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NEUROPHARMACOLOGY

Differential Regulation of Mesolimbic 3^*/62^,* and 42^,* Nicotinic Acetylcholine Receptor Sites and Function after Long-Term Oral Nicotine to Monkeys

The Parkinson's Institute, Sunnyvale, California (S.E.M., N.P., T.B., M.Q.); Department of Radiology, Johns Hopkins University School of Medicine, Baltimore, Maryland (H.F.); and Department of Biology and Psychiatry, University of Utah, Salt Lake City, Utah (J.M.M.)

Received March 11, 2006; accepted April 17, 2006.

	Abstract

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Because the mesolimbic dopamine system plays a critical role in nicotine addiction/reinforcement and because nicotinic receptors regulate dopamine release, we initiated a study to evaluate the long-term effects of nicotine (>6 months at the final dose) on nicotinic acetylcholine receptor (nAChR) sites and function in the nucleus accumbens of nonhuman primates. Nicotine was given in the drinking water as this mode of administration is long-term but intermittent, thus resembling smoking in this aspect. We determined the effects of nicotine treatment on function and binding of the 3/62^* and 42^* nAChRs subtypes in nucleus accumbens, a region directly implicated in the addictive effects of nicotine. To evaluate function, we measured nicotine and K⁺-evoked [³H]dopamine release from nucleus accumbens synaptosomes. Changes in 42^* and 3/62^* nAChRs were measured using ¹²⁵I-epibatidine, [¹²⁵I]A85380 [5-[¹²⁵I]iodo-3(2(S)-azetidinylmethoxy) pyridine] and ¹²⁵I--conotoxin MII autoradiography. Chronic nicotine treatment, which led to plasma nicotine levels in the range of smokers, significantly increased nucleus accumbens 42^* nAChR sites and function compared with control. By contrast, this treatment did not significantly change 3/62^* nAChR sites or evoked dopamine release in this region compared with control. Thus, these data are distinct from previous results in striatum in which the same nicotine treatment paradigm decreased striatal 3/62^* nAChR sites and function. The finding that long-term nicotine treatment selectively modulates 42^* and not 3/62^* nAChR expression in primate nucleus accumbens is consistent with the results of studies in nicotinic receptor mutant mice implicating the 42^* nAChR subtype in nicotine-mediated addiction.

Diverse populations of nAChRs are distributed throughout the mesolimbic dopamine pathway. 42^*, 62^*, and 7^* nAChR subtypes have been identified on dopamine cell bodies in the ventral tegmental area and nerve terminals in the nucleus accumbens (Le Novere et al., 1996; Quik et al., 2000, 2001; Klink et al., 2001; Champtiaux et al., 2003; Wooltorton et al., 2003). Although there are multiple nAChR populations in the mesolimbic dopamine system, the 42^* subtype seems primarily responsible for mediating nicotine's reinforcing effects. This contention is based on studies using nAChR null mutant mice, which demonstrated a critical role for the 2 subunit in nicotine self-administration and conditioned place preference (Picciotto et al., 1998; Klink et al., 2001; Walters et al., 2006). Moreover, 2 subunit re-expression in the ventral tegmental area of 2 null mutant mice led to a recovery of nicotine self-administration (Maskos et al., 2005). The selective activation of 4^* nAChRs in mice expressing a mutant form of the receptor indicates that receptors containing 4^* are sufficient for nicotine-induced reward, tolerance, and sensitization (Tapper et al., 2004).

Most studies concerning the reinforcing effects of nicotine use acute administration of nicotine. However, because smoking is a chronic behavior that leads to long-term adaptive changes in the brain, knowledge of the chronic changes is essential for understanding smoking behavior and implementing effective treatments for smoking cessation. One consequence of long-term nicotine exposure is an altered regulation of central nervous system nicotinic receptors in both animal models and smokers. The 42^* nAChR population is increased in rodent brain, with chronic nicotine treatment, and is also elevated in the brains of smokers (Marks et al., 1983; Schwartz and Kellar, 1983; Benwell et al., 1988). Other neuronal nAChR subtypes seem to respond in a different manner with nicotine administration, with a down-regulation of 62^* nAChRs (Nguyen et al., 2003; Lai et al., 2005; Mugnaini et al., 2006) and no change in 3^* subtypes (Ulrich et al., 1997; Davila-Garcia et al., 2003).

These differential changes in nAChR subtypes with long-term nicotine treatment may lead to complex effects on receptor-mediated activity, particularly in brain regions in which several nAChR subtypes regulate the same function. For instance, striatal dopamine release is modulated by both 42^* and 62^* nAChRs in rodents (Champtiaux et al., 2003; Salminen et al., 2004b) and in monkeys (McCallum et al., 2006). Recent studies have shown that binding sites and function of these two distinct subtypes are differentially regulated in striatum, with long-term nicotine exposure resulting in an increase in 42^* and a simultaneous decrease in 62^* nAChR sites and function (Salminen et al., 2004a; Lai et al., 2005; McCallum et al., 2006; Mugnaini et al., 2006).

The objective of the present study was to investigate whether chronic nicotine administration resulted in a similarly complex regulation of nAChR binding sites and function in the nucleus accumbens, a region critically involved in regulating the reinforcing effects of nicotine. To approach this, we measured both 42^* and 3/62^* nAChR-mediated function and binding sites after nicotine treatment in the nucleus accumbens of nonhuman primates, an animal model with a close behavioral resemblance to man. Results show that only 42^* and not 3/62^* nAChR sites and function were altered with chronic nicotine treatment, suggesting that 42^* nAChRs play a more prominent role in modulating the effects of long-term nicotine treatment in nucleus accumbens.

