Collins and Marks (1996); the preponderance of evidence from studies that have investigated chronic nicotine administration in mice suggests that increases in receptor number following chronic nicotine are associated with tolerance development. The effects of chronic nicotine delivery in the rat however are less clear, since both tolerance and sensitization have been reported. Glutamate, the major excitatory amino acid neurotransmitter in the CNS, has been shown to play a critical role in behavioural adaptations to repeated drug treatments (reviewed by Trujillo and Akil 1995). Some studies have shown that MK801, a non-competitive Nmethyl-D-aspartate (NMDA) receptor antagonist can block behavioural adaptations to amphetamine and cocaine (Schenk et al. 1993; Stewart and Druhan 1993). Similar studies performed with nicotine have demonstrated that MK801 also prevents the development of both tolerance and sensitization to nicotine in rats (Shoaib and Stolerman 1992, 1996; Shoaib et al. 1994). Chronic treatment with nicotine shifted the doseresponse curve for the hyper-locomotion upwards and MK801 blocked the development of this sensitization (Shoaib and Stolerman 1992). In vivo microdialysis experiments have shown that MK801 can also prevent the development of sensitization to the increases in extracellular dopamine that nicotine produces in the nucleus accumbens (Shoaib et al. 1994). The purpose of the present studies was to extend these findings by investigating whether MK801 could attenuate nicotine-induced increases in nicotinic receptor binding. The hypothesis that MK801 may attenuate behavioural sensitization by preventing the

up- regulation of nicotinic receptors was tested by measuring receptor numbers in the same individuals using autoradiographic binding with [3H]-cytisine and [3H]MK801.

Materials and methods Subjects Male Sprague-Dawley rats (Charles River, Wilmington, Mass., USA) weighing 250–350 g were housed three per cage and maintained on a 12-h light / dark cycle with lights on at 0800 hours. Food and water were available ad libitum. Animals used in this study were maintained in facilities fully accredited by the American Association for the Accreditation of Laboratory Animal Care (AAALAC) and all experimentation was conducted in accordance with the guidelines of the Institutional Care and Use Committee of the Addiction Research Center, National Institute on Drug Abuse, NIH, and the Guide for Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council, Department of Health, Education and Welfare, Publication (NIH) 85–23, revised 1985.

Locomotor activity experiments The locomotor activating effects of nicotine were assessed in Plexiglass activity chambers (40 × 40 × 35 cm) (Columbus Instruments, Columbus, Ohio, USA). A microcomputer interfaced to the activity chambers recorded movement and by calculation determined the distance travelled in cm by the animal during a particular recording period (5 min). The distance travelled was taken as ambulatory activity exhibited by subjects.

Design of the experiment Four groups of 12 rats were used in this study. During chronic treatment, rats were injected IP with saline or MK801 (0.3 mg / kg) followed 30 min later by SC injections of saline or nicotine (0.4 mg / kg) in their home cages. These injections were given every day for 7 days. Following drug pretreatment, tests of locomotor activity were performed on 2 days. Each rat was tested in a photocell chamber for 60 min, beginning immediately after an injection of saline or nicotine (0.4 mg / kg SC). The first test took place 24 h after the last pretreatment and drug and saline were tested in a counterbalanced order within each group. Four rats from each treatment group (two rats from each counterbalanced-order subgroup) were randomly selected for quantitative autoradiographic analysis of neurotransmitter receptor binding.

Tissue preparation Immediately following the last test, animals were decapitated and once the brains were removed, they were frozen in isopentane ([35°C) and then stored at [70°C for at least 24 h. The brains were then sectioned (16 µM thick) on a Jung CM1800 cryostat and thaw-mounted onto glass sides that were coated with chrome alum and gelatin to ensure tissue adherence. Coronal sections were collected from the level of the anterior olfactory nucleus, caudally through the medial vestibular nucleus. Adjacent sets of sections were prepared so that the binding of [3H]-cytisine and [3H]- MK801 could be directly compared.

