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NEUROPHARMACOLOGY

Chronic Nicotine Differentially Regulates 6- and 3-Containing Nicotinic Cholinergic Receptors in Rat Brain

Department of Pharmacology and Physiology, George Washington University, Washington, DC (D.C.P., A.B.G.); Department of Pharmacology, Georgetown University, Washington, DC (D.M., J.C.P., K.J.K.); and Department of Psychiatry, University of Utah, Salt Lake City, Utah (J.M.M.)

Received February 13, 2007; accepted April 18, 2007.

	Abstract

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We investigated the effects of chronic nicotine on 6- and 3-containing nicotinic acetylcholine receptors (nAChRs) in two rat brain regions using three methodological approaches: radioligand binding, immunoprecipitation, and nicotine-stimulated synaptosomal release of dopamine. Nicotine was administered by osmotic minipumps for 2 weeks. Quantitative autoradiography with [¹²⁵I]-conotoxin MII to selectively label 6^* nAChRs showed a 28% decrease in binding in the striatum but no change in the superior colliculus. Immunoprecipitation of nAChRs labeled by [³H]epibatidine in these two regions showed that chronic nicotine increased 4- and 2-containing nAChRs by 39 to 67%. In contrast, chronic nicotine caused a 39% decrease in 6-containing nAChRs in striatum but no change in superior colliculus. No changes in 3-containing nAChRs were seen in either region after chronic nicotine. The decreased expression of 6-containing nAChRs persisted for at least 3 days, recovering to baseline by 7 days after removal of the pumps. There was a small but significant decrease in total nicotine-stimulated dopamine release in striatal synaptosomes after nicotine exposure. However, the component of dopamine release that was resistant to -conotoxin MII blockade was unaffected, whereas dopamine release that was sensitive to blockade by -conotoxin MII was decreased by 56%. These findings indicate that the 6^* nAChR is regulated differently from other nAChR subtypes, and they suggest that the inclusion of a 3 subunit with 6 may serve to inhibit nicotine-induced down-regulation of these receptors.

Much recent attention has focused on a class of nAChRs sensitive to the cone snail toxin -conotoxin MII (-CtxMII). Although originally described as selective for 32 nAChRs (Cartier et al., 1996), more recent evidence indicates that the selectivity extends to nAChRs containing 6^* subunits (Whiteaker et al., 2000; Champtiaux et al., 2002; Zoli et al., 2002). This nAChR subtype is localized largely to catecholaminergic regions, visual structures, and the habenular-peduncular pathway (Le Novère et al., 1996; Quik et al., 2000; Whiteaker et al., 2000; Champtiaux et al., 2002). Although not the major nAChR subtype in striatum, 6-containing nAChRs contribute disproportionately to nicotine-stimulated release of dopamine (Kulak et al., 1997; Kaiser et al., 1998; Salminen et al., 2004), and they are selectively affected in Parkinson's disease (Quik et al., 2004). Our original attempts to determine the effect of chronic nicotine on these 3/6^* receptors used an indirect method to deduce subtype populations based on differential sensitivity of [¹²⁵I]epibatidine autoradiography to the competing ligands cytisine and A-85380; we found little evidence for change in most brain regions, including striatum and superior colliculus (Nguyen et al., 2003). Subsequent studies addressed this problem using more direct binding and functional methods. Parker et al. (2004), using an antibody directed at the 6 subunit and homogenate binding of [¹²⁵I]epibatidine and [¹²⁵I]-CtxMII, reported that 6 receptors in rat brain were disproportionately increased by long-term self-administration of nicotine. However, more recent studies reported a decrease in striatal [¹²⁵I]-CtxMII binding in nicotine-treated rats (Mugnaini et al., 2006) and in both nicotine-stimulated dopamine release and [¹²⁵I]-CtxMII striatal binding in nicotine-treated mice (Lai et al., 2005).

In this study, we used a paradigm of long-term nicotine exposure that we previously demonstrated up-regulates both 7^* and 42^* nAChR binding sites in a wide variety of rat brain regions but has no effect on 34^* receptors (Nguyen et al., 2003; Rasmussen and Perry, 2006). We used three different approaches to assess the effect of nicotine on 3/6^* nAChRs: binding of [¹²⁵I]-CtxMII and [¹²⁵I]A-85380, immunoprecipitation with a battery of subunit-selective antibodies, and determination of -CtxMII-sensitive and -CtxMII-resistant striatal dopamine release. Our results show that chronic nicotine exposure results in down-regulation of 6^* nAChR numbers and function in rat striatum but not in superior colliculus. We were surprised to find that nAChRs containing 3 subunits in these regions seem to be unaffected by nicotine exposure, suggesting a complex regulatory mechanism for 6-containing nAChRs.

