Up-Regulation of Brain Nicotinic Acetylcholine Receptors in the Rat during Long-Term Self-Administration of Nicotine: Disproportionate Increase of the 6 Subunit

In male rats continually self-administering nicotine (approximately 1.5 mg free base/kg/day), we found a significant increase of nicotinic acetylcholine receptors (nAChRs) labeled by epibatidine (Epb) in 11 brain areas. A large increase of highaffinity Epb binding sites was apparent in the ventral tegmentum/substantia nigra, nucleus tractus solitarii, nucleus accumbens, thalamus/subthalamus, parietal cortex, hypothalamus, and amygdala. A smaller but significant up-regulation of highaffinity Epb sites was seen in the piriform cortex, hippocampus, caudate/putamen, and cerebellar cortex. The up-regulation of nAChRs, shown by immunoadsorption and Western blotting, involved 4, 6, and 2 subunits. As a consequence of longterm self-administration of nicotine, the 6 immunoreactive (IR) binding of either labeled Epb or I-conotoxin MII increased to a much greater extent than did 4 or 2 IR binding of Epb. In addition, the 2 IR binding of Epb was consistently enhanced to a greater extent than was 4. These findings may reflect a larger surface membrane retention of 6-containing and, to some degree, 2-containing nAChRs compared with 4-containing nAChRs during long-term self-administration of nicotine. Nicotine, one of the most widely abused addictive alkaloids, activates a family of pentameric nicotinic acetylcholine receptors (nAChRs) known to transport cations. This affects the release of neurotransmitters (Gallardo and Leslie, 1998; Fu et al., 2000a) and influences diverse functions, including feeding, arousal, endocrine regulation, nociception, and aspects of cognition. Cell membrane nAChRs accumulate in various paradigms of long-term exposure to nicotine or other nAChR modulators. This occurs in the central nervous system of primates, including humans (Perry et al., 1999); of rodents, including mice (Marks et al., 1983; Pauly et al., 1996) and rats (Schwartz and Kellar, 1983; Mugnaini et al., 2002); as well as in mammalian cells expressing cloned nAChRs (Wang et al., 1998). No substantial up-regulation of mRNAs encoding the major nAChR subunits was detected in the brains of rodents that exhibited major increases in nAChRs after long-term treatment with nicotine (Pauly et al., 1996; Mugnaini et al., 2002). The increases in nAChRs are thus not likely to depend primarily on enhanced de novo synthesis, but they could be linked to nAChR desensitization. A largely reversible partial desensitization of nAChRs in response to nicotine was documented in rodent brain (Damsma et al., 1989; Sharp and Matta, 1993). In view of the low nAChR endocytosis and recycling in homeotherms (Higgins and Berg, 1988) and the generally limited control of concentration of cell membrane constituents, aggregation or compartmentalization of nAChRs could lead to the accumulation of membrane receptors during long-term nicotine treatment. In rodent forebrain, the principal nAChRs contain 2 subunits ( 2* nAChRs) and 4 subunits ( 4* nAChRs) (Flores et al., 1992); 7 receptors are also well represented (Kaiser and Wonnacott, 2000). In several areas, there is significant expression of 6* (Champtiaux et al., 2002; Zoli et al., 2002) and 3* (Azam et al., 2002) nAChRs. The release of dopamine by nicotine (Damsma et al., 1989; Fu et al., 2000a) seems to be associated with 6* (Champtiaux et al., 2002), 3* (Kulak This work was supported by National Institutes of Health grants DA03977 (to B.M.S.), NS11323 (to J.M.L.), and MH53631 (to J.M.M.). ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; Bgtx, -bungarotoxin; Cntx, -conotoxin MII; Epb, epibatidine; VTA/SN, the ventral tegmentum area/substantia nigra; IR, immunoreactive; HEK, human embryonic kidney; 2* nAChR, nicotinic acetylcholine receptor containing 2 subunits; 4* nAChR, nicotinic acetylcholine receptor containing 4 subunits. 