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Pharmacology of -Conotoxin MII-Sensitive Subtypes of Nicotinic Acetylcholine Receptors Isolated by Breeding of Null Mutant Mice

Institute for Behavioral Genetics, University of Colorado, Boulder, Colorado (O.S., J.A.D., E.L., M.C., A.C.C., M.J.M., S.R.G.); and Departments of Biology and Psychiatry, University of Utah, Salt Lake City, Utah (J.M.M.)

Received October 4, 2006; accepted March 6, 2007

	Abstract

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Subtypes of nicotinic acetylcholine receptors (nAChR) containing 6 subunits comprise 25 to 30% of the presynaptic nAChRs expressed in striatal dopaminergic terminals in rodents and 70% in monkeys. This class of receptors, potentially important in nicotine addiction, binds -conotoxin MII (-CtxMII) with high affinity and is heterogeneous, consisting of several subtypes in mice, possibly an important consideration for the design of compounds that selectively activate or antagonize the 6 subclass of nAChRs. Selected-null mutant mice were bred to generate isolated subtypes of 62* nAChRs expressed in vivo for assessing pharmacology of 62* nAChRs. Binding to striatal membranes and function in synaptosomes from (4–/–)(3+/+) and (4–/–)(3–/–) mice were measured and compared with wild-type (4+/+)(3+/+) mice. Gene deletions (4 and 3) decreased binding of ¹²⁵I--CtxMII without affecting affinity for -CtxMII or inhibition of -CtxMII binding by epibatidine or nicotine. Deletion of the 4 subunit substantially increased EC₅₀ values for both nicotine- and cytisine-stimulated -CtxMII-sensitive dopamine release from striatal synaptosomes. A further increase in EC₅₀ values was seen upon the additional deletion of the 3 subunit. The data indicate that one -CtxMII-sensitive nAChR subtype, prevalent on wild-type dopaminergic terminals, has the lowest EC₅₀ for a nicotine-mediated function so far measured in mice. In conclusion, the gene deletion strategy enabled isolation of 6* subtypes, and these nAChR subtypes exhibited differential activation by nicotine and cytisine.

Identifying the nAChR subtypes that modulate dopamine release is of interest because dopamine plays a vital role in reinforcing effects of nicotine (Dani and Heinemann, 1996). Early pharmacological studies established that nicotinic agonists elicit robust increases in [³H]dopamine release from rodent striatal synaptosomes (Rapier et al., 1988, 1990; Grady et al., 1992). The discovery that -conotoxin MII (-CtxMII) is a partial inhibitor established that at least two nAChR subtypes modulate [³H]dopamine release (Grady et al., 1997; Kulak et al., 1997). Approximately 25 to 30% of agonist-evoked [³H]dopamine release from striatal synaptosomes is mediated by -CtxMII-sensitive nAChRs in rodent (Kulak et al., 1997; Kaiser et al., 1998; Salminen et al., 2004). A larger percentage (70%) is -CtxMII-sensitive in monkey (McCallum et al., 2005).

Expression of multiple nAChR subtypes in dopaminergic neurons is suggested by detection of mRNA for eight nAChR subunits (3–7, 2–4) in dopamine cell bodies (Klink et al., 2001; Azam et al., 2002). Studies with nAChR subunit-null mutant mice demonstrate that certain subunits play vital roles in those nAChRs that modulate dopamine release. Agonist-induced dopamine release is totally lost from striatal synaptosomes (Grady et al., 2001) and slices (Zhou et al., 2001) obtained from 2-null mutant mice, establishing that the 2 subunit plays an essential role in all nAChR subtypes that modulate dopamine release. Although -CtxMII binds with high affinity to both 6*- and 32*-nAChRs (Cartier et al., 1996; McIntosh et al., 2004) (* indicates the possibility of additional subunits; Lukas et al., 1999), this binding, as well as -CtxMII-sensitive agonist-stimulated dopamine release, is totally absent in dopaminergic neurons of 6-null mutant mice (Champtiaux et al., 2002, 2003), establishing that 6* rather than 3* nAChRs modulate dopamine release. The 3 gene deletion substantially decreases the -CtxMII-sensitive component of dopamine release, indicating that the 3 subunit is important in nAChRs that bind -CtxMII with high affinity (Cui et al., 2003). We have analyzed the effects of 4, 5, 7, 2, 3, and 4 gene deletion on -CtxMII-sensitive and -resistant dopamine release (Salminen et al., 2004). Results with 2 and 3 deletions replicated those in our previous reports (Grady et al., 2001; Cui et al., 2003). Deleting the 4 gene totally eliminated the -CtxMII-resistant component of dopamine release as well as 50 to 60% of the -CtxMII-sensitive component, whereas deleting the 5 subunit decreased maximal -CtxMII-resistant dopamine release. We concluded that 42 and 452 nAChRs modulate the -CtxMII-resistant component of dopamine release and that 6432, 632, and perhaps 62 nAChRs modulate the -CtxMII-sensitive component of dopamine release. Using an immunochemical approach to assess expression of 6-containing nAChRs, Gotti et al., (2005) also concluded that the -CtxMII-sensitive nAChRs expressed in dopaminergic neurons are comprised of 6432, 63b2, and 62 nAChRs.