	Materials and Methods

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Materials. Striatum and superior colliculus used to determine the nAChR subunit profiles were dissected from brains of adult Sprague-Dawley rats purchased from Zivic Miller laboratories (Portersville, PA). The brain regions were dissected on ice, quickly refrozen on dry ice, and stored at -80°C for later use. Rabbit antisera directed at bacterially expressed fusion proteins containing partial sequences of the cytoplasmic domains of nAChR 2, 4, 5, 3, and 4 subunits were kind gifts from Drs. Scott Rogers and Lorise Gahring (University of Utah, Salt Lake City, UT). These antisera have been described previously (Flores et al., 1992; Rogers et al., 1992). An antibody directed at the C-terminal peptide sequence of the rat nAChR 3 subunit was affinity purified from rabbit serum. This antibody has been described previously (Yeh et al., 2001). A monoclonal antibody, mAb 270, to the chick 2 subunit was made from hybridoma stocks (American Type Culture Collection, Manassas, VA). This mAb was originally developed and characterized by Whiting and Lindstrom (1987). The specificity of most of the antibodies in the immunoprecipitation procedures was reported previously (Hernandez et al., 2004; Marritt et al., 2005; Turner and Kellar, 2005). For the 6 subunit, the following peptide was synthesized (ResGen, Inc., Carlsbad, CA) from the cytoplasmic loop region of the rat nAChR 6 subunit: 5'-GVKDPKTHTKRPAKVKFTHRKEPKLLKEC-3' (Champtiaux et al., 2003). Rabbits were immunized with this peptide (Lampire Biologicals, Pipersville, PA), and the antibody produced was then affinity purified from the rabbit serum. Evidence for the specificity of this antibody in immunoprecipitation assays is provided under Results. For simplicity, in this article, we use the term antibody to refer to unpurified antisera, as well as to affinity purified antisera and monoclonal antibody.

Protein G-Sepharose beads were purchased from Amersham Biosciences Corporation (Piscataway, NJ). Normal rabbit serum (NRS) was purchased from Calbiochem (San Diego, CA). All other chemicals unless otherwise noted were obtained from Sigma Aldrich (St. Louis, MO). [³H]Epibatidine ([³H]EB; 55 Ci/mmol) and [¹²⁵I]A-85380 (2200 Ci/mmol) were obtained from PerkinElmer Life and Analytical Sciences (Boston, MA). [7,8-³H]Dopamine (40–60 Ci/mmol) was obtained from GE Healthcare (Piscataway, NJ). [¹²⁵I]-CtxMII was synthesized by a reported method (Whiteaker et al., 2000). Tyr⁰--CtxMII (25 nmol) was dissolved in 25 µl of dH₂O; to this was added NH₄ acetate (40 µl of 0.3 M, pH 5.3) and 10 µCi of Na¹²⁵I (22 µl; PerkinElmer Life and Analytical Sciences). The reaction was initiated by addition of chloramine-T (40 µl of 0.4 mM), followed by incubation for 10 min at room temperature. The reaction was terminated by addition of ascorbic acid (65 µl, 0.5 M) followed by trifluoroacetic acid (0.8 ml, 0.1%). The reaction mixture was then purified by high-performance liquid chromatography using an analytical Vydac C18 reversed-phase column. A 50-min linear gradient was run from 75% solvent A (0.1% trifluoroacetic acid) to 75% solvent B (0.09% trifluoroacetic acid, 60% acetonitrile) at 1 ml/min; absorbance was monitored 215 nm. Fractions were collected in polypropylene tubes containing 10 µl of 20 mg/ml lysozyme to decrease adsorption; fractions were then concentrated to dryness by vacuum centrifugation and resuspended in 40% methanol and stored at -40°C until use. This protocol readily separates unreacted Tyr⁰--CtxMII from the monoiodo and di-iodo forms (Whiteaker et al., 2000); only the monoiodo form was utilized and based on the purification was assumed to be maximally iodinated (approximately 2200 Ci/mmol).

Animal Treatment. Osmotic minipumps (Alzet model 2002; Durect Corporation, Cupertino, CA) were filled with sterile saline or with nicotine hydrogen tartrate in saline, at concentrations calculated to achieve a dose of 6 mg/kg/day, calculated as nicotine free base (37 µmol/kg). We have previously found that this dose produced blood levels of 0.56 µM for nicotine and 3.5 µM for cotinine (Nguyen et al., 2004), which is comparable with levels achieved in humans who are moderate to heavy smokers (Rose et al., 1999). Others have reported that this same protocol using half the dose (i.e., 3 mg/kg/day free base) achieved brain levels of nicotine of approximately 1.5 µM (Mugnaini et al., 2006); nicotine has been shown to accumulate in the brain over time with continuous dosing (Ghosheh et al., 2001).