0026-895X/04/6503-611–622$20.00 MOLECULAR PHARMACOLOGY Vol. 65, No. 3 Copyright © 2004 The American Society for Pharmacology and Experimental Therapeutics 2906/1128610 Mol Pharmacol 65:611–622, 2004 Printed in U.S.A. 611 at A PE T Jornals on Jne 2, 2017 m oharm .aspeurnals.org D ow nladed from et al., 2001), and 7 receptors (Fu et al., 2000b). Indeed, nicotine-induced locomotor activity, associated with enhanced striatal dopaminergic activity, is selectively decreased by 6* antisense oligonucleotides (Le Novere et al., 1999). Because the regulation of dopaminergic neurotransmission by acetylcholine may involve 6* receptors at the level of dopamine terminals (Champtiaux et al., 2003), an increase in this subtype of nAChR during long-term exposure to nicotine may alter dopamine release. In cell lines, up-regulation of surface nAChRs by nicotine was shown to include both 3* (Meyer et al., 2001; Ridley et al., 2002) and 6*, as well as 4* (Nelson et al., 2003) and 7 receptors (Quik et al., 1996). Oocyte-expressed 3* receptors are less sensitive than the 4* receptors to down-regulation (Fenster et al., 1997) and desensitization (Hsu et al., 1996) by nicotinic agonists. This may also pertain to 6* receptors, because 3 and 6 subunits are structurally and pharmacologically similar (Champtiaux et al., 2002; Kulak et al., 2002). Also, 4* receptors may show a larger constitutive removal from cell membrane than 3* receptors (Cooper et al., 1999) and presumably 6* receptors. Thus, nAChRs containing different subunits may not increase proportionally during long-term treatment with nicotine. In view of the above findings, our experimental objectives were (1) to measure changes in nAChR expression related to long-term nicotine self-administration, especially in brain areas known for important interactions between nicotinic cholinergic and catecholaminergic neurotransmission and (2) to determine whether nicotine self-administration selectively up-regulates the nAChR subunits that are preferentially involved in the activation of catecholaminergic systems. Our results show a larger up-regulation of 6* (and also of 2*) relative to 4* nAChRs in several major brain areas of rats continually self-administering nicotine. Materials and Methods Chemicals and Antibodies. Chemicals were obtained from Sigma Chemical (St. Louis, MO) and Sigma/RBI (Natick, MA). Radioactive chemicals were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). -Conotoxin MII was synthesized and labeled with I by J. M. McIntosh. Mouse monoclonal antibody to bacterially expressed human 6 nAChR subunit (mAb350) and rat monoclonal antibodies against rat 4 (mAb299) and 2 (mAb295) subunits were raised by J. Luo and J. M. Lindstrom. To remove any 3 antibody-like immunoprotein, mAb350 was pretreated with human 3 nAChR subunit immobilized on CH Sepharose 4B (Kuryatov et al., 2000). This antibody does not cross-react with 4 2 or 3 2 nAChRs expressed in oocytes (Kuryatov et al., 2000). The polyclonal rabbit antibodies to bacterially expressed human 3 (sc-5590), 4 (sc-5591), 7 (sc-5544), and 2 (sc-11372) subunits were purchased from Santa Cruz Biochemicals (Santa Cruz, CA). These antibodies were all raised against large ( 100 residues) C-terminal peptides from the respective subunits, and specific blocking peptides are not available. However, neither the monoclonal 6 antibody nor the polyclonal 4 and 2 antibodies produced significant immunoadsorption of 3 4 nAChRs expressed in HEK-293 cells (Xiao et al., 1998) (see Results). Also, saturating quantities of the polyclonal 7 antibody did not affect the immunoadsorption of any of the above antibodies; conversely, the immunoadsorption of I-bungarotoxin bound to solubilized 7 receptors from four brain areas was not modified by saturating amounts of the above 3, 4, 6, or 2 antibodies (see Results). Animals and Treatments. Male Lewis rats (2.5 months of age; initial body weight, 220–240 g) were obtained from Harlan (Indianapolis, IN), and male DBA mice (6–7 weeks old) were from the National Cancer Institute (Bethesda, MD). All treatments were in accord with the animal care protocols approved by the Animal Care and Use Committee of the University of Tennessee Health Science Center at Memphis. Nicotine self-administration was performed according to our protocols published previously (Fu et al., 2001). Briefly, 7 d after acclimation to a reverse-light cycle and