Studying the pharmacology of 6*-nAChRs has been hampered by the difficulty of expressing 62-nAChRs in Xenopus laevis oocytes (Kuryatov et al., 2000). However, under conditions in which some expression was achieved, addition of 6to 4 and 2 subunits did not change EC₅₀ values for activation by ACh or nicotine, and chimeric 6/3 expression with 2 increased EC₅₀ values compared with 42-nAChRs (Kuryatov et al., 2000). These results contrast with our measurements for agonist-stimulated dopamine release, where the -CtxMII-sensitive 62*-nAChRs exhibited significantly lower EC₅₀ values than the -CtxMII-resistant 42*-nAChRs (Salminen et al., 2004). Recombinant receptors may not have the same pharmacological selectivity as native receptors (Nicke et al., 2003). On the other hand, the more complex 6432 nAChR, not yet studied in oocytes, may be responsible for the increased sensitivity to agonists observed for dopamine release.

The studies described here used wild-type, 4-null mutant and 4/3 double-null mutant mice to characterize the pharmacology of 62* nAChRs. The sequential gene deletion strategy is designed to simplify 6* expression from that of wild-type (6432, 632, and 62) to that of 4-null mutant mice (632 and 62), to that of the 4/3 double mutation (62), thereby allowing the characterization of the pharmacological properties of functional receptors assembled with normal processing and expressed in appropriate dopaminergic cells.

	Materials and Methods

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Materials. 7,8-[³H]Dopamine was obtained from PerkinElmer Life and Analytical Sciences (Boston, MA) (specific activity, 40–60 Ci/mmol). HEPES and sucrose were products of Roche Applied Science (Indianapolis, IN). Sigma-Aldrich (St. Louis, MO) was the source for the following compounds: acetylcholine iodide (ACh), ascorbic acid, aprotinin, atropine sulfate, A-85380, bovine serum albumin (BSA), cytisine, dihydro--erythroidine (DHE), diisopropyl fluorophosphate, (–)-epibatidine hydrochloride, EDTA, EGTA, leupeptin trifluoroacetate, methyllycaconitine (MLA), (–)-nicotine tartrate, nomifensine, pargyline, polyethylenimine, pepstatin A, and phenylmethylsulfonyl fluoride. -CtxMII was synthesized as described previously (Cartier et al., 1996), as was ¹²⁵I--CtxMII (Whiteaker et al., 2000) (specific activity, 2200 Ci/mmol). All other chemicals were reagent grade. OptiPhase SuperMix scintillation fluid was purchased from PerkinElmer Life Sciences and Analytical Sciences–Wallac Oy (Turku, Finland).

Animals. Animal care and experimental procedures were in accordance with the guidelines and approval of the Animal Care and utilization Committee of the University of Colorado, Boulder. Mice used in this study were bred and maintained at the Institute for Behavioral Genetics, University of Colorado (Boulder, CO). Mice were weaned at 25 days of age and housed with same-sex littermates. A 12-h light/dark cycle (lights on from 7 AM to 7 PM) at 22°C was used. Mice had free access to food (Teklad Rodent Diet; Harlan, Madison, WI) and water. DNA was extracted from tail clippings, taken at 40 days of age, using the DNeasy kit from QIAGEN (Valencia, CA) and analyzed by polymerase chain reaction for assignment of genotype (Salminen et al., 2004).