Male Sprague-Dawley rats (225–275 g; Hilltop Lab Animals, Scottdale, PA) were anesthetized with isoflurane and the minipumps inserted into a s.c. pocket via a small incision made over the shoulders. While under anesthesia, animals were administered buprenorphine (0.1 mg/kg s.c.) for postoperative pain. The wound was closed with clips, and the area was swabbed with antiseptic. After recovery from anesthetic (10–30 min), animals were returned to individual cages. Fourteen days after minipump implantation, animals were lightly anesthetized with isoflurane and either sacrificed by decapitation, or, to measure the reversibility of effects of nicotine on 6-containing receptors, the minipumps were removed and the rats were then sacrificed 1, 3, 7, or 30 days later. In those recovery experiments, the saline control rats were sacrificed as a group on day 14, so the results do not take into account any developmental changes that might take place over the following 30 days. However, because these rats were young adults, it is unlikely that such changes contributed significantly to the results. Animal use and procedures were approved by the George Washington University Medical Center Institutional Animal Care and Use Committee.

Autoradiography. After decapitation, brains were rapidly removed and frozen on dry ice. Frozen coronal brain sections (16 µm) were cut and mounted onto Superfrost Plus slides (Fisher Scientific, Newark, DE) and stored at -80°C until use. Autoradiographic methods were adapted from the work of Whiteaker et al. (2000). Sections were preincubated for 15 min in buffer 1 (20 mM HEPES, pH 7.5, 144 mM NaCl, 1.5 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, 1 mM phenylmethylsulfonyl fluoride, 0.1% bovine serum albumin) at room temperature. This was followed by incubation for 60 min at room temperature in buffer 2 (buffer 1 plus 5 mM EGTA, 5 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml peptidase A, 10 µg/ml leupeptin) containing 0.8 nM [¹²⁵I]-CtxMII. Adjacent sections were incubated in the same buffer with 100 µM nicotine added to determine nonspecific binding. Slides were then rinsed for 5 min at room temperature in buffer 1, followed by 10 min in buffer 1 on ice, then sequential dips in ice-cold 5 mM HEPES and H₂O followed by rapid air-drying. For [¹²⁵I]A-85380, slides were first preincubated in buffer 1 for 15 min at room temperature, followed by incubation for 60 min at room temperature in buffer 1 with 0.6 nM [¹²⁵I]A-85380 plus 100 nM unlabeled -CtxMII, followed by two 5-min rinses in buffer 1, a water dip, and rapid air-drying. Adjacent sections were incubated with the addition of 100 µM nicotine to determine nonspecific binding. After overnight desiccation, the sections were apposed to film (Kodak BioMax MR; Eastman Kodak, Rochester, NY) for 3 to 5 days along with ¹²⁵I standards (Amersham, Arlington Heights, IL); film was developed in an automatic developer. Film images were digitized and quantitative densitometric analysis of binding was done using the Loats Inquiry digital densitometry system (Loats Associates, Winchester, MD). Quantification of binding was done by comparison with standard curves constructed from ¹²⁵I standards; regions were identified by comparison with the rat brain atlas of Paxinos and Watson (1998). Nonspecific binding in adjacent sections was subtracted from the total binding in the paired section to calculate specific binding. Values for specific binding of nicotine- and saline-treated animals were compared using Student's t test with a Bonferroni correction. Statistically significant differences between means were accepted at p < 0.05.

Immunoprecipitation Assays. Brain tissues were homogenized in 50 mM Tris HCl buffer (pH 7.4 at 24°C), centrifuged twice at 35,000g for 10 min, and the membrane pellets were resuspended in fresh buffer. The receptors were solubilized by incubating the homogenates in 2% Triton X-100 with gentle rotation for 3 h at 4 °C. After centrifuging the mixture at 35,000g for 10 min, aliquots of the clear supernatant from striatum (equivalent to 6 mg of tissue) and superior colliculus (equivalent to 4 mg of tissue) were added to sample tubes containing 1.5 nM [³H]EB. One of the subunit-specific antibodies at an optimal concentration, which had been determined previously, or an equivalent volume of NRS, was added to each sample tube. The final volume of the assay was 150 µl. The samples were then rotated gently overnight at 4°C. After the addition of 50 µl of a 50% slurry of protein G-Sepharose beads, rotation at 4°C was continued for another hour. The samples were then centrifuged at 12,000g for 1 min, and the supernatants were removed and filtered over GF/B filters that had been prewet with 0.5% polyethylenimine. Radioactivity on the filters was then measured by liquid scintillation counting. The remaining pellets were washed by resuspension in 1.2 ml of 50 mM cold Tris HCl buffer, pH 7.0, followed by centrifugation at 12,000g for 1 min. The pellets were then dissolved in 200 µl of 0.1 N NaOH, and the radioactivity was quantified by liquid scintillation counting.