123 [3H]-cytisine binding Prior to all binding experiments, brain sections were slowly warmed to room temperature under a vacuum. For [3H]-cytisine binding, sections were pre-incubated for 30 min at 4°C in a Krebs-RingerHEPES buffer (KRH) of the following composition : NaCl (118 mM), KCl (4.8 mM), CaCl2 (2.5 mM), MgSO4 (1.2 mM), HEPES (20 mM), NaOH (10 mM) (Pauly and Collins 1993). The sections were then transferred to KRH buffer containing 5 nM [3H]cytisine (New England Nuclear; specific activity = 42 Ci /mmol) and incubated for 120 min at 4°C. The slides were then washed twice in KRH (4°C, 10 s each), twice in dilute KRH (× 0.1, 4°C, 10 s each) and once in ice cold deionized water (10 seconds). The sections were then gently dried with ambient air flow from a desktop fan. Non-specific binding was determined by incubating adjacent sections in [3H]-cytisine with the inclusion of 1 µM unlabelled nicotine. Under these conditions, the signal obtained for non-specific binding did not exceed film background.

[3H]-MK801 binding The methods described by Porter and Greenamyre (1994) were used for [3H]-MK801 binding, with minor modifications. Sections were pre-incubated for 30 min at room temperature in a 5 mM TRISHCL buffer containing 2.5 mM CaCl2. The sections were then transferred to TRIS HCL buffer containing 10 nM [3H]-MK801 (New England Nuclear; specific activity = 25 Ci/mmol), 5 µM spermidine, 100 µM glycine and 5 µM glutamic acid and incubated for 90 min at room temperature. The slides were then washed three times in 5 mM TRIS-HCL (4°C, 20 min each), once in dilute TRIS-HCL (× 0.1, 4°C, 10 s) and once in ice cold deionized water (10 s). After washing, all slides were gently air-dried and then stored overnight in a desiccator at room temperature. Unlabelled MK801 10 µM was used for non-specific binding. The binding signal in the presence of 10 µM unlabelled MK801 did not exceed film background. All slides were then exposed to tritium-sensitive Amersham Hyperfilm (3H) in Wolf X-ray cassettes that contained calibrated tissue paste 3H standards prepared from whole brain homogenates (Pauly and Collins 1993). Both [3H]-cytisine and [3H]-MK801 sections were exposed to film for 4 weeks. Films were processed in Kodak D-19 developer (5 min), indicator stop bath (30 s) and Kodak rapid fixer (5 min).

Quantification of receptor binding Receptor binding in specific brain nuclei was quantified using NIH Image software and a Power Macintosh based image analysis system (Scion LG-3 frame grabber, Sony XC-77 CCD camera and a Northern Lights desktop illuminator). Molar quantities of ligand bound were determined using values interpolated from the optical density versus tissue radioactivity standard curve. No corrections were made for tritium quenching. Binding in each brain region was quantified bilaterally on three to four sections per animal. Binding values are expressed as nCi bound per mg of wet tissue weight (SEM).

Statistics Locomotor activity data was analyzed using a two-factor analysis of variance (ANOVA) with repeated measures for time course. Differences from controls were determined using Tukey’s post hoc protected t-tests (GBSTAT, Silver Spring, Md., USA). Autoradiographic binding values were expressed as a percentage of control binding to compensate for extensive regional variation in receptor density. An arc-sin transformation of the percentage data

was performed, followed by a two-way ANOVA. Student Neuman-Keul post hoc tests were used to evaluate the effects of each individual treatment.

Results Locomotor activity Figure 1 shows the time course of locomotor activity in four groups of rats. The mean activity scores for 12 rats following injections of nicotine (0.4 mg/kg SC) and saline (1 ml / kg SC) are shown in rats pre-treated for 7 days with either saline (S-S group), nicotine (0.4 mg /kg SC, S-N group), the same dose of nicotine plus MK801 (0.3 mg / kg IP, MKN group), and MK801 (0.3 mg /kg IP, MK-S group) alone. Data following administration of saline to all groups are examined first. A two-factor ANOVA for repeated measures indicated no significant differences between the four groups [F(3,287) = 1.22, NS] with no interaction with changes in time course [F(33,287) = 0.97, NS]. However, significant changes in activity over time was observed [F(11, 287) = 58.8, P < 0.0001]. In view of the similarity of baseline activity, the effects of nicotine challenge on locomotor activity for the four groups were assessed using the same analysis. A two-factor ANOVA for repeated measures for nicotine-induced activity revealed significant differences between the four groups [F(3,287) = 3.73, P < 0.05] which interacted with time course of activity [F(33,287) = 1.89, P < 0.01]. Significant changes were observed in time course [F(11,287) = 58.8, P < 0.0001]. Post hoc analyses supported the observation of greater responsiveness to nicotine in the S-N group compared to the other groups (Fig. 1). On closer examination of the results using a two-factor ANOVA for repeated measures for each group, an analysis that took into consideration the baseline scores, only the S-N group revealed an overall significant effect of the nicotine challenge [F(1,143) = 9.9, P < 0.02] while the other groups failed to show significant differences [S-S: F(1,143) = 0.16, NS, MK-S : F(1,143) = 1.81, NS, MK-N : F(1,143) = 0.62, NS]. In rats that had no previous exposure to nicotine or MK801 (S-S), nicotine produced moderate increases in activity over the latter half of the test period (Fig. 1). These increases were augmented in rats pretreated with nicotine (S-N group), an effect that persisted throughout the 60 min test session. This sensitized response to nicotine was not apparent in animals treated with MK801 and nicotine (MK-N, Fig. 1). In rats pretreated with MK801 alone for 7 days (MK-S), the administration of nicotine produced increases in activity that were similar to the pattern of activity for rats pretreated with saline only (group S-S).