handling, all rats received jugular cannulae under xylazine-ketamine anesthesia and then were immediately housed in individual environmental enclosures without shaping, conditioning, or food deprivation. After an additional 3 d of recovery, rats were randomly assigned to treatment groups, and the jugular lines were filled either with nicotine bitartrate (4 mM in heparinized saline; 50 l delivered over 0.81 s per 300 g of body weight in one self-administration) or saline without nicotine. The self-administration of nicotine proceeded as detailed previously (Fu et al., 2001). Stable nicotine self-administration, defined as 3 consecutive days showing more than 40 active lever presses per day at less than 15% variance, and with active (green-lit) lever-press counts significantly greater than those of inactive lever, was achieved within 6.5 1.0 (mean S.E.M.) days. At that time, the approximate dosage of self-administered nicotine free base was 1.5 mg/kg/day (Fig. 1). After 18 days of self-administration, the animals were anesthetized with isoflurane before decapitation by guillotine. Brains were promptly excised and frozen in dry ice before storage for not more than 14 days at 80°C. Tissue Collection. The rat or mouse forebrains were sliced (at 7°C) into 1-mm thick sections. Selected areas were then excised according to the coordinates of Paxinos and Watson (1986) and Franklin and Paxinos (1997), respectively. The following areas were collected from rat brains: parietal cortex, the piriform/entorhinal cortex, the caudate/putamen (from 0 to 2 mm anterior to bregma), nucleus accumbens (2 mm centered about anterior commissure and 0–2 mm anterior to bregma), amygdala (medial to entorhinal/piriform cortex, from approximately 3 to 6 mm posterior to bregma), hypothalamus (from optic chiasm to mammillary nuclei, 1–5 mm posterior to bregma), thalamus (including subthalamic area; approximately 2–3 mm inferior to corpus callosum and 3 mm lateral to midline, 1–6 mm posterior to bregma), hippocampus (from 4 to 6 mm posterior to bregma), ventral tegmental area/substantia nigra (VTA/ SN; ventral horizontal cut, 4–6 mm posterior to bregma), the nucleus tractus solitarii area (1 mm inferior and 2 mm lateral to the fourth ventricle, 11–13 mm posterior to bregma), and rostral cerebellar cortex. With mouse brains, the thalamus/subthalamus was collected from 0.5 to 3 mm posterior to bregma, 1 to 2 mm inferior to corpus callosum, and approximately 2.5 mm lateral to midline. The hippocampus was taken from 3 to 4 mm posterior to bregma, and parietal/somatosensory cortex at 1 mm from bregma. Excised tissues were immediately stored at 80°C and then processed to obtain particulates within 7 days. Isolation of Particulates. The isolation of particulates was done at 0 to 4°C, essentially as described previously by Marks et al. (1998). The tissue homogenate was made in ice-cold Epb assay buffer, using a motor-driven Teflon pestle at 800 rpm. The homogenate was sedimented for 12 min at 20,000gmax, and the pellets were resedimented before storage at 80°C. The Epibatidine Binding Assay. The protocol described by Marks et al. (1998) was generally followed, including the same binding buffer. Particulates or dispersed cells were resedimented twice from this buffer before assays. For competition assays with Iepibatidine (2170 Ci/mmol) (PerkinElmer Life and Analytical Sciences), 50 pM was used, yielding binding parameters similar to those obtained from saturation binding of I-Epb or [H]Epb (see Results). To obtain numerically adequate binding, the input of [H]epibatidine (56 Ci/mmol) (PerkinElmer Life and Analytical Sciences) in competition assays was 500 pM. Duplicate assays at 0, 10, 30, 100, 300, and 1000 pM unlabeled Epb and at 30 M ( )-nicotine (for nonspecific binding) were done for quantitative comparisons between animals self-administering nicotine versus saline. For saturation 612 Parker et al. at A PE T Jornals on Jne 2, 2017 m oharm .aspeurnals.org D ow nladed from assays, the Ior H-labeled Epb input was 10 to 1000 pM using 8 to 12 concentrations. The protein concentration was 0.025 mg/ml in I-Epb assays and 0.1 mg/ml in [H]Epb assays. The assay volume