	Materials and Methods

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Animals and Treatments. The experiments with drug-naive female squirrel monkeys (n = 13) (Saimiri sciureus) were performed as described previously (McCallum et al., 2006), according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee at the Parkinson's Institute. Monkeys, ranging in weight from 0.5 to 0.7 kg, were obtained from Osage Research Primates (Osage Beach, MO) and from the Primate Research Laboratory (University of South Alabama, Mobile, AL). The animals' history, together with their general appearance, suggested that they were in late adulthood (10-15 years). They were immediately quarantined in accordance with California State regulations. The animals were housed in a room with a 13:11-h light/dark cycle and given food once daily (at quantities to maintain body weight) with free access to water. Quarantine was followed by a 1-month acclimation period. The animals were subsequently given either 1% saccharin (n = 7) or nicotine (free base) plus 1% saccharin (n = 6) in their drinking water, with both groups run in parallel. In brief, nicotine was increased by 50 µg/ml-increments over a 3-month period up to 650 µg/ml, a concentration at which the animals were maintained for an additional 6 to 8 months. This treatment regimen resulted in plasma nicotine and cotinine levels of 8.3 ± 2.6 ng/ml (n = 6) and 275 ± 84.3 ng/ml (n = 6), respectively, as reported previously (McCallum et al., 2006). Nicotine levels were within the reported range (5-50 ng/ml) detected in smokers (Hukkanen et al., 2005). Fluid intake was not significantly different between the two groups. Nicotine was removed from the drinking water 24 h before death. The animals were euthanized according to the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association by injecting 1.5 ml of euthanasia solution intraperitoneally (390 mg of sodium pentobarbital and 50 mg of phenytoin sodium/ml) followed by 1.5 ml/kg of the same solution intravenously.

Tissue Preparation. The brains were removed from the skull and divided along the midline with a dissecting knife. One half was placed in a Plexiglas monkey brain mold and sectioned into 6-mm blocks, which were each quickly frozen on a glass slide in isopentane on dry ice and stored at -80°C for autoradiographic studies. The other half was cut into 2-mm-thick blocks. The nucleus accumbens was hand-dissected from block A15-A14 for the measurement of dopamine and metabolites and preparation of synaptosomes for the [³H]dopamine uptake and release experiments.

Determination of Dopamine and Its Metabolites. Approximately 5 mg of tissue was dissected from the nucleus accumbens from each animal and extracted into 250 µl of 0.4 N perchloric acid. Dopamine and its metabolites, homovanillic acid and dihydroxyphenylacetic acid (DOPAC), were determined using high-pressure liquid chromatography coupled to electrochemical detection (Quik et al., 2001). The rate of dopamine turnover was calculated as the ratio of dopamine to its metabolites (dopamine/{DOPAC + homovanillic acid}).

Uptake and Release of [³H]Dopamine. The nucleus accumbens (10-15 mg wet weight) was homogenized in 2 ml of ice-cold 0.32 M sucrose buffered with 5 mM HEPES, pH 7.5. Synaptosomes were prepared, and [³H]dopamine uptake measured as described previously (Grady et al., 2002; McCallum et al., 2005, 2006). In brief, synaptosomes (250 µg of tissue/ml) were preincubated at 37°C for 10 min in buffer containing 128 mM NaCl, 2.4 mM KCl, 3.2 mM CaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 25 mM HEPES, pH 7.5, 10 mM glucose, 1 mM ascorbic acid, and 0.01 mM pargyline. This was followed by incubation (2 or 10 min) in the same buffer also containing 0.5 µCi of [³H]dopamine (final concentration of 100 nM). Uptake was terminated by the addition of ice-cold buffer followed by vacuum-filtration through type A/E glass fiber filters. After washing, radio-activity on the filters was determined using liquid scintillation counting.

[³H]Dopamine release was done by preincubation (37°C) of another aliquot of the same synaptosomal preparation as above for 10 min followed by a 5-min incubation with 100 nM [³H]dopamine (Grady et al., 2002; McCallum et al., 2005, 2006). An aliquot of synaptosomes (0.5-1 mg of tissue) was placed onto A/E glass-fiber filters. After a 10-min baseline period, release was stimulated by an 18-s exposure to nicotine or K⁺. Filters were perfused with or without 50 nM -conotoxin MII for 3 min just before nicotine exposure to determine 42^* and 3^*/62^* nAChR-mediated release. Fifteen 18-s fractions were collected. Radioactivity was determined by liquid scintillation counting.

[³H]Dopamine release was quantified as described previously (McCallum et al., 2005) by plotting release as fraction number against counts per minute, using a curve-fitting algorithm of SigmaPlot 5.0 for MicroSoft Disk Operating System (MS-DOS) (Jandel Scientific, San Rafael, CA) to fit the data. Basal release was assessed from fractions just before and after the stimulated release. Release was normalized to the tissue wet weight to obtain units of counts per minute/milligram of tissue. R_max and EC₅₀ values were obtained from dose-response curves by fitting data to a nonlinear regression equation in SigmaPlot 2001 for Windows (SPSS Inc., Chicago, IL).

Receptor Autoradiography. Sections (20 µm) were cut using a cryostat at -15°C. After thaw-mounting onto Superfrost Plus slides, sections were air-dried and stored at -80°C. Nonspecific binding was determined using 100 µM nicotine or 100 nM epibatidine. After each experiment, slides were exposed to Kodak MR film (Eastman Kodak (Rochester, NY) with ¹²⁵I standards for 2 to 5 days, depending on the radioligand used.