124

Fig. 1 Tests on locomotor activity after a saline (¡) and nicotine (l) challenge in rats (n = 12) pretreated for 7 days with MK801 or saline (MK or S) and nicotine or saline (N or S) for 7 days. The figure shows the effects of a saline challenge and nicotine injection tested over 2 days in a counterbalanced order. The sensitisation that develops with nicotine pretreatment is shown in the S-N group. Co-administration of MK801 30 min prior to chronic nicotine injections prevents the development of sensitisation (MK-N). Pretreatment with the non-competitive NMDA antagonist MK801 alone (MK-S group) failed to modify nicotine-induced activity. Each point represents the mean ± SEM. Significant differences (P < 0.05) in response to nicotine according to a two-way ANOVA for repeated measures followed by Tukey’s protected t-test are shown for between-group comparisons against the saline-saline (S-S) treated group (#) and within-subject comparisons of the nicotine challenge against saline (*)

Quantification of [3H]-cytisine binding Table 1 shows the effects of chronic exposure of saline (S-S), nicotine (S-N), MK801 (MK-S) or a combination of nicotine and MK801 (MK-N) on brain [3H]-cytisine binding in 21 brain regions (MK-N). Representative autoradiographs of [3H]-cytisine binding from each treatment group are shown in Figs. 2 and 3. Chronic treatment with nicotine for 7 days increased [3H]-cytisine binding in 15 of the 21 regions analyzed. On average, [3H]-cytisine binding increased by 14% in the nicotine-saline group as compared to the saline-saline group. However, the magnitude of the

changes following nicotine treatment varied across different brain regions. For example, [3H]-cytisine binding increased by approximately 30 % in the olfactory tubercle and mesencephalic central gray, while no appreciable increases were observed in the substantia nigra, dentate gyrus or the interpeduncular nucleus. An ANOVA of the four groups revealed significant differences in binding between S-N, MK-S and MK-N treated groups [F(2,62) = 9.86, P < 0.002]. Post hoc tests indicated significant differences between S-N versus MK-N (P < 0.05) and S-N versus MK-S groups (P < 0.05). When a two-factor ANOVA was performed for each brain region, significant effects of nicotine treatment were evident in the subiculum, substantia nigra, cortex 1,2, cortex 3–6, ventral posteromedial thalamus, ventrlateral geniculate nucleus, frontal cortex, central gray, nucleus accumbens, olfactory tubercle and zona incerta. These increases in binding were not found in groups co-treated with MK801. In general, pretreatment with MK801 attenuated nicotineinduced increases in [3H]-cytisine binding, reaching statistical significance in six of the 15 regions. For example, in the nucleus accumbens, chronic nicotine treatment increased [3H]-cytisine binding by 17%; this up-regulation was attenuated to 3 % of control in rats treated chronically with MK801 and nicotine (Table 1). Chronic administration of MK801 failed to significantly modify binding of [3H]-cytisine in all 21 regions (Table 1).

125 Table 1 The effects of chronic saline (S-S), nicotine (S-N), MK801 (MK-S) or the combination of MK801 and nicotine (MK-N) on brain regional binding of [3H]-cytisine. Autoradiographs were produced and analysed as previously described. Data represent nCi [3H]-cytisine bound per mg wet tissue weight (mean ± SEM; n = 4 per group). Animals that received chronic nicotine injections tended to have increased numbers of receptors in some brain regions. MK801 alone had no effect on [3H]-cytisine binding. When MK801 was administered prior to each daily nicotine injection, nicotine-induced receptor up-regulation was attenuated. (*Different from S-S, + different from S-N)