The counts precipitated in tubes containing NRS, which was used as control for nonspecific precipitation, were subtracted from those in the pellets immunoprecipitated with antibodies. For determination of subunit profile, the calculated number of radiolabeled nAChRs immunoprecipitated by each antibody was compared with the total number of [³H]EB-labeled receptors, as measured in both the supernatants and the pellets after immunoprecipitation, and the data are presented as the percentage of the total nAChRs immunoprecipitated. For comparison of the number of nAChRs immunoprecipitated by specific antibodies in tissues from saline- and nicotine-treated rats, data are presented as the calculated number of [³H]EB-labeled nAChRs per mg tissue, which was carefully weighed before homogenization while still frozen.

A one-sample Student's t test was used to determine whether residual values in immunoprecipitation assays were different from 0. Statistical analyses of the differences between group means were assessed using Student's t test. The recovery of 6-containing receptors to control levels after exposure to nicotine was evaluated by regression analysis to determine the reversibility of the nicotine effects and to estimate its time course.

[³H]Dopamine Release. The methods for measurements of [³H]dopamine release were adapted from the work of Grady et al. (1997). Immediately following decapitation, striatal tissue was removed and placed into ice-cold 0.32 M sucrose buffered with 5 mM HEPES at pH 7.5. Tissue was homogenized with 10 to 20 strokes of a Teflon homogenizer and then centrifuged for 20 min at 12,000g. Pellets were resuspended in 1.6 ml of uptake buffer (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 10 µM pargyline) to create the synaptosomal mixture. This mixture was then incubated for 10 min at 37°C before addition of 100 nM [³H]dopamine, followed by an additional 5-min incubation at 37°C. All subsequent steps were done at room temperature.

Synaptosomes (80-µl aliquots) were applied to glass fiber filters and perfused with perfusion buffer (uptake buffer containing 0.1% bovine serum albumin and 10 µM nomifensine) at 1 ml/min for 10 min before beginning collection of fractions. For some samples, the second 5 min of this perfusion included 50 nM -CtxMII; this concentration and time were previously shown by Grady et al. (Salminen et al., 2004) to yield optimal inhibition of the 6-receptor component of release. Fractions were then collected every 18 s for 4 min; nicotine-stimulated [³H]dopamine release was obtained by perfusing with different concentrations of nicotine in perfusion buffer for a total of 1 min during the collection period. The radioactivity of the fractions as well as that remaining on the filter after perfusion was measured by scintillation counting.

Radioactivity in fractions immediately before and after the stimulated peak was used to calculate basal release as a single exponential decay (SigmaPlot 2001; SPSS, Inc., Chicago, IL). This basal release was then subtracted from the values in the peak (fractions that exceeded the baseline by 10% or more). Peak values were then summed and expressed as a percentage of total counts (sum of all fractions collected plus filter radioactivity). Nicotine-stimulated release data were fit to a sigmoidal dose-response curve using Prism 4.0 (GraphPad Software, San Diego, CA), and E_max and EC₅₀ values were calculated. Comparison of treatment groups (i.e., ± nicotine) was accomplished by an F test of the parameters generated from the fitted curves (differences accepted at p < 0.05).

	Results

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Receptor Autoradiography. We used quantitative autoradiography to measure the effects of nicotine treatment on nAChR binding sites in the striatum and the superficial gray layer of the superior colliculus, two regions previously demonstrated to have relatively high fractions of 6/3^* receptor subtypes (Whiteaker et al., 2000; Zoli et al., 2002; Salminen et al., 2004; Gotti et al., 2005a,b). Adjacent sections from striatum and superior colliculus were labeled with either [¹²⁵I]-CtxMII or [¹²⁵I]A-85380 in the presence of 100 nM -CtxMII. Although [¹²⁵I]-CtxMII can bind to both 6^* and 32^* nAChRs (Cartier et al., 1996; Whiteaker et al., 2000, 2002), in both of these brain regions, [¹²⁵I]-CtxMII binding seems to represent 6-containing receptors predominantly (Champtiaux et al., 2002; Whiteaker et al., 2002). [¹²⁵I]A-85380 binds to all 2-containing receptors (Mukhin et al., 2000; Xiao and Kellar, 2004), but by including -CtxMII to mask binding to 62^* and 32^* nAChRs, it labels predominantly 42^* nAChRs in these brain regions.