¹²⁵I--Conotoxin MII autoradiography was performed as described previously (Quik et al., 2001). Thawed sections were preincubated at 22°C for 15 min in 20 mM HEPES buffer, pH 7.5, containing 144 mM NaCl, 1.5 mM KCl, 2 mM CaCl₂, 1 mM MgSO4, 0.1% bovine serum albumin, and 1 mM phenylmethylsulfonyl fluoride. They were then incubated for 1 h with 0.5 nM ¹²⁵I--conotoxin MII in buffer with 0.5% bovine serum albumin, 5 mM EDTA, 5 mM EGTA, and 10 µg/ml each of aprotinin, leupeptin, and pepstatin A, followed by washing in HEPES buffer.

[¹²⁵I]A85380 [5-[¹²⁵I]iodo-3(2(S)-azetidinylmethoxy) pyridine, 0.2 nM, 1450 Ci/mmol] binding to brain sections was done at 22°C for 60 min. The buffer consisted of 50 mM Tris, pH 7.0, 120 mM NaCl, 5 mM KCl, 2.5 mM CaCl₂, and 1.0 mM MgCl₂, with or without 100 nM -conotoxin MII. Sections were washed in buffer as described previously (Kulak et al., 2002).

For [¹²⁵I]epibatidine binding, slides were preincubated at 22°C for 30 min in buffer containing 50 mM Tris, pH 7.5, 120 mM NaCl, 5 mM KCl, 2.5 mM CaCl₂, and 1.0 mM MgCl₂. This was followed by a 40-min incubation with 0.015 nM ¹²⁵I-epibatidine in the presence or absence of -conotoxin MII (100 nM). Sections were washed as described previously (McCallum et al., 2006). Optical density measurements from the autoradiograms were determined using ImageQuant (Amersham Biosciences Inc., Piscataway, NJ). They were converted to nCi/mg tissue using standard curves generated from ¹²⁵I standards, with optical density readings within the linear range of the film. The receptor binding values for any one animal were averaged from two to four independent experiments for each radioligand.

Data Analyses. Statistical analyses were done with the Prism program (GraphPad Software, Inc., San Diego, CA). Student's unpaired t test was used for comparison between the two different treatment groups, as indicated in the legends. Two-way repeated measures analysis of variance (ANOVA), followed by Bonferroni post hoc tests, was used for comparison of dose-response curves for the two treatment groups, with the independent variables being treatment versus nicotine concentration used in the release assay. Values are expressed as the mean ± S.E.M. of the indicated number of animals. Values were considered statistically significantly different when p < 0.05.

Materials. [³H]Dopamine (30-60 Ci/mmol) and [¹²⁵I]epibatidine (2200 Ci/mmol) were obtained from PerkinElmer Life and Analytical Sciences (Boston, MA). ¹²⁵I standards were from Amersham Biosciences Inc. Saccharin and nicotine were from Sigma (St. Louis, MO); A/E glass-fiber filters were from Gelman, Inc. (Ann Arbor, MI), Econosafe cocktail was from RPI (Mt. Prospect, IL). Kodak MR film was from Amersham Biosciences Inc., and Superfrost Plus slides were from Fisher (Pittsburgh, PA).

	Results

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Effect of Long-Term Oral Nicotine Treatment on the Level of Dopamine and Metabolites in Nucleus Accumbens. Experiments were first done to investigate the effect of chronic nicotine treatment on the levels of dopamine and its metabolites DOPAC and homovanillic acid in nucleus accumbens tissue from control and nicotine-treated monkeys (Table 1). Although there was no effect of nicotine treatment on dopamine levels in accumbens (p > 0.05), the metabolite homovanillic acid but not DOPAC was significantly decreased (p < 0.01). This resulted in a slightly higher, although not significant, rate of dopamine turnover in the brains of nicotine-treated animals as reflected in the dopamine to metabolites ratio (Table 1).

View this table: [in this window] [in a new window]   TABLE 1 Effect of long-term nicotine treatment on the levels of dopamine and its metabolites in nucleus accumbens Monkeys were administered nicotine in the drinking water for 1 year. Dopamine, DOPAC, and homovanillic acid were then measured in the nucleus accumbens using high-performance liquid chromatography. The turnover is defined as the ratio of dopamine/DOPAC + homovanillic acid. Each value represents the mean ± S.E.M. of the indicated number of animals.

Effect of Long-Term Oral Nicotine Treatment on Synaptosomal [³H]Dopamine Uptake. Since nicotine exposure altered [³H]dopamine uptake characteristics in striatum (McCallum et al., 2006), we determined both maximal and rate of [³H]dopamine uptake into synaptosomes prepared from nucleus accumbens of treated and control animals (Table 2). Nicotine treatment had no effect on either measure in nucleus accumbens. Maximal uptake of [³H]dopamine, expressed as femtomoles/microgram of tissue, was determined by incubating synaptosomes with [³H]dopamine at equilibrium (10 min); it was similar in both control and nicotine-treated animals. The rate of [³H]dopamine uptake (femtomole/microgram of tissue/minute) was determined after a 2-min incubation period when uptake was half-maximal; no differences were observed between the two treatment groups (Table 2).

View this table: [in this window] [in a new window]   TABLE 2 Effect of long-term oral nicotine on synaptosomal [3H]dopamine uptake Monkeys were administered nicotine for 1 year as described under Materials and Methods. [3H]Dopamine uptake into synaptosomes prepared from the nucleus accumbens was then determined. The rate and maximal uptake were determined at 2 and 10 min of incubation with [3H]dopamine, respectively. Each value represents the mean ± S.E.M. of the indicated number of animals.