Brain region

S-S

S-N

MK-S

MK-N

Caudate putamen Cerebral Cx (layers 1,2) Cerebral Cx (layers 3-6) Cingulate Cx Dentate gyrus Dorsolateral geniculate n. Frontal Cx Interpeduncular n. Medial geniculate n. Medial habenula Mesencephalic central gray Nucleus accumbens Olfactory tubercle Posterolateral thalamic n. Retrosplenial Cx Subiculum Substantia nigra Superior colliculus Ventral posteromedial thal. Ventrolateral geniculate n. Zona incerta

0.80 ± 0.07 0.68 ± 0.05 0.64 ± 0.04 0.94 ± 0.04 0.65 ± 0.06 1.03 ± 0.01 0.85 ± 0.05 1.77 ± 0.09 0.93 ± 0.01 1.05 ± 0.01 0.44 ± 0.05 0.75 ± 0.07 0.52 ± 0.06 1.01 ± 0.02 1.05 ± 0.01 0.80 ± 0.02 0.86 ± 0.01 1.04 ± 0.01 0.92 ± 0.02 0.91 ± 0.03 0.25 ± 0.02

0.92 ± 0.02* 0.82 ± 0.01* 0.76 ± 0.03* 1.01 ± 0.01* 0.74 ± 0.03 1.06 ± 0.01* 0.96 ± 0.01* 1.92 ± 0.08 0.98 ± 0.03* 1.07 ± 0.01* 0.56 ± 0.05* 0.88 ± 0.02* 0.67 ± 0.04* 1.05 ± 0.01 1.05 ± 0.01 0.91 ± 0.01* 0.91 ± 0.03 1.05 ± 0.01 1.01 ± 0.01* 0.98 ± 0.02* 0.40 ± 0.06*

0.87 ± 0.03 0.69 ± 0.03 0.62 ± 0.06 0.96 ± 0.03 0.70 ± 0.04 1.05 ± 0.01 0.87 ± 0.03 1.97 ± 0.04 0.92 ± 0.03 1.08 ± 0.01 0.51 ± 0.03 0.74 ± 0.05 0.53 ± 0.06 1.04 ± 0.01 1.04 ± 0.01 0.85 ± 0.01 0.88 ± 0.02 1.05 ± 0.00 0.97 ± 0.02 0.96 ± 0.02 0.34 ± 0.05

0.82 ± 0.05+ 0.70 ± 0.05+ 0.69 ± 0.04 0.97 ± 0.02 0.71 ± 0.05 1.03 ± 0.02 0.89 ± 0.02 1.78 ± 0.09 0.93 ± 0.02 1.06 ± 0.01 0.46 ± 0.05 0.77 ± 0.04+ 0.56 ± 0.04+ 1.02 ± 0.02 1.05 ± 0.01 0.81 ± 0.03+ 0.84 ± 0.02 1.04 ± 0.01 0.96 ± 0.02 0.92 ± 0.03 0.33 ± 0.05+

Quantification of [3H]-MK801 binding Fig. 2A–D Autoradiographs depicting the effects of chronic injections of A S-S, B S-N, C MK-S and D MK-N on rat brain [3H]cytisine binding in forebrain regions. Brain [3H]-cytisine binding was increased in most brain regions after chronic treatment with nicotine. Chronic treatment with the combination of MK801 and nicotine (MK-N) prevented the nicotinic receptor up-regulation. NA Nucleus accumbens, CP caudate putamen, CX3 cerebral cortex layer 3

Table 2 shows the effects of chronic exposure of saline (S-S), nicotine (S-N), MK801 (MK801-S) or a combination of nicotine and MK801 on brain [3H]-MK801 binding in 21 brain regions. Representative autoradiographs of [3H]-MK801 binding from each treatment group are shown in Figs 4 and 5. Despite significantly higher levels of binding to [3H]-MK801 throughout the

126

Fig. 3A–D Autoradiographs depicting the effects of chronic injections of A S-S, B S-N, C MK-S and D MK-N on rat brain [3H]cytisine binding in midbrain regions. Brain [3H]-cytisine binding was increased in most brain regions after chronic treatment with nicotine. Chronic treatment with the combination of MK801 and nicotine prevented the nicotinic receptor up-regulation. SUB Subiculum, MG medial geniculate nucleus, SN substantia nigra

MK801 binding to decrease in the MK-S group. However, due to the large degree of variability between the different brain regions, statistical tests failed to confirm this.