The 4-null mutant mice, originally obtained from the laboratory of John Drago (Ross et al., 2000), were bred onto C57BL/6 background for 1 generation, and the 3-null mutants, from the laboratory of Stephen Heinemann (Cui et al., 2003), were bred onto C57BL/6 background for nine generations. Wild-type mice [the (4+/+)(3+/+) genotype] were littermates of the 4-null mutant mice [the (4–/–)(3+/+) genotype] and both were generated by breeding mice heterozygous for the 4 subunit [the (4+/–)(3+/+) genotype] and were of mixed genetic background. Mice with the double 4- and 3-null mutations [the (4–/–)(3–/–) genotype] were generated by first breeding mice of the (4–/–)(3+/+) genotype with mice of the (4+/+)(3–/–) genotype to generate mice of the (4+/–)(3+/–) genotype. These mice were subsequently bred together and pups were genotyped for both null mutations. This procedure generated both (4–/–)(3+/+) and (4–/–)(3–/–) mice that were littermates and on a mixed background with a greater contribution of C57BL/6 than the wild-type and 4-null mutants described above. No differences in binding or functional measures were found between the mixed background wild-type mice and C57BL/6 mice or between the two mixed backgrounds of the (4–/–)(3+/+) mice. The double knockout mice generated were subsequently bred for one generation to produce sufficient numbers of defined double-knockout mice.

Synaptosome Preparation for Release Experiments. After a mouse was sacrificed by cervical dislocation, its brain was removed and placed immediately on an ice-cold platform. The striatum (ST), olfactory tubercle (OT), and/or nucleus accumbens (NAcc) were dissected. Tissues from each mouse were homogenized in 0.5 ml of ice-cold 0.32 M sucrose buffered with 5 mM HEPES, pH 7.5. A crude synaptosomal pellet was prepared by centrifugation at 12,000g for 20 min. The pellets were resuspended in "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 0.01 mM pargyline using 1.6 ml for ST or OT from one mouse and 0.8 ml for NAcc.

Uptake of [³H]Dopamine. Synaptosomes were incubated at 37°C in uptake buffer for 10 min before addition of 100 nM [³H]dopamine (1 µCi for every 0.2 ml of synaptosomes), and the suspension was incubated for an additional 5 min.

Perfusion and Release. All experiments were conducted at room temperature using methods described previously (Grady et al., 1997, 2001) with modifications for collection into 96-well plates. In brief, aliquots of synaptosomes (80 µl) were distributed onto filters and perfused with the perfusion buffer (uptake buffer containing 0.1% BSA and 1 µM nomifensine) at 0.7 ml/min for 10 min before fractions were collected. For experiments using ACh as agonist, the synaptosomes were treated with diisopropyl fluorophosphate (10 µM) during the last 5 min of the uptake procedure, and atropine (1 µM) was added to the perfusion buffer. Fractions (0.1 ml) were collected into 96-well plates every 10 s using a Gilson FC204 fraction collector with a multicolumn adapter (Gilson, Inc., Middleton, WI). Radioactivity was determined by scintillation counting using a 1450 MicroBeta Trilux scintillation counter (Perkin Elmer Life and Analytical Sciences–Wallac Oy) after addition of 0.15 ml of OptiPhase SuperMix scintillation cocktail. Instrument efficiency was 40%.

Membrane Preparation for ¹²⁵I--CtxMII Binding. Each mouse was sacrificed by cervical dislocation, and the brain was removed and placed on an ice-cold platform. Olfactory tubercles, striatum, and superior colliculus were dissected. Samples were homogenized in ice-cold 2x physiological buffer (288 mM NaCl, 3 mM KCl, 4 mM CaCl₂, 2 mM MgSO₄, and 40 mM HEPES, pH 7.5) using a glass-Teflon tissue grinder. The homogenate was then incubated with 1 mM phenylmethylsulfonyl fluoride at 22°C for 15 min to inactivate endogenous serine proteases and centrifuged at 20,000g (15 min, 4°C, RC-2B centrifuge; Sorvall, Newton, CT). The pellet was resuspended in distilled water and centrifuged as before. The pellet was washed once more by resuspension and centrifugation and then resuspended in a final volume of distilled water (0.250 ml for individual regions). Protein concentrations in the membrane preparations were measured using the method of Lowry et al. (1951), with BSA as the standard.