Long-Term Oral Nicotine Treatment Increases 42^* but Not 3/62^* Receptor-Mediated [³H]Dopamine Release in Monkey Nucleus Accumbens. We next determined the effect of long-term nicotine treatment on nicotine-evoked [³H]dopamine release from synaptosomes prepared from the nucleus accumbens of control and nicotine-treated monkeys (Fig. 1). We differentiated accumbal dopamine release mediated by 42^* nAChRs from that component mediated by 3/62^* nAChRs using the selective 3/62^* antagonist, -conotoxin MII. In control animals, the proportion of release mediated by 3/62^* nAChRs (75%) was appreciably greater than that mediated by 42^* nAChRs (25%), with similar proportions in nicotine-treated monkeys (Fig. 1). This differs from the relative proportions of release observed in the rodent nucleus accumbens for which <50% is mediated by -conotoxin MII-sensitive nAChRs (Grady et al., 2002).

View larger version (27K): [in this window] [in a new window]   Fig. 1. Long-term oral nicotine treatment increases 42* nAChR-mediated [3H]dopamine release in monkey nucleus accumbens with no effect on 3/62* nAChR-evoked function. Monkeys were administered nicotine in the drinking water for 1 year. There was a significant increase in Rmax values for 42* but not 3/62* nAChR-mediated [3H]dopamine release with chronic nicotine treatment (left panels). A comparison of nicotine-evoked release at concentrations ranging from 3 x 10-8 to 3 x 10-4 M showed that there was a significant increase in 42* nAChR-evoked release from synaptosomes from monkeys chronically treated with nicotine compared with controls [top right panel, two-way ANOVA, F(1,63) = 6.73, p < 0.01]. Data represent the mean ± S.E.M. of five to six animals. Significance of difference from control using Student's t test, *, p < 0.05.

Previously, we had reported that long-term nicotine treatment to monkeys resulted in differential effects on nAChR subtype-evoked dopamine release in monkey striatum, such that the 42^* nAChR-mediated component was increased and that portion of release mediated by 3/62^* nAChRs was decreased (McCallum et al., 2006). In nucleus accumbens, however, a different pattern emerged following oral nicotine treatment. As in striatum, the component of dopamine release mediated by 42^* nAChRs was increased in the nucleus accumbens of nicotine-treated monkeys compared with controls (Fig. 1, top panels). In treated animals, the R_max for nicotine-evoked release was 2927 ± 185 cpm/mg (n = 5), a 27% increase compared with the R_max value for nicotine-evoked dopamine release in control animals (2142 ± 192 cpm/mg; n = 7; p < 0.05) (Fig. 1).

Whereas 42^* nAChR-mediated release was increased in nucleus accumbens, long-term nicotine treatment was associated with no change in the R_max of -conotoxin MII-sensitive dopamine release or that mediated by 3/62^* nAChRs. In fact, a small (14%) but nonsignificant increase in the R_max value obtained from nicotine-treated animals (8077 ± 641; n = 5) was observed compared with the R_max calculated for dopamine release in control animals (6924 ± 762; n = 7) (Fig. 1, bottom panels). These findings are in contrast to the significant declines in this component of dopamine release observed in primate and rodent striatum from nicotine-treated animals (Salminen et al., 2004a; Lai et al., 2005; McCallum et al., 2006).

Effect of Long-Term Nicotine Treatment on Baseline and K⁺-Evoked Dopamine Release. Long-term nicotine exposure may have affected basal release and thus modulated the increase in nicotine-evoked release observed for the 42^*-mediated component. To rule out this possibility, basal release was calculated by taking the mean of several fractions of release before and after the stimulated release in the presence and absence of -conotoxin MII. There was no effect of nicotine treatment on basal release, with comparable values between nicotine-treated and control animals (p > 0.05; Table 3). Likewise, there was no significant change in basal release in the presence of -conotoxin MII in either treatment group (p > 0.05; Table 3).

View this table: [in this window] [in a new window]   TABLE 3 Effect of long-term nicotine treatment on baseline and K+-evoked dopamine release [3H]Dopamine release from nucleus accumbens synaptosomes was determined as described under Materials and Methods. Baseline release was measured by averaging the fractions immediately before and after stimulus-evoked release in the absence and presence of -conotoxin MII (5 x 10-8 M). K+-stimulated release was done in the presence of 20 mM K+. Results show that chronic oral nicotine treatment does not alter baseline and K+-stimulated release. Values represent the mean ± S.E.M. of the indicated number of animals.

Next, we evaluated the effect of extended oral nicotine treatment on [³H]dopamine release evoked by 20 mM K⁺.No differences in mean K⁺-evoked dopamine release were observed between the two treatment groups (p > 0.05; Table 3). This suggests that the observed changes in nicotine-evoked release with long-term nicotine treatment were selectively mediated via nAChR activation and were not the result of a generalized effect of chronic nicotine treatment on the exocytotic machinery.

Long-Term Nicotine Treatment Increases 42^* but Not 3/62^* nAChR Sites in Monkey Nucleus Accumbens. Nicotinic receptor sites in the nucleus accumbens were measured using ¹²⁵I-epibatidine, which labels 2^* through 6^* nAChRs, and [¹²⁵I]A85380, which binds to 2^* subtypes. To distinguish between 42^* and 3/62^* nAChRs using these ligands, unlabeled -conotoxin MII was added during the binding experiments to allow for the measurement of both -conotoxin MII-resistant (42^*) and -conotoxin MII-sensitive (3/62^*) ¹²⁵I-epibatidine and [¹²⁵I]A85380 binding sites. In addition, 3/62^* nAChR sites were measured directly using ¹²⁵I--conotoxin MII.