Discussion brain, there were no significant differences between the four treatment groups. Considerable levels of [3H]MK801 binding were observed in the dentate gyrus (Fig. 5), caudate (Fig. 4), cortex, septum (Fig. 5) and subiculum (Fig. 5). There was a tendency for [3H]Table 2 The effects of chronic saline (S-S), nicotine (S-N), MK801 (MK-S) or the combination of MK801 and nicotine (MK-N) on brain regional binding of [3H]-MK801. Autoradiographs were produced and analysed as previously described. Data represent the nCi of [3H]-MK801 bound per mg wet tissue weight (mean ± SEM; n = 4 per group). ANOVA results suggest that there were no treatment differences in [3H]-MK801 binding

As previously reported (Shoaib and Stolerman 1992; Shoaib et al. 1994), MK801 attenuated the development of behavioural sensitization to nicotine in rats. This confirmation provided a starting point to test our hypothesis that MK801 attenuates behavioural

Brain region

S-S

S-N

MK-S

MK-N

Caudate putamen Dentate gyrus Interpeduncular n. Medial geniculate n. Mesencephalic central gray Nucleus accumbens Olfactory tubercle Parietal Cx Retrosplenial Cx Septum Subiculum Substantia nigra Superior colliculus Ventral posteromedial thalamus Zona incerta

2.76 ± 0.31 6.90 ± 0.75 0.80 ± 0.09 2.50 ± 0.30 0.90 ± 0.10 3.22 ± 0.34 3.04 ± 0.27 4.14 ± 0.29 2.47 ± 0.26 3.74 ± 0.56 2.22 ± 0.17 0.65 ± 0.10 1.90 ± 0.17 1.88 ± 0.17 0.70 ± 0.09

2.64 ± 0.18 6.59 ± 0.24 0.70 ± 0.06 2.16 ± 0.21 0.76 ± 0.06 3.06 ± 0.22 2.90 ± 0.48 3.99 ± 0.22 2.13 ± 0.16 3.48 ± 0.18 2.05 ± 0.16 0.59 ± 0.05 1.63 ± 0.01 1.94 ± 0.17 0.62 ± 0.08

2.21 ± 0.10 6.91 ± 0.30 0.79 ± 0.07 2.11 ± 0.21 0.83 ± 0.07 2.51 ± 0.12 2.84 ± 0.11 3.51 ± 0.10 2.18 ± 0.16 3.12 ± 0.06 2.10 ± 0.17 0.64 ± 0.05 1.74 ± 0.13 1.74 ± 0.10 0.67 ± 0.08

2.35 ± 0.13 6.49 ± 0.31 0.65 ± 0.06 1.83 ± 0.08 0.70 ± 0.06 2.80 ± 0.10 2.44 ± 0.36 3.47 ± 0.13 1.97 ± 0.07 3.26 ± 0.09 1.82 ± 0.07 0.49 ± 0.04 1.56 ± 0.06 1.57 ± 0.07 0.52 ± 0.06

127

Fig. 4A–D Autoradiographs depicting the effects of chronic injections of A S-S, B S-N, C MK-S and D MK-N on rat brain [3H]MK801 binding in forebrain regions. Brain [3H]-MK801 binding remained unchanged in all brain regions after chronic treatment with either MK801 or nicotine (MK-N). CP Caudate putamen, OT olfactory tubercle

sensitization by preventing nicotinic receptor up-regulation. Chronic treatment with nicotine significantly increased cytisine binding in the identical paradigm that produced sensitization to the locomotor activating effects of nicotine. Nicotine-induced increases in cytisine binding were significantly attenuated in animals pretreated with MK801. In contrast, chronic administration of MK801 alone for 7 days failed to produce any changes in the behavioural responsiveness to nicotine and binding to cytisine or MK801. The first part of the experiment examined the effects of MK801 on locomotor activity. In saline-pretreated rats, nicotine produced a small degree of locomotor activation, a response that was augmented in nicotinetreated rats. This adaptation to nicotine was attenuated when MK801 was administered 30 min before each daily dose of nicotine. The magnitude of sensitization observed in the present study was a little weaker than that reported in previous studies (Shoaib and Stolerman 1992; Shoaib et al. 1994). The short period of chronic treatment along with the single test challenge with nicotine may explain this apparent difference from previous studies (Shoaib and Stolerman 1992; Shoaib et al. 1994).