¹²⁵I--CtxMII Binding to Membranes. Membrane binding of ¹²⁵I--CtxMII was performed as described in Salminen et al. (2005). Samples were incubated in 1.2 ml of siliconized polypropylene tubes arranged in a 96-well format, using 40 to 50 µg of membrane protein per tube. The binding buffer contained 144 mM NaCl, 1.5 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, 20 mM HEPES, pH 7.5, and was supplemented with 0.1% (w/v) BSA, 5 mM EDTA, 5 mM EGTA, and 10 mg/ml each of aprotinin, leupeptin trifluoroacetate, and pepstatin A to protect the ligand from endogenous proteases. The small reaction volume (30 µl) minimized the amount of tissue and radioisotope required. All incubations progressed for 2 h at 22°C. Six replicates were used to determine the total and nonspecific (in the presence of 1 µM epibatidine) binding. When inhibition of ¹²⁵I--CtxMII binding was measured, various concentrations of competing ligands were included in triplicate wells. After the 2-h incubation, each sample was diluted with 1 ml of binding buffer containing 0.1% (w/v) BSA and incubated for 4 min at 22°C to reduce nonspecific binding to the membrane preparation. Binding reactions were terminated by filtration of samples onto a Unifilter^R GF/B filter (Whatman Inc., Clifton, NJ) treated with 5% (w/v) nonfat dry milk for 30 min, using a modified Inotech Cell Harvester (Inotech Biosystems, Rockville, MD). Samples were subsequently washed with four changes of ice-cold binding buffer containing 0.1% (w/v) BSA. Washes were performed at 30-s intervals, each lasting approximately 5 s. All filtration steps were performed at 4°C. Bound ligand was quantified by gamma counting at 60% efficiency, using a 1450 MicroBeta Trilux scintillation counter (Perkin Elmer Life and Analytical Sciences–Wallac Oy) after addition of 25 µl of Optiphase SuperMix scintillation cocktail to each well.

Data Analysis. Data were analyzed using the SigmaPlot 5.0 and SPSS for DOS (SPSS Inc., Chicago, IL). Perfusion data were plotted as counts per minute versus fraction number. Fractions collected before and after the peak were used to calculate baseline as a single exponential decay. The calculated baseline was subtracted from the experimental data. Fractions that exceeded baseline by 10% or more were summed to give total released counts per minute. Counts per minute released above baseline were normalized to baseline to give units of release [(counts per minute – baseline)/baseline] (Grady et al., 1997, 2001). (Note that because of the modifications required to use 96-well plates, the data are normalized to a 10-s baseline rather than an 18-s baseline that was used in previous studies from our laboratory. This results in higher R_max values for normalized data.)

Agonist dose response data were fit to the Hill equation (Grady et al., 2001). IC₅₀ values for inhibition of release were calculated by fitting the data to a single site, partial-inhibition equation [release = R/((1 + [An]/IC₅₀) + C)] where R is the maximum amount of release that was inhibited by the antagonist, C is the amount of release that could not be inhibited by the antagonist and [An] is the antagonist concentration) (Grady et al., 2001). For competitive antagonists, K_i values were estimated from IC₅₀ values using the equation: K_i = IC₅₀/(1 + EC₅₀/[Ag]) where [Ag] is concentration of agonist.

Results for inhibition of binding were calculated using a one-site fit: B = B₀/[1+(I/IC₅₀)] where B is ligand bound at inhibitor concentration I,B₀ is the binding in the absence of inhibitor, and IC₅₀ is the concentration of inhibitor required to reduce binding to 50% of B₀.

Statistical significance was assessed from the R_max,EC₅₀, and IC₅₀ values by one-way (genotype) or two-way (genotype and brain region) analysis of variance with Tukey's post hoc test. The EC₅₀ and IC₅₀ data were calculated using log transformation of the data.