When -conotoxin MII-resistant sites (42^* nAChRs) were examined in the nucleus accumbens of nicotine-treated and control monkeys, significant increases in binding were found after long-term nicotine treatment (Fig. 2, top panels). -Conotoxin MII-resistant ¹²⁵I-epibatidine sites in monkeys given oral nicotine increased by 33% compared with controls (p < 0.05). Likewise, -conotoxin MII-resistant [¹²⁵I]A85380 sites showed a substantial increase of 72% in nicotine-treated animals compared with controls (p < 0.001). The 42^* sites measured using [¹²⁵I]A85380 were significantly greater (p < 0.01) than those determined using ¹²⁵I-epibatidine in nucleus accumbens of animals chronically treated with oral nicotine.

View larger version (24K): [in this window] [in a new window]   Fig. 2. Nicotine treatment increases 42*-nAChR sites in monkey nucleus accumbens with no change in the 3/62* nAChR subtype. Monkeys were administered nicotine as described under Materials and Methods. 42* nAChR binding sites (Ctx-res) were determined by measuring binding of 125I-epibatidine (epi, top left panel) and [125I]A85380 (top right panel) in the presence of -conotoxin MII (10-7 M). 3/62* nAChR binding sites (Ctx-sens) were determined by measuring the difference between total binding and binding in the presence of -conotoxin MII (10-7 M) for 125I-epibatidine (bottom left panel) and [125I]A85380 (bottom middle panel). Binding of 125I--conotoxin MII is shown in the bottom right panel. Values represent the mean ± S.E.M. of five to seven animals. Significance of difference from control using Student's t test, *, p < 0.05; ***, p < 0.001.

	Discussion

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The present results are the first to show the consequences of extended nicotine treatment (1 year) delivered in the drinking water on nicotinic receptor sites and function in nonhuman primate nucleus accumbens, a region subject to a complex regulation by multiple nAChR subtypes (Quik et al., 2000, 2001; Klink et al., 2001; Champtiaux et al., 2003; Wooltorton et al., 2003). Moreover, our examination of the nAChR regulation of the nucleus accumbens, a critical region in nicotine-mediated addiction, is distinct from similar studies in rodents in which the focus is frequently on other dopaminergic brain regions. We found that long-term nicotine administration resulted in an up-regulation of 42^* nAChRs in the nucleus accumbens that was associated with a corresponding increase in 42^* receptor-evoked [³H]dopamine release. In contrast, long-term nicotine treatment did not change nucleus accumbens 3/62^* receptor binding or receptor-evoked dopamine release. Since 42^* and 3/62^* are the predominant nAChR populations on dopamine terminals in the nucleus accumbens, these data indicate that the primary receptor influenced by chronic nicotine treatment in this region is the 42^* population. This preferential up-regulation of 42^* nAChRs may suggest that this subtype is more important in nicotine-mediated reward in nonhuman primates. This interpretation is consistent with previous work in nAChR mutant mice, suggesting that 42^* nAChRs are important in nicotine addiction (Picciotto et al., 1998; Klink et al., 2001; Tapper et al., 2004; Maskos et al., 2005; Walters et al., 2006). Enhanced expression of 42^* nAChRs on presynaptic dopaminergic terminals may lead to an elevated release of dopamine in the mesolimbic system, a cellular mechanism that may underlie nicotine reinforcement.

The question arises regarding the molecular basis for the differential regulation of the 4^* and 6^* nAChR subtypes by nicotine in the mesolimbic compared with the nigrostriatal dopaminergic system. Evidence from experiments in cell culture suggests that nicotine can act as a pharmacological chaperone to cause up-regulation by inducing an active or desensitized receptor conformation (most likely of high affinity) that assembles more efficiently (Sallette et al., 2004; Kuryatov et al., 2005). In the mesolimbic system there may be no limiting amount of any subunit. Thus, nicotine may enhance assembly of high-affinity 42^* nAChRs without decreasing the formation of other nAChRs, such as the 3/6^* subtypes that may not desensitize and thus be of lower affinity. In the nigrostriatal pathway, the amount of 2 subunit may be limiting such that nicotine exposure could upregulate assembly of the high-affinity 42^* nAChRs only at the expense of the lower affinity 3/6^* nAChRs.

In our studies, we used -conotoxin MII-resistant ¹²⁵I-epibatidine or [¹²⁵I]A85380 binding to identify 42^* receptors. This was based on previous work showing that ¹²⁵I-epibatidine binds to 2-6^* nAChRs (Davila-Garcia et al., 1997), whereas -conotoxin MII interacts with 3/62^* subtypes (Quik and McIntosh, 2006). ¹²⁵I-Epibatidine sites measured in the presence of -conotoxin MII would therefore most likely represent 42^* subtypes since the two major subtypes in this region are the 42^* and 3/62^* (Quik et al., 2001; Kulak et al., 2002). Likewise, because [¹²⁵I]A85380 labels 2^* nAChRs (Kulak et al., 2002) and -conotoxin MII inhibits binding to 3/62^* subtypes, -conotoxin MII-resistant [¹²⁵I]A85380 binding sites provide an index of 42^* nAChRs. Unexpectedly, although both -conotoxin MII-resistant ¹²⁵I-epibatidine and [¹²⁵I]A85380 binding detect 42^* subtypes, nicotine treatment led to an appreciably greater increase in the sites identified with [¹²⁵I]A85380 compared with ¹²⁵I-epibatidine. These results are consistent with those in rodent striatum (Lai et al., 2005), which also demonstrate a larger increase in 42^* sites identified with [¹²⁵I]A85380. These data most likely suggest that [¹²⁵I]A85380 and ¹²⁵I-epibatidine bind to different 42^* nAChR populations containing 2 and/or 3 subunits in monkey striatum (Quik et al., 2005) and 5 and/or 4 subunits in rodent striatum (Zoli et al., 2002). Previous work also suggests that [¹²⁵I]A85380 and ¹²⁵I-epibatidine bind to different nAChR subtypes in spinal cord, although their composition remains unknown (Khan et al., 2001).