Representative autoradiographs of these two radioligands from these two brain regions in saline- and nicotine-treated rats are shown in Figs. 1 and 2, and the results from quantitative analyses of the autoradiographic study are shown in Table 1. In the striatum from nicotine-treated rats, [¹²⁵I]-CtxMII binding was reduced by 28% (p < 0.01). In the superior colliculus, in contrast, no significant change in [¹²⁵I]-CtxMII binding was found. Binding of [¹²⁵I]A-85380 in the presence of 100 nM unlabeled -CtxMII, which represents primarily 42^* nAChRs, was significantly increased in the striatum from nicotine-treated rats; in the superior colliculus, there was a trend toward an increase that did not quite reach statistical significance.

View larger version (134K): [in this window] [in a new window]   Fig. 1. Representative autoradiographic images from coronal rat brain sections through striatum. A to C, [125I]-CtxMII binding. D and E, [125I]A-85380 binding in the presence of 100 nM -CtxMII. A and D, saline-treated animal, total binding. B and E, nicotine-treated animal, total binding. C and F, nonspecific binding.

View larger version (105K): [in this window] [in a new window]   Fig. 2. Representative autoradiographic images from coronal rat brain sections through superior colliculus. A to C, [125I]-CtxMII binding. D and E, [125I]A-85380 binding in the presence of 100 nM -CtxMII. A and D, saline-treated animal, total binding. B and E, nicotine-treated animal, total binding. C and F, nonspecific binding. SuG, superficial gray layer, superior colliculus.

View this table: [in this window] [in a new window]   TABLE 1 Autoradiographic binding in striatum and superior colliculus in rats treated chronically with saline or nicotine Values represent means ± S.E.M. of specific binding (femtomoles per milligram) of [125I]-CtxMII or [125I]A-85380 in the presence of 100 nM unlabeled -CtxMII, measured in the striatum and the superficial gray layer of the superior colliculus (n = 7–8).

Immunoprecipitation. The specificity of the 6 antibody for immunoprecipitation of nAChRs is shown in Table 2. It immunoprecipitated 23 and 28% of the nAChRs in the striatum and superior colliculus, respectively, whereas in the thalamus, less than 4% of the receptors were immunoprecipitated. No nAChRs were immunoprecipitated with the 6 antibody in the hippocampus, cerebral cortex, or cerebellum, where no gene transcript or 6 subunit protein has been reported, but which do express 2, 3, 4, 5, 2, and 4 subunits of nAChRs. These results indicate that that this 6 antibody is highly selective for the 6 subunit of nAChRs.

View this table: [in this window] [in a new window]   TABLE 2 Specificity of the 6 antibody

We first determined the nAChR subunit profiles in rat striatum and superior colliculus by immunoprecipitation with antibodies selective for different nAChR subunits. As shown in Fig. 3, 4 and 2 subunits are the predominant subunits in striatum as well as superior colliculus, but both regions also have a significant fraction of heteromeric nAChRs containing the 6 subunit, 23 and 28% of the total nAChRs in striatum and superior colliculus, respectively. This is consistent with earlier studies using similar immunoprecipitation procedures (Zoli et al., 2002; Gotti et al., 2005b), as well as with the relatively high level of [¹²⁵I]-CtxMII binding in these two regions, as shown in Figs. 1 and 2 and also found previously (Champtiaux et al., 2002). A smaller but significant percentage of the nAChRs also contain the 3 subunit, which is associated primarily with the 62^* subtype(s) in these brain regions (Champtiaux et al., 2003; Cui et al., 2003; Gotti et al., 2005b). In addition, significant levels of receptors containing 3 and 5 subunits were detected in striatum and superior colliculus. No receptors containing 2 or 4 subunits were detected in striatum or superior colliculus. The absence of the 4 subunit in these brain areas indicates that both regions require 2 subunits to form agonist-binding sites for all their heteromeric nAChRs.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Subunit profiles of heteromeric nAChRs in striatum and superior colliculus. nAChRs from striatum (A) and superior colliculus (B) were solubilized, labeled with [3H]EB and immunoprecipitated with each of the subunit-specific antibodies shown. Nonspecific immunoprecipitation was measured with NRS and has been subtracted. Data are mean ± S.E.M. from three to six experiments.