	Results

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ST, OT, and superior colliculus (SC) contain significant ¹²⁵I--CtxMII binding (Whiteaker et al., 2000; Salminen et al., 2005); therefore, these three brain areas were assayed for -CtxMII binding in wild-type mice (4+/+)(3+/+), mice with the 4-null mutation (4–/–)(3+/+), and mice with the double-null mutation (4–/–)(3–/–). ¹²⁵I--CtxMII binding was detectable in all three regions of all three genotypes (Fig. 1), but binding was reduced considerably after 4 gene deletion (4–/–)(3+/+) and double 4 and 3 gene deletions, (4–/–)(3–/–). Binding in ST of (4–/–)(3+/+) mice and of (4–/–)(3–/–) mice was reduced to 47% and 20%, respectively, of that of wild-type mice. In OT, binding in (4–/–)(3+/+) mice and in (4–/–)(3–/–) mice was 38% and 34%, respectively, of that of wild-type mice. In SC, residual ¹²⁵I--CtxMII binding in (4–/–)(3+/+) mice and (4–/–)(3–/–) mice was 56% and 12%, respectively, of that of wild-type mice.

View larger version (19K): [in this window] [in a new window]   Fig. 1. -CtxMII binding in ST, OT, and SC for three genotypes. 125I--CtxMII membrane binding was measured using a single ligand concentration of 0.5 nM in ST, OT, and SC membranes prepared from (4+/+)(3+/+), (4–/–)(3+/+), and (4–/–)(3–/–) mice. *, significant difference from wild type, P < 0.05; +, significant difference from 4 subunit deletion, P < 0.05.

The inhibition of ¹²⁵I--CtxMII (0.5 nM) binding by nicotine (Fig. 2a) and epibatidine (Fig. 2b) were measured for each of the three genotypes, using membrane preparations from the three regions combined. Data are presented as percentage of uninhibited controls. Controls were in agreement with the binding data of Fig. 1, with binding to membranes of the (4–/–)(3+/+) mixed regions at 49 ± 6% of wild-type and to (4–/–)(3–/–) at 18 ± 3% of wild-type binding. IC₅₀ values for inhibition of ¹²⁵I--CtxMII binding by nicotine and epibatidine were not affected by deletion of either the 4 subunit or both 4 and 3 subunits (Fig. 2c).

View larger version (17K): [in this window] [in a new window]   Fig. 2. Inhibition of 125I--CtxMII binding by nicotine and epibatidine in three genotypes of mice. Mouse brain particulate fractions from combined ST, OT, and SC were incubated at 22°C with 0.5 nM 125I--CtxMII. In all cases nonspecific binding was determined by including 1 µM unlabeled epibatidine. Each point represents the mean ± S.E.M. of 5–6 separate determinations. All data are presented as percentage of control binding to membranes of the same genotype. Inhibition by nicotine (a) and epibatidine (b) are shown. No significant differences in IC50 values with genotype were detected (c).

View larger version (21K): [in this window] [in a new window]   Fig. 3. Inhibition of agonist-induced DA release by -CtxMII in three genotypes. Aliquots of synaptosomes were exposed to varying concentrations of -CtxMII, as indicated, for 5 min immediately preceding exposure to 10 µM ACh for 10s. For a, data are expressed as percentage of control release (no -CtxMII). Curves shown are fits to single-site partial inhibition for (4+/+)(3+/+) [release = 29/((1+[An]/IC50)+71)] and, for (4–/–)(3+/+) and (4–/–)(3–/–), to single-site full inhibition [release = 100/(1+[An]/IC50)]. For b, DA release via -CtxMII-sensitive nAChRs for the three genotypes are compared. Only wild-type mice showed -CtxMII-resistant activity, which, for these experiments, was 17.80 ± 2.32 units of dopamine release (data not shown). c presents a comparison of IC50 values for -CtxMII for the three genotypes. *, significantly different from wild type, P < 0.05; +, significantly different from 4-null mutant, P < 0.05.