The second part of the experiment examined the effects of MK801 on radioligand binding to [3H]-cytisine and [3H]-MK801 in the same groups of rats used for the locomotor tests. As previously reported with chronic nicotine treatment and nicotinic receptor upregulation (Hulihan-Giblin et al. 1990; El Bizri and Clarke 1994; Zhang et al. 1994), 7 days of daily nicotine administration produced increases in the number of nicotinic binding sites in a number of brain regions. This upregulation was observed with [3H]-cytisine binding, a ligand known to bind to the a4b2 isoform of nicotinic receptors with high affinity similar to nicotine. An average increase of 14 % was observed for all the 21 brain regions examined. In a similar manner to the behavioural data, this biochemical adaptation to nicotine was blocked when MK801 was administered 30 min before each daily dose of nicotine. Chronic treatment of MK801 alone failed significantly to modify binding to cytisine. The increase in [3H]-cytisine binding most likely reflects an increase in the Bmax, since ligand binding was performed only at a single isotope concentration, there is a possibility that differences in receptor affinity could explain these differences. Our results demonstrate that a short regimen of chronic nicotine treatment consisting of daily injections for 7 days can produce upregulation of nicotine receptors in rat brain. In a pilot experiment, this regimen caused changes in [3H]-cytisine binding to the a4b2 nAChR but not [125I]-bungarotoxin binding to the a7 nAChR. This finding is consistent with other studies which have shown that receptors require extended

128

Fig. 5A–D Autoradiographs depicting the effects of chronic injections of A S-S, B S-N, C MK-S and D MK-N on rat brain [3H]MK801 binding in midbrain regions. Brain [3H]-MK801 binding remained unchanged in all brain regions after chronic treatment with either MK801 or nicotine (MK-N). SC Superior colliculus, DG dentate gyrus, MG medial geniculate nucleus

treatment periods of high nicotine doses to induce receptor upregulation. [3H]-cytisine binding was significantly increased by the chronic nicotine regimen employed in approximately 50% of the brain regions quantified. [3H]-Cytisine binding in animals that received MK801 and nicotine was not different from control levels in any brain region. It is possible that receptor upregulation in some of these regions, for example the nucleus accumbens and caudate putamen may be relevant in mediating the locomotor activating properties of nicotine. Microinjection and lesion studies have shown that the nucleus accumbens represents a locus that mediates the locomotor hyperactivity properties of nicotine (Clarke et al. 1988; Museo and Wise 1990), an effect thought to be produced via the release of dopamine in the terminal region (Imperato et al. 1986). The small but significant increases in nicotinic receptors observed in the present study are consistent with previous studies using similar regimens of chronic nicotine exposure. Treating rats twice per day with 0.45 mg/kg nicotine for 18 days, Zhang et al. (1994) measured increases of 15–20% in rat brain binding. El-Bizri and Clarke (1994), treating rats with twice daily

injections of 0.6 mg/kg nicotine for 12 days, observed a 19% increase in cortical nicotine binding. Similarly, Hulihan-Giblin et al. (1990), treating rats twice daily with 0.8 mg/kg for 10 days reported an increase of 30% in the cortex. Thus our protocol of giving a lower dose of nicotine for a shorter period of time would be expected to produce smaller increases in receptor number. Nevertheless, significant increases were measured in 15 of the 21 regions analyzed. MK801 significantly attenuated receptor increases in each brain region quantified. From the present experiment, it may not be surprising to find a close association between behavioural and biochemical observations with nicotine. A similar relationship was observed from a series of studies conducted in mice (Marks et al. 1985). A time course study revealed a close association between the response to nicotine and the number of brain nicotinic receptors. The best correlation was observed with activity measured in a Y-maze. Chronic infusion of nicotine to mice resulted in tolerance developing to locomotor activity 4 days from the start of the infusion and this was closely associated with the time course for increases in brain nicotinic receptors to develop. Further, the loss of tolerance after the chronic drug treatment and the return of the increased nicotine binding to baseline levels supported the hypothesis that adaptations to behavioural effects of nicotine were linked to changes at the receptor level. However, the effects of nicotine on physiological measures such as heart rate and body temperature did not show a strong association with the number of brain nicotinic receptors (Marks et al. 1985).