Results from immunoprecipitation assays in striatum from saline- and nicotine-treated rats are shown in Fig. 4. Because nAChRs containing 4 and 2 subunits are expressed at a much higher level than the nAChRs containing other subunits in striatum, the data for the receptors containing these predominant subunits and the less prevalent 6 and 3 subunits are presented in separate graphs with appropriate scales for easier visualization (Fig. 4, A and B). Chronic exposure to nicotine increased the number of striatal nAChRs immunoprecipitated by the antibodies to 4 and the 2 subunits by 40% each (Fig. 4A), consistent with the quantitative autoradiographic data for [¹²⁵I]A-85380 binding shown in Table 1. In contrast, immunoprecipitation with the antibody directed at the 6 subunit demonstrated a 40% decrease in the striatal nAChRs containing 6 subunits in nicotine-treated rats (Fig. 4B); this too is consistent with the quantitative autoradiographic data for [¹²⁵I]-CtxMII binding in striatum (Table 1). Interestingly, no change was detected in the number of nAChRs containing 3 subunits in the nicotine-treated rats (Fig. 4B), indicating that chronic exposure to nicotine did not affect the 3-containing nAChRs in the striatum.

View larger version (20K): [in this window] [in a new window]   Fig. 4. Effects of 14 days chronic nicotine exposure on nAChRs in rat striatum labeled by [3H]EB and immunoprecipitated by various antibodies. A, nAChRs immunoprecipitated by 4 and by 2 antibodies (n = 5 for each). B, nAChRs immunoprecipitated by 6 and 3 antibodies (n = 18 for each). Note different y-axis scale in A and B. Different from saline controls: **, p < 0.01; ***, p < 0.001.

View larger version (20K): [in this window] [in a new window]   Fig. 5. Effects of 14 days chronic nicotine exposure on nAChRs in rat superior colliculus labeled by [3H]EB and immunoprecipitated by various antibodies. A, nAChRs immunoprecipitated by 4 and by 2 antibodies (n = 5 for each). B, nAChRs immunoprecipitated by 6 and 3 antibodies (n = 18 for each). Note different y-axis scale in A and B. Different from saline controls, ***, p < 0.001.

View larger version (23K): [in this window] [in a new window]   Fig. 6. Recovery from effects of chronic nicotine exposure on nAChRs in rat striatum labeled by [3H]EB and immunoprecipitated by 6 antibodies. After 14 days of treatment by osmotic minipumps, animals (n = 5–6) were either sacrificed (saline; Nic + 0), or pumps were removed, followed by sacrifice at indicated days (+1, +3, +7, +30). Different from saline controls, *, p < 0.05.

View larger version (12K): [in this window] [in a new window]   Fig. 7. Effects of chronic nicotine exposure on release of [3H]dopamine from rat striatal synaptosomes. Data points represent percentage of total tissue [3H]dopamine per fraction, ±S.E.M.; n = 9 for each treatment group. Lines represent best fit of data to a sigmoidal dose-response curve. A, total release; for saline-treated, Emax, 3.85 ± 0.13; EC50, 0.13 µM; for nicotine-treated, *, Emax, 3.33 ± 0.13; EC50, 0.14 µM. B, -CtxMII-resistant release (in presence of 50 nM -CtxMII); for saline-treated, Emax, 2.39 ± 0.10; EC50, 0.12 µM; for nicotine-treated, Emax, 2.53 ± 0.11; EC50, 0.18 µM. C, -CtxMII-sensitive release (calculated by A - B for each nicotine concentration); for saline-treated, Emax, 1.95 ± 0.14; EC50, 5.9 µM; for nicotine-treated, *, Emax, 0.90 ± 0.14; *,EC50, 0.91 µM. *, Different from saline controls, p < 0.05.

	Discussion

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nAChR up-regulation during chronic nicotine exposure has long been viewed as somewhat of a paradox, and we now know sensitivity to up-regulation in vivo varies by receptor subtype, with 42^* receptors being highly sensitive, 7^* receptors less sensitive, and 34^* receptors virtually unaffected (Flores et al., 1997; Dávila-García et al., 2003; Nguyen et al., 2003). The development of -CtxMII greatly facilitated the characterization of 6-containing nAChRs and aided studies of their regulation. In the striatum, these receptors assemble with 2 subunits (Salminen et al., 2005); thus, they might be expected to share sensitivity to up-regulation with the 42^* subtype. In fact, the initial report of regulation of 6^* nAChRs by chronic nicotine found an increase in these receptors in rat brain following 18 days of nicotine self-administration (Parker et al., 2004). However, subsequent studies in mice (Lai et al., 2005) and rats (Mugnaini et al., 2006) found a decrease in [¹²⁵I]-CtxMII binding in brain following chronic nicotine exposure. To further address this question, we administered nicotine to rats chronically and measured 6 nAChRs three ways: by autoradiography with [¹²⁵I]-CtxMII, by quantitative immunoprecipitation, and by nicotine-stimulated dopamine release from striatal synaptosomes. All three measurements yielded similar results, which lead to the conclusion that chronic nicotine exposure in this model causes either a decrease or no change in 6^* nAChRs, depending on brain region. Furthermore, although 3 subunits are frequently associated with 6-containing nAChRs (Cui et al., 2003; Gotti et al., 2005a), immunoprecipitation studies indicate that the receptor subtypes containing 3 subunits are not decreased by nicotine treatment.