was 0.40 ml, and reactions were incubated for 90 min at 23 to 24°C. Under these conditions, 95% saturation of 50 pM I-Epb or 500 pM [H]Epb and 500 pM I-Epb binding was achieved in less than 15 and 2 min, respectively. The time points for binding kinetics, measured by filtration, were 1, 3, 6, 10, 20, 30, 45, 60, and 90 min. In kinetic as well as in some saturation experiments, the binding was terminated by filtration using Whatman GF/C filters (Whatman, Clifton, NJ) (presoaked in 0.3% polyethylenimine) and washing with ice-cold assay buffer. No consistent differences in binding were apparent between the centrifugation and filtration protocols. The competition and most saturation assays were terminated by spinning for 12 min at 20,000gmax at 4°C followed by rinsing with ice-cold assay buffer. The tube bottoms or filters containing bound I-Epb were counted in a MicroMedic -scintillation counter (Valeant Pharmaceuticals International, Costa Mesa, CA). For counting of [H]Epb, the pellets were solubilized, or the filters were soaked overnight in 2% sodium dodecylsulfate/10 mM Tris-HCl, pH 8.8, followed by the addition of liquid scintillation solvent and then underwent counting in a Beckman LS-3801 counter (Beckman Coulter, Fullerton, CA). Receptors solubilized in 2% Triton X-100 were quantified by polyethylene glycol precipitation (Parker et al., 1998b). The Binding of I-Labeled -Conotoxin MII and -Bungarotoxin. -Conotoxin MII (Cntx) was synthesized and monoiodinated (specific activity, 2170 Ci/mmol) as described previously (Whiteaker et al., 2000b), and I-labeled -bungarotoxin (Bgtx) (specific activity, 140 Ci/mmol) was obtained from PerkinElmer Life and Analytical Sciences. Assay inputs of Cntx and Bgtx were 0.4 and 1.0 nM, respectively. Nonspecific binding for Cntx was defined at 10 M ( )-cytisine plus 100 M ( )-nicotine (because nicotine alone in some cases did not displace all binding that was sensitive to cytisine). For Bgtx, nonspecific binding was defined at 1 M unlabeled Bgtx. The particulates were resedimented once from the binding buffer described by Whiteaker et al. (2000b) before incubation at 0.4 mg protein/ml protein for 2 (Cntx) and 5 (Bgtx) h at 24°C. The assay was terminated using Whatman GF/C filters presoaked in 1% polyethylenimine and rapid washing with ice-cold binding buffer. Nonspecific binding of Cntx and Bgtx was 40 to 60% of total binding. Immunoadsorption of Nicotinic Receptors. At 2% Triton X-100 with inputs of solubilized protein lower than 0.2 mg/ml, the polyclonal rabbit antibodies to C-terminal portions of human 3, 4, and 2 nAChR subunits (expressed in Escherichia coli) produced saturating immunoadsorption of Epb-labeled nAChRs at immunoglobulin concentrations lower than 6 g/ml; the mouse and rat monoclonal antibodies produced saturating immunoadsorption lower than 30 g of immunoglobulin/ml. Particulates were processed at 0 to 4°C as described by Wang et al. (1998). Briefly, particulates were solubilized at 2% Triton X-100 and 0.4 to 0.5 mg protein/ml, and after 90 min in ice, they were centrifuged for 12 min at 20,000gmax. Tracers (1 nM [H]Epb, 200 pM I-Epb, 500 pM I-Cntx, or 1 nM I-Bgtx) were added to the supernatant and incubated for 16 to 18 h at 4°C. Duplicate aliquots containing 25 g of extracted protein were mixed, in the final volume of 0.20 ml, with the appropriate antibody, using 1, 3, and 6 l of each antibody. This corresponded to 0.2, 0.6, and 1.2 g of immunoglobulin for the polyclonal antibodies, or 1, 3, and 6 g of immunoglobulin for the monoclonal 6 antibody, and produced a complete saturation of receptor immunoadsorption for all antibodies (see Results) (Fig. 5A). The mixtures were rotated with protein A/protein G agarose (Santa Cruz Biochemicals) for 8 h at 4°C. Correction for nonspecific adsorption was obtained by incubating the largest input of the respective antibody with 10 M cytisine plus 100 M ( )-nicotine for Epb or Cntx tracers and with 1 M unlabeled Bgtx plus 100 M nicotine for the Bgtx tracer. The nonspecific adsorption was always less than 2% of total agarose gel radioactivity with Epb tracers (with typical “signal-to-noise” ratios better than 100 for 3 and 6 and