129

Associations between binding and behaviour are difficult to resolve, particularly when the time course of changes is considered. For example, in rats, Stolerman et al. (1974) reported that sensitized responses to nicotine persist for extended periods after cessation of the pretreatment schedule. It is likely that extended sensitization persists for longer than the time required for return to baseline of neuronal nAChR number; however, this has not been tested empirically. Another feature that complicates comparisons to other studies is the fact that mice generally do not express sensitized responses to nicotine. Therefore, it becomes difficult to relate changes such as sensitization to the receptor level. Measurement of both behaviour and binding over different times, as shown for mice (Marks et al. 1985), will substantially increase our understanding of the biochemical basis of behavioural sensitization. Recent studies using in vivo microdialysis clearly indicate the expression of neurochemical sensitization correlates with behavioural responsiveness (Marshall et al. 1997). The mechanism of action of MK801 in preventing behavioural adaptation is unclear. Our previous work using behavioural and biochemical measures indicated a possible brain region implicated in producing these changes in responsiveness to nicotine. In vivo microdialysis experiments suggested the nucleus accumbens was a possible locus for the action of MK801 (Shoaib et al. 1994). Our previous report showed that both MK801 and D-CPPene, a competitive NMDA receptor antagonist, could attenuate sensitization to the dopamine-releasing effects of nicotine in the nucleus accumbens. The findings from the present binding experiment support this view, since up-regulation of nicotinic bindings sites in the nucleus accumbens was prevented by MK801 pretreatment and numerous MK801 binding sites were observed in the nucleus accumbens. In addition to blocking the ion channels of NMDA receptors (Wong et al. 1986; Heuttner and Bean 1988), MK801 is also capable of blocking the ion channels of nicotinic receptors (Ramoa et al. 1990; Amador and Dani 1991). However, we have argued that the effects of MK801 on behavioural adaptations to nicotine is more likely due to an antagonist effect at NMDA receptors rather than nicotinic blockade (Shoaib et al. 1994; Shoaib and Stolerman 1996). Interestingly, mecamylamine, a non-competitive antagonist of nicotine receptors when administered concurrently with nicotine failed to prevent the up-regulation of nicotinic binding sites in mice (Pauly et al. 1996). Rather, there was an additive increase in receptor levels in many of the brain regions. It would be interesting to evaluate alterations in behavioural sensitivity to nicotine in animals that received chronic nicotine, mecamylamine or the combination of the drugs. In summary, the present findings suggest that MK801 prevents adaptation to the behavioural effects

of nicotine via the prevention of upregulation of the isoform of nicotinic receptors. Specifically, the present results support the notion that receptor up-regulation may be responsible for the increased responsiveness to nicotine. Our previous report on functional changes measured using in vivo microdialysis support this view. Taken together, it seems reasonable to speculate that MK801 prevents adaptations to chronic nicotine administration by disrupting processes underlying the upregulation of central nicotinic receptors. The possibility remains, however, that nicotine-induced receptor upregulation and the expression of behavioural sensitization to nicotine occur, simultaneously but are physiologically unrelated events.

References Amador M, Dani JA (1991) MK-801 inhibition of nicotinic acetylcholine receptor channels. Synapse 7 : 207–215 Benwell MEM, Balfour DJK, Anderson JM (1988) Evidence that tobacco smoking increases the density of ([)-[3H]nicotine binding sites in human brain. J Neurochem 50 : 1243–1247 Clarke PBS, Fu DS, Jakubovic A, Fibiger HC (1988) Evidence that mesolimbic dopaminergic activation underlies the locomotor stimulant action of nicotine in rats. J Pharmacol Exp Ther 246 : 701–708 Collins AC, Marks MJ (1996) Are nicotinic receptors activated or inhibited following chronic nicotine treatment? Drug Dev Res 38 : 231–242 El-Bizri H, Clarke PBS (1994) Regulation of nicotinic receptors in rat brain following quasi-irreversible nicotinic blockade by chlorisondamine and chronic treatment with nicotine. Br J Pharmacol 111 : 917–925 Goudie AJ, Emmett-Oglesby MW (1989) Psychoactive drugs : tolerance and sensitisation. Humana Press, Clifton, New Jersey Huettner JE, Bean BP (1988) Blockade of NMDA-activated current by the anticonvulsant MK-801 : selective binding to open channels. Proc Natl Acad Sci USA 85 : 1307–1311 Hulihan-Giblin BA, Lumpkin MD, Kellar KJ (1990) Effect of chronic administration of nicotine on prolactin release in the rat: inactivation of prolactin response by repeated injections of nicotine. J Pharmacol Exp Ther 252 : 21–25 Imperato A, Mulas A, Di Chiara G (1986) Nicotine preferentially stimulates dopamine release in the limbic system of freely moving rats. Eur J Pharmacol 132 : 337–338 Ksir CJ, Hakan RL, Kellar KJ (1987) Chronic nicotine and locomotor activity : influences of exposure dose and test dose. Psychopharmacology 92 : 25–29 Marks MJ, Burch JB, Collins AC (1983) Effects of chronic nicotine infusion on tolerance development and nicotinic receptors. J Pharmacol Exp Ther 226 : 817–825 Marks MJ, Stitzel JA, Collins AC (1985) Time course study of the effects of chronic nicotine infusion on drug response and brain receptors. J Pharmacol Exp Ther 235 : 619–628 Marshall DL, Redfern PH, Wonnacott S (1997) Presynaptic nicotinic modulation of dopamine release in the three ascending pathways studied by in vivo microdialysis : comparison of naive and chronic nicotine-treated rats. J Neurochem 68 : 1511–1519 Morrison CF, Stephenson JA (1972) The occurrence of tolerance to a central depressant effect of nicotine. Br J Pharmacol 46 : 151–156 Museo E, Wise RA (1990) Microinjections of a nicotinic agonist into dopamine terminal fields: effects on locomotion. Pharmac Biochem Behav 37 : 113–116