The decrease in binding we detected by [¹²⁵I]-CtxMII autoradiography in rat striatum is consistent with that reported in mice after 1 to 6 weeks of nicotine given in drinking water (Lai et al., 2005) and in rats after 2 weeks of nicotine infused at 3 mg/kg/day via minipump (Mugnaini et al., 2006). In contrast to the decrease in the striatum, binding of [¹²⁵I]-CtxMII in the superior colliculus was unaffected by chronic nicotine exposure. The difference in the responsiveness to nicotine of putative 6-containing receptors in the striatum and superior colliculus confirms studies reported previously (Mugnaini et al., 2006). This difference is unlikely to be due to a difference in cellular location of the 6-containing receptors because in both regions these receptors seem to be located exclusively on incoming axon terminals (Zoli et al., 2002; Gotti et al., 2005b; Cox et al., 2006). In contrast to its effect to decrease striatal 6-containing receptors, nicotine treatment increased striatal binding of [¹²⁵I]A-85380, which in the presence of excess -CtxMII labels predominantly 42^* nAChRs.

To further delineate the 6-containing nAChR subtype(s) affected by nicotine treatment, we immunoprecipitated [³H]EB-labeled nAChRs with subunit-selective antibodies. We detected 6-containing receptors in significant numbers in both striatum and superior colliculus but few or none in the other regions surveyed, which is consistent with other studies (Whiteaker et al., 2000; Champtiaux et al., 2002). The effects of nicotine on 6-containing nAChRs measured by immunoprecipitation are entirely consistent with the autoradiography results; that is, down-regulation in striatum but no change in superior colliculus. In contrast, the 42 nAChRs were markedly increased in both brain regions. The immunoprecipitation studies showed also that the down-regulation of 6 nAChRs in the striatum persists for at least 3 days after stopping nicotine administration. Interestingly, Mugnaini et al. (2006), using autoradiographic measurements, reported that nicotine-induced down-regulation of 6 nAChRs lasted longer in dopamine terminal fields than in cell body regions. This difference could reflect the time required to transport newly synthesized receptors down the axons.

An important new finding revealed by our immunoprecipitation studies is that nicotine treatment does not change the density of the nAChRs containing 3 subunits. A majority of the 3 subunits are associated with 6 and 2 subunits in both the rat striatum and superior colliculus (Champtiaux et al., 2003; Gotti et al., 2005a,b). Thus, nicotine's differential regulation of nAChRs containing 6 subunits versus those containing 3 subunits suggests that the population of 62^* receptors labeled by [¹²⁵I]-CtxMII is heterogeneous. Consistent with this, we found more nAChRs containing 6 subunits than 3 subunits in both brain regions. This agrees with results from an earlier study in the rat striatum (Zoli et al., 2002) but differs from a study in superior colliculus (Gotti et al., 2005b). Moreover, although knocking out the 3 subunit in mice decreased a majority of the [¹²⁵I]-CtxMII binding sites in the striatum and a smaller fraction in the midbrain (Cui et al., 2003), a substantial fraction of these sites remained. Together, these data suggest that [¹²⁵I]-CtxMII labels at least two populations of 62^* nAChRs in the brain, one with the 3 subunit incorporated and the other without. Our immunoprecipitation studies distinguish between these 3-containing receptors in the striatum and suggest that the 623^* subtype does not down-regulate in response to nicotine in vivo.

The mechanisms underlying the decrease in 6-containing nAChRs are not yet known, but Lindstrom and colleagues (Tumkosit et al., 2006) have suggested that the decrease in 62^* receptors and the increase in 42 receptors, as seen in the striatum, could be related; thus, if the availability of the 2 subunit is limiting, and nicotine increases assembly of 42 nAChRs (Kuryatov et al., 2005; Sallette et al., 2005), the density of 62^* nAChRs might be decreased because of depletion of the pool of 2 subunits (Tumkosit et al., 2006). In heterologous cell models, the presence of the 3 subunit increases the expression of 623 receptors, suggesting that the 3 subunit stabilizes and/or allows more efficient assembly of this receptor subtype (Tumkosit et al., 2006). This might then explain why 3-containing nAChRs in the striatum are not decreased by nicotine.