better than 1000 for 4 and 2 subunits), but it represented up to 30% of total gel radioactivity with I-Cntx or I-Bgtx tracer. The assays were terminated by dilution and sedimentation for 1 min at 1000gmax followed by three resedimentations Fig. 1. A comparison of lever-pressing activity in self-administration of nicotine or saline by rats over 18 days after habituation. Six animals were compared in each group. The brains of these animals were used in receptor assays shown in Fig. 3. The mean numbers of lever presses per day ( S.E.M.) for the four data sets were analyzed by two-way analysis of variance (see Materials and Methods). The difference between treatments was highly significant (F 51.6, p 0.001), whereas the difference across the time points (F 1.34, p 0.164) or that in interaction between the type of treatment and the length of treatment (F 1.29, p 0.087) was not significant. The data were further compared in Scheffe’s tests, and the differences significant at 95% confidence between the nicotine self-administering set and all other sets are indicated in the graph by asterisks. Up-Regulation of Brain nAChR Subunits by Nicotine 613 at A PE T Jornals on Jne 2, 2017 m oharm .aspeurnals.org D ow nladed from from fresh assay buffer. [H]Epb-labeled pellets were dispersed in 2% sodium dodecyl sulfate/0.01 M Tris-HCl, pH 8.8, before liquid scintillation counting. Immunoblotting of Nicotinic Receptors. The washed particulates were solubilized and extracted at 4°C in radioimmunoprecipitation assay buffer (Santa Cruz Biochemicals), the extracts were cleared (30 min at 30,000gmax and 4°C), and the supernatants were mixed with an equal volume of a denaturation buffer (Bio-Rad, Hercules, CA), heated for 30 min at 98°C, and then resedimented as described above. Aliquots of the supernatant were electrophoresed in polyacrylamide gels, electroeluted onto nitrocellulose, and saturated with a blotting buffer (Blotto B; Bio-Rad). The membranes were then incubated for 16 h at 4°C in 0.14 M NaCl/10 mM Tris-HCl, pH 7.4, containing 2 g/ml of one of the rabbit polyclonal antibodies (Santa Cruz Biochemicals) to human 3, 4, or 2 nAChR subunits or 6 g/ml of the mouse monoclonal antibody to human 6 subunit. After washing and a 90-min exposure to goat anti-rabbit or anti-mouse immunoglobulins coupled to horseradish peroxidase (1:2000 to 1:5000 dilution) and final washing, membranes were exposed to Luminol bioluminescent reagent (Santa Cruz Biochemicals), apposed to Kodak BR Biomax film (Eastman Kodak, Rochester, NY), and the negatives were scanned. Data Analysis. To analyze the frequency of nicotine and saline self-administration, the mean number of lever presses per day were compared by two-way analysis of variance, followed where appropriate by Scheffe’s test, using SPSS statistical software, version 11.0 (SPSS Inc., Chicago, IL). Evaluation of immunoadsorption with multiple data-point profiles was done by nonlinear fitting to MichaelisMenten equation (Parker and Waud, 1971) followed by t tests on means of the binding parameters. Grain density in scans of Western blot profiles was evaluated using OptiQuant software (PerkinElmer Life and Analytical Sciences). The means of receptor binding parameters were compared in two-tailed Student’s or Dunnett’s t tests (Zar, 1984). The quantitative comparisons on six competition points (see The Epibatidine Binding Assay section under Materials and Methods) were done with least-square Scatchard fits, because nonlinear Scatchard fitting with only six concentration points tends to result in large variations; the least-squares Scatchard linearization using analytical second derivatives is routine in similar evaluations (Segel, 1974; Marks et al., 1998). The nonlinear curve fits generated by either the LIGAND program (Munson and Rodbard, 1980) or the curve-fitting facility of the SigmaPlot program (version 8.0; SPSS Inc.) were used to check for multiple components in competition profiles with eight or more concentration points. Hill slopes (nH) were calculated using Scatchard estimates of Bmax in the logarithmic Hill equation (Segel, 1974). Sequence comparisons were done in FASTA programs (Pearson, 2000).