Because ACh-evoked dopamine release was measurable in all three genotypes, the pharmacology of this nAChR-mediated function could be investigated. Concentration-effect curves for nicotine-stimulated -CtxMII-sensitive dopamine release were determined in synaptosomes prepared from ST (Fig. 4a), NAcc (Fig. 4b), and OT (Fig. 4c) of the wild-type, 4-null mutant and 43 double-null mutant genotypes. Gene deletions altered both maximal responses and EC₅₀ values for nicotine-stimulated -CtxMII-sensitive dopamine release similarly in each brain region (two-way analysis of variance revealed no significant genotype by brain region interactions). As was the case for the ACh-stimulated release (Fig. 3), maximal nicotine-stimulated -CtxMII-sensitive dopamine release for both the 4-null- and the 4/3 double-null-mutants was significantly lower than that of wild-type mice in each brain region (Fig. 4d). In addition, a significant and substantial increase in EC₅₀ values was observed after 4, and 4 plus 3, gene deletions (Fig. 4e). EC₅₀ values for nicotine were 4- to 7-fold higher in (4–/–)(3+/+) than in wild-type mice. EC₅₀ values for (4–/–)(3–/–) mice were an additional 4- to 7-fold higher than for mice with only the 4 subunit gene deletion. Consequently, EC₅₀ values for the (4–/–)(3–/–) mice were 25- to 34-fold higher for nicotine-stimulated dopamine release than those for -CtxMII-sensitive nicotine-stimulated dopamine release in wild-type mice.

View larger version (35K): [in this window] [in a new window]   Fig. 4. Nicotine dose-response curves for -CtxMII-sensitive DA release determined from synaptosomes prepared from ST, NAcc and OT of three genotypes. Dopamine release was stimulated by an 18s exposure to the indicated concentrations of nicotine. For the (4+/+)(3+/+) genotype, release was determined with and without a 5 min exposure to -CtxMII (50 nM) immediately preceding the nicotine exposure. Data shown is determined by difference (total release - -CtxMII-resistant release). Curves are from fits of the data to the Hill equation. Comparison of maximum release and the EC50 values for these agonists are shown in d and e, respectively. *, significantly different from wild type, P < 0.05; +, significantly different from 4-null mutant, P < 0.05.

Dose response curves for stimulation of -CtxMII-sensitive dopamine release by four other nicotinic agonists were determined in wild-type, 4-null mutant, and 4/3 double-null mutant genotypes. These assays were confined to ST because there seemed to be no significant regional differences when nicotine was used as test compound. Data for A-85380, acetylcholine, epibatidine, and cytisine are presented in Fig. 5a, b, c, and d, respectively. The effects of deletion of the 4 and 4 plus 3 subunits on maximal nAChR-stimulated dopamine release were similar for each agonist (Figs. 4d and 5f). The largest effect for each agonist was elicited by deletion of the 4 subunit. This deletion eliminated all -CtxMII resistant release and significantly reduced the -CtxMII sensitive release. R_max values in 4–/– mice for epibatidine (48%), ACh (49%) and A85380 [GenBank] (44%) were less than half those of WT mice, whereas R_max values for both cytisine (63%) and nicotine (65%) were slightly less affected by 4 gene deletion. Agonist-stimulated -CtxMII-sensitive dopamine release was further reduced by deletion of the 3 subunit using nicotine with NAcc synaptosomes and using ACh with ST synaptosomes. With other agonists and regions, this further decrease was not statistically significant. In mice with both 4 and 3 gene deletions, R_max values were approximately 40% of those measured for WT mice for epibatidine, ACh, and A85380 [GenBank] and 50% for cytisine and nicotine.

View larger version (23K): [in this window] [in a new window]   Fig. 5. Dose-response curves for agonist-stimulated -CtxMII-sensitive dopamine release determined from synaptosomes prepared from ST of three genotypes. Dopamine release was stimulated by an 18-s exposure to the indicated concentrations of agonist. For the (4+/+)(3+/+) genotype, release was determined with and without a 5-min exposure to -CtxMII (50 nM) immediately preceding the agonist exposure. Data shown is determined by difference (total release – -CtxMII-resistant release). Curves are from fits of the data to the Hill equation. A comparison of the EC50 and Rmax values for the four agonists by genotype is shown in e and f, respectively. *, significantly different from wild type, P < 0.05; +, significantly different from 4-null mutant, P < 0.05.

For nicotine and cytisine, the two agonists for which gene deletions elicited large changes in EC₅₀, the dose response data were subjected to further analysis to estimate EC₅₀ values for individual nAChR subtypes. The assumption here is that a change in EC₅₀ value by gene deletion indicates that the EC₅₀ value for the subtype removed differs significantly from the remaining subtypes. Minimal effect of gene deletion on EC₅₀ value means the subtypes have similar EC₅₀ values. Subtraction of