130 Pauly JR, Collins AC (1993) An autoradiographic analysis of nicotinic cholinergic receptors following chronic corticosterone treatment. Neuroendocrinology 57:262–271 Pauly JR, Marks MJ, Stitzel JA, Gross SD, Collins AC (1991) Chronic nicotine infusions and regulation of CNS nicotinic cholinergic receptors: an autoradiographic study. J Pharmacol Exp Ther 258: 1127–1136 Pauly JR, Marks MJ, Robinson SF, van de Kamp JL, Collins AC (1996) Chronic nicotine and mecamylamine treatment increase brain nicotinic receptor binding without accompanying changes in 4 or 2 mRNA. J Pharmacol Exp Ther 278:361–369 Porter RHP, Greenamyre JT (1994) Inhibition of autoradiographic MK-801 binding by an endogenous factor. Neurosci Lett 169 : 105–108 Ramoa AS, Alkondon M, Aracava Y, Irons J, Lunt GG, Desphande SS, Wonnacott S, Aronstam RS, Albuquerque EX (1990) The anticonvulsant MK-801 interacts with the peripheral and central nicotinic acetylcholine receptor ion channels. J Pharmacol Exp Ther 254:71–82 Schenk S, Valadez A, McNamara C, House DT, Higley D, Bankson MG, Horger BA (1993) Development and expression of sensitization to cocaine’s reinforcing properties: role of NMDA receptors. Psychopharmacology 111:332–338 Schwartz RD, Kellar KJ (1985) In vivo regulation of (3H)acetylcholine recognition sites in brain by nicotinic cholinergic drugs. J Neurochem 45:427–433 Shoaib M, Stolerman IP (1992) MK801 attenuates behavioural adaptation to chronic nicotine administration in rats. Br J Pharmacol 105:514–515

Shoaib M, Stolerman IP (1996) The NMDA antagonist dizocilpine (MK801) attenuates tolerance to nicotine in rats. J Psychopharmacol 10 : 214–218 Shoaib M, Benwell MEM, Akbar MT, Stolerman IP, Balfour DJK (1994) Behavioural and neurochemical adaptations to nicotine in rats : influence of NMDA antagonists. Br J Pharmacol 111 : 1073–1080 Stewart J, Druhan JP (1993) Development of both conditioning and sensitization of the behavioural activating effects of amphetamine is blocked by the non-competitive NMDA receptor antagonist. Psychopharmacology 110 : 125–132 Stolerman IP, Bunker P, Jarvik ME (1974) Nicotine tolerance in rats : role of dose and dose interval. Psychopharmacologia 34 : 317–324 Trujillo KA, Akil H (1995) Excitatory amino acids and drugs of abuse : a role for N-methyl-D-aspartate receptors in drug tolerance, sensitization and physical dependence. Drug Alcohol Depend 38 : 139–154 Wong EF, Kemp JA, Priestley T, Knight AR, Woodruff GN, Iversen LL (1986) The anticonvulsant MK-801 is a potent Nmethyl-D-aspartate antagonist. Proc Natl Acad Sci USA 83 : 7104–7108 Zhang X, Gong Z, Nordberg A (1997) Effects of chronic treatment with (+)- and ([)-nicotine on nicotinic acetylcholine receptors and N-methyl-D-aspartate receptors in rat brain. Brain Res 644 : 32–39