The superior colliculus also seems to contain a larger fraction of nAChRs with 6 subunits than 3 subunits, again indicating that some 6 receptors contain 3 subunits and some do not. Yet, there was no measurable change in either [¹²⁵I]-CtxMII binding sites or nAChRs immunoprecipitated by the 6 or 3 antibodies, suggesting that even in the absence of the 3 subunit, some 6-containing receptors are resistant to nicotine-induced down-regulation. Most of the nAChRs in the superior colliculus are on retinal ganglion cell axons that innervate the superficial layers of the colliculus (Gotti et al., 2005b; Cox et al., 2006). Therefore, the absence of a nicotine-induced decrease in 6-containing receptors in the superior colliculus even while 42 receptors are increased suggests that these receptors are expressed by different retina ganglion cells.

Because stimulation of nAChRs in dopamine terminal regions enhances dopamine release, we assessed this activity in striatal synaptosomes prepared from rats treated with nicotine or saline. Our finding that 38% of the nicotine-stimulated dopamine release in saline-treated animals is blocked by -CtxMII is consistent with reports of from 30 to 70% block in rodents and monkeys (Kaiser et al., 1998; Champtiaux et al., 2003; Salminen et al., 2004; McCallum et al., 2005). Neurotoxin studies demonstrate that 6-containing nAChRs in this region are localized to dopamine terminals, whereas non-6 nAChRs are found on multiple cell types (Zoli et al., 2002; Champtiaux et al., 2003). This probably explains why the proportion of -CtxMII-sensitive dopamine release is higher than would be expected by the overall proportion of 6-containing nAChRs. Our finding of a 54% decrease in the E_max for nicotine-stimulated dopamine release confirms that the decrease in 6^* receptor number is matched by a change in receptor function in striatum. Likewise, in mice treated chronically with oral nicotine, -CtxMII-sensitive striatal dopamine release declined 65% (Lai et al., 2005).

Chronic nicotine exposure had essentially no effect on -CtxMII-resistant dopamine release. Similar results were reported in mice exposed to chronic oral nicotine (Lai et al., 2005). This may seem surprising in light of the increase in 42^* nAChRs; however, a large fraction of these receptors are not located on dopamine terminals (Zoli et al., 2002; Champtiaux et al., 2003) and thus do not directly mediate dopamine release. Furthermore, a significant fraction of striatal 42^* receptors coexpress the 5 subunit, and virtually all of these 425 receptors are located on dopamine terminals (Zoli et al., 2002; Champtiaux et al., 2003). Interestingly, we recently found that 42 nAChRs that contain the 5 subunit are not up-regulated by nicotine treatment in vivo (Perry et al., 2005). Thus, despite the overall increase in striatal 42^* nAChRs induced by chronic nicotine, it seems likely that the 42^* nAChRs on dopamine terminals are not up-regulated by chronic nicotine. In fact, the nicotine-induced release of dopamine in vivo seems to be mediated by 42 nAChRs in the cell body areas (Corrigall et al., 1994; Nisell et al., 1997; Champtiaux et al., 2003).

In conclusion, we have used both receptor autoradiography and subunit-selective immunoprecipitation to demonstrate that chronic exposure of rats to nicotine leads to a decrease in 6-containing nAChRs in the striatum but not in the superior colliculus. The presence of the 3 subunit and perhaps other subunits in the 6-containing receptors seems to modulate the regulatory effects of nicotine on these receptors. The decrease in striatal 6^* nAChRs persisted for at least 3 days following termination of nicotine and was accompanied by a decline in -CtxMII-sensitive dopamine release in striatal synaptosomes, demonstrating the functional significance of the decrease in receptor number. The shift in the nicotinic receptor profile following such exposure may be relevant to nicotine dependence and its treatment.

	Acknowledgements

 
We thank Ashleigh Keller for superb technical assistance. We thank Barry B. Wolfe and Robert P. Yasuda (Georgetown University, Washington, DC) and Scott Rogers and Lorise Gahring of University of Utah (Salt Lake City) for sharing antibodies.

	Footnotes

 
This study was supported by the National Institutes of Health [Grants DA015767 (to D.C.P.), DA012976 (to K.J.K.), and MH53631 and DA12242 (to J.M.M.)].

D.C.P. and D.M. contributed equally to this work.

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

doi:10.1124/jpet.107.121228.

ABBREVIATIONS: nAChR, neuronal nicotinic acetylcholine receptor; -CtxMII, -conotoxin MII; A-85380, 5-iodo-3-(2(S)-azetidinylmethoxy)pyridine; NRS, normal rabbit serum; EB, epibatidine.

Address correspondence to: Kenneth J. Kellar, Department of Pharmacology, Georgetown University School of Medicine, 3900 Reservoir Road NW, Washington DC 20057. E-mail: kellark{at}georgetown.edu

	References

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