Our results showing that 3^*/62^* nAChR sites and function in the mesolimbic dopaminergic system were not altered by long-term nicotine exposure were somewhat surprising because this subtype mediates a large proportion of nicotine-evoked release (75%) in monkey nucleus accumbens. One possibility is that 3^*/62^* nAChRs in the nucleus accumbens are more resistant to modification resulting from chronic drug exposure than are 42^* nAChRs. Such a hypothesis is consistent with evidence that 3^*/6^* nAChRs are more resistant to nicotine-induced up-regulation than are 4^* nAChRs in rats (Ulrich et al., 1997; Davila-Garcia et al., 2003; Nguyen et al., 2003). These observations do not rule out the possibility that the 3/6^* subtype is important in nicotine-induced addictive processes but that its involvement in this process is not reflected by an up- or down-regulation of receptor sites or function.

Not only are 3/62^* and 42^* nAChRs in the nucleus accumbens controlled in a different manner with long-term nicotine treatment but, in addition, regulation of 3/62^* nAChRs with nicotine exposure is different in the nucleus accumbens compared with striatum (summarized in Table 4). Although nicotine treatment did not change 3/62^* nAChR binding or nicotine-mediated dopamine release in the nucleus accumbens, this same treatment down-regulated both 3/62^* nAChR sites and function in monkey striatum (McCallum et al., 2006). In contrast, 42^* nAChR number and function are up-regulated in both striatum and nucleus accumbens with nicotine treatment. A point of note is that changes in receptor binding and function correlate well in both nucleus accumbens and striatum when considered separately for the two subtypes, that is, 3/62^* versus 42^* nAChRs. This close correspondence is no longer observed in either region when changes in total nAChR sites are compared with total nicotine-evoked function after nicotine treatment (Table 4), most likely because the change in receptor subtype selective sites or function with the greatest magnitude predominates.

View this table: [in this window] [in a new window]   TABLE 4 Summary of the effect of long-term oral nicotine treatment on 42* and 3/62* nAChR sites and function in the mesolimbic and nigrostriatal dopaminergic pathway Oral nicotine treatment increased 42* nAChR-evoked dopamine (DA) release and binding in both the nucleus accumbens and striatum. However, comparison of the data shows that 3/62* nAChR sites and function are differentially regulated, with no change in striatal 3/62* sites and function in nucleus accumbens but a decrease in striatum (McCallum et al., 2006). 2-6* refers to function or binding performed in the absence of -conotoxin MII.

Differential changes in mesolimbic and nigrostriatal nAChR sites and function in response to nicotine administration that we observed in a synaptosomal preparation have also been observed in vivo. For instance, microdialysis studies in rodents have shown that nicotine causes a greater output of dopamine in the nucleus accumbens compared with striatum (Di Chiara and Imperato, 1988; Benwell and Balfour, 1997; Janhunen and Ahtee, 2004). Janhunen and Ahtee (2004) observed differential effects on striatal and accumbal extracellular dopamine with epibatidine, such that epibatidine produced a smaller output of dopamine in the nucleus accumbens compared with the striatum, whereas the effects of nicotine on dopamine output were similar in these two regions. These differences in nicotine-mediated dopamine output observed using microdialysis data might be due to the presence of varying nAChR subtypes with unique characteristics in the nucleus accumbens compared with the striatum. For instance, the 3/62^* nAChR populations present in nucleus accumbens may be slower to desensitize following nicotine exposure than their counterparts in the striatum. This may have relevance for the development of new pharmacotherapies for smoking cessation.

To our knowledge, changes in 3/62^* nAChR-mediated function in nucleus accumbens after nicotine treatment have not been determined in rodent brain, although 3/62^* nAChR sites and function in the mouse striatum have previously been shown to decrease with chronic nicotine treatment (Salminen et al., 2004a; Lai et al., 2005). Receptor changes in rodent striatum following long-term nicotine treatment have been somewhat inconsistent, with studies reporting both increases and decreases in 3/62^* nAChRs (Nguyen et al., 2003; Parker et al., 2004; Mugnaini et al., 2006). These discrepancies among studies may be attributed to species differences with respect to the composition and/or proportion of these receptors expressed in the nucleus accumbens, as well as the methods used for identification of 3/62^* nAChRs.

In summary, the present results show that chronic nicotine treatment selectively up-regulates 42^* nAChR sites and function in the nucleus accumbens of nonhuman primates, whereas in striatum, the up-regulation of 42^* nAChRs is accompanied by a down-regulation of 3^*/62^* sites and function. As the nucleus accumbens is a critical component of the reward pathway, these findings may indicate a greater involvement of 42^* nAChRs in nicotine-mediated reward systems than 3/62^* nAChRs.

	Footnotes

 
This work was supported by National Institutes of Health Grants NS42091 and NS47162 (to M.Q.) and MH53631 (to J.M.M.)

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.104414.

ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; DOPAC, dihydroxyphenylacetic acid; [¹²⁵I]A85380, 5-[¹²⁵I]iodo-3(2(S)-azetidinylmethoxy) pyridine; ANOVA, analysis of variance.

^* denotes nicotinic receptors containing the indicated and/or subunit and also additional undefined subunits.

Address correspondence to: Dr. Maryka Quik, The Parkinson's Institute, 1170 Morse Ave., Sunnyvale, CA 94089-1605. E-mail: mquik{at}parkinsons-institute.org

	References

Top Abstract Materials and Methods Results Discussion References

 

Benwell ME and Balfour DJ (1997) Regional variation in the effects of nicotine on catecholamine overflow in rat brain. Eur J Pharmacol 325: 13-20.[CrossRef][Medline]

Benwell ME, Balfour DJ, and Anderson JM (1988) Evidence that tobacco smoking increases the density of (-)-[³H]nicotine binding sites in human brain. J Neurochem 50: 1243-1247.[Medline]

Buisson B and Bertrand D (2002) Nicotine addiction: the possible role of functional upregulation. Trends Pharmacol Sci 23: 130-136.[CrossRef][Medline]

Champtiaux N, Gotti C, Cordero-Erausquin M, David DJ, Przybylski C, Lena C, Clementi F, Moretti M, Rossi FM, Le Novere N, et al. (2003) Subunit composition of functional nicotinic receptors in dopaminergic neurons investigated with knockout mice. J Neurosci 23: 7820-7829.[Abstract/Free Full Text]

Corrigall WA, Coen KM, and Adamson KL (1994) Self-administered nicotine activates the mesolimbic dopamine system through the ventral tegmental area. Brain Res 653: 278-284.[CrossRef][Medline]

Davila-Garcia MI, Musachio JL, and Kellar KJ (2003) Chronic nicotine administration does not increase nicotinic receptors labeled by [¹²⁵I]epibatidine in adrenal gland, superior cervical ganglia, pineal or retina. J Neurochem 85: 1237-1246.[CrossRef][Medline]

Davila-Garcia MI, Musachio JL, Perry DC, Xiao Y, Horti A, London ED, Dannals RF, and Kellar KJ (1997) [¹²⁵I]IPH, an epibatidine analog, binds with high affinity to neuronal nicotinic cholinergic receptors. J Pharmacol Exp Ther 282: 445-451.[Abstract/Free Full Text]

Di Chiara G and Imperato A (1988) Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats. Proc Natl Acad Sci USA 85: 5274-5278.[Abstract/Free Full Text]

Grady SR, Murphy KL, Cao J, Marks MJ, McIntosh JM, and Collins AC (2002) Characterization of nicotinic agonist-induced [³H]dopamine release from synaptosomes prepared from four mouse brain regions. J Pharmacol Exp Ther 301: 651-660.[Abstract/Free Full Text]

Hukkanen J, Jacob P 3rd, and Benowitz NL (2005) Metabolism and disposition kinetics of nicotine. Pharmacol Rev 57: 79-115.[Abstract/Free Full Text]

Janhunen S and Ahtee L (2004) Comparison of the effects of nicotine and epibatidine on the striatal extracellular dopamine. Eur J Pharmacol 494: 167-177.[CrossRef][Medline]

Khan IM, Stanislaus S, Zhang L, Taylor P, and Yaksh TL (2001) A-85380 and epibatidine each interact with disparate spinal nicotinic receptor subtypes to achieve analgesia and nociception. J Pharmacol Exp Ther 297: 230-239.[Abstract/Free Full Text]

Klink R, de Kerchove d'Exaerde A, Zoli M, and Changeux JP (2001) Molecular and physiological diversity of nicotinic acetylcholine receptors in the midbrain dopaminergic nuclei. J Neurosci 21: 1452-1463.[Abstract/Free Full Text]

Kulak JM, Musachio JL, McIntosh JM, and Quik M (2002) Declines in different 2^* nicotinic receptor populations in monkey striatum after nigrostriatal damage. J Pharmacol Exp Ther 303: 633-639.[Abstract/Free Full Text]

Kuryatov A, Luo J, Cooper J, and Lindstrom J (2005) Nicotine acts as a pharmacological chaperone to up-regulate human 42 acetylcholine receptors. Mol Pharmacol 68: 1839-1851.[Abstract/Free Full Text]

Lai A, Parameswaran N, Khwaja M, Whiteaker P, Lindstrom JM, Fan H, McIntosh JM, Grady SR, and Quik M (2005) Long-term nicotine treatment decreases striatal 6^* nicotinic acetylcholine receptor sites and function in mice. Mol Pharmacol 67: 1639-1647.[Abstract/Free Full Text]

Le Novere N, Zoli M, and Changeux JP (1996) Neuronal nicotinic receptor 6 subunit mRNA is selectively concentrated in catecholaminergic nuclei of the rat brain. Eur J Neurosci 8: 2428-2439.[CrossRef][Medline]

Marks MJ, Burch JB, and Collins AC (1983) Genetics of nicotine response in four inbred strains of mice. J Pharmacol Exp Ther 226: 291-302.[Abstract/Free Full Text]

Maskos U, Molles BE, Pons S, Besson M, Guiard BP, Guilloux JP, Evrard A, Cazala P, Cormier A, Mameli-Engvall M, et al. (2005) Nicotine reinforcement and cognition restored by targeted expression of nicotinic receptors. Nature (Lond) 436: 103-107.[CrossRef][Medline]

McCallum SE, Parameswaran N, Bordia T, Fan H, Tyndale RF, Langston JW, McIntosh JM, and Quik M (2006) Increases in 4^* but not 3^*/6^* nicotinic receptor sites and function in the primate striatum following chronic oral nicotine treatment. J Neurochem 96: 1028-1041.[CrossRef][Medline]

McCallum SE, Parameswaran N, Bordia T, McIntosh JM, Grady SR, and Quik M (2005) Decrease in 3^*/6^* nicotinic receptors but not nicotine-evoked dopamine release in monkey brain after nigrostriatal damage. Mol Pharmacol 68: 737-746.[Abstract/Free Full Text]

Mugnaini M, Garzotti M, Sartori I, Pilla M, Repeto P, Heidbreder CA, and Tessari M (2006) Selective down-regulation of [¹²⁵I]Tyr--conotoxin MII binding in rat mesostriatal dopamine pathway following continuous infusion of nicotine. Neuroscience 137: 565-572.[CrossRef][Medline]

Nguyen HN, Rasmussen BA, and Perry DC (2003) Subtype-selective up-regulation by chronic nicotine of high-affinity nicotinic receptors in rat brain demonstrated by receptor autoradiography. J Pharmacol Exp Ther 307: 1090-1097.[Abstract/Free Full Text]

Parker SL, Fu Y, McAllen K, Luo J, McIntosh JM, Lindstrom JM, and Sharp BM (2004) Up-regulation of brain nicotinic acetylcholine receptors in the rat during long-term self-administration of nicotine: disproportionate increase of the 6 subunit. Mol Pharmacol 65: 611-622.[Abstract/Free Full Text]

Picciotto MR, Zoli M, Rimondini R, Lena C, Marubio LM, Pich EM, Fuxe K, and Changeux JP (1998) Acetylcholine receptors containing the 2 subunit are involved in the reinforcing properties of nicotine. Nature (Lond) 391: 173-177.[CrossRef][Medline]

Quik M and McIntosh JM (2006) Striatal 6^* nicotinic acetylcholine receptors: potential targets for Parkinson's disease therapy. J Pharmacol Exp Ther 316: 481-489.[Abstract/Free Full Text]

Quik M, Polonskaya Y, Gillespie A, Jakowec M, Lloyd GK, and Langston JW (2000) Localization of nicotinic receptor subunit mRNAs in monkey brain by in situ hybridization. J Comp Neurol 425: 58-69.[CrossRef][Medline]

Quik M, Polonskaya Y, Kulak JM, and McIntosh JM (2001) Vulnerability of ¹²⁵I--conotoxin MII binding sites to nigrostriatal damage in monkey. J Neurosci 21: 5494-5500.[Abstract/Free Full Text]

Quik M, Vailati S, Bordia T, Kulak JM, Fan H, McIntosh JM, Clementi F, and Gotti C (2005) Subunit composition of nicotinic receptors in monkey striatum: effect of treatments with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine or L-DOPA. Mol Pharmacol 67: 32-41.[Abstract/Free Full Text]

Sallette J, Bohler S, Benoit P, Soudant M, Pons S, Le Novere N, Changeux JP, and Corringer PJ (2004) An extracellular protein microdomain controls up-regulation of neuronal nicotinic acetylcholine receptors by nicotine. J Biol Chem 279: 18767-18775.[Abstract/Free Full Text]

Salminen O, Grady SR, Collins AC, McIntosh JM, and Marks MJ (2004a) Chronic nicotine infusion down regulates -conotoxin MII-sensitive nicotinic acetylcholine receptors in C57bl/6 mice, in Program No. 48.21. Abstract Viewer/Itinerary Planner (online); 2004 October 23-27; San Diego, CA. Society for Neuroscience, Washington, DC.

Salminen O, Murphy KL, McIntosh JM, Drago J, Marks MJ, Collins AC, and Grady SR (2004b) Subunit composition and pharmacology of two classes of striatal presynaptic nicotinic acetylcholine receptors mediating dopamine release in mice. Mol Pharmacol 65: 1526-1535.[Abstract/Free Full Text]

Samaha AN and Robinson TE (2005) Why does the rapid delivery of drugs to the brain promote addiction? Trends Pharmacol Sci 26: 82-87.[Medline]

Schwartz RD and Kellar KJ (1983) Nicotinic cholinergic receptor binding sites in the brain: regulation in vivo. Science (Wash DC) 220: 214-216.[Abstract/Free Full Text]

Tapper AR, McKinney SL, Nashmi R, Schwarz J, Deshpande P, Labarca C, Whiteaker P, Marks MJ, Collins AC, and Lester HA (2004) Nicotine activation of 4^* receptors: sufficient for reward, tolerance and sensitization. Science (Wash DC) 306: 1029-1032.[Abstract/Free Full Text]

Ulrich YM, Hargreaves KM, and Flores CM (1997) A comparison of multiple injections versus continuous infusion of nicotine for producing up-regulation of neuronal [³H]epibatidine binding sites. Neuropharmacology 36: 1119-1125.[CrossRef][Medline]

Walters CL, Brown S, Changeux JP, Martin B, and Damaj MI (2006) The 2 but not 7 subunit of the nicotinic acetylcholine receptor is required for nicotine-conditioned place preference in mice. Psychopharmacology (Berl) 184: 339-344.[CrossRef][Medline]

Wonnacott S, Sidhpura N, and Balfour DJ (2005) Nicotine: from molecular mechanisms to behaviour. Curr Opin Pharmacol 5: 53-59.[CrossRef][Medline]

Wooltorton JR, Pidoplichko VI, Broide RS, and Dani JA (2003) Differential desensitization and distribution of nicotinic acetylcholine receptor subtypes in midbrain dopamine areas. J Neurosci 23: 3176-3185.[Abstract/Free Full Text]

Zoli M, Moretti M, Zanardi A, McIntosh JM, Clementi F, and Gotti C (2002) Identification of the nicotinic receptor subtypes expressed on dopaminergic terminals in the rat striatum. J Neurosci 22: 8785-8789.[Abstract/Free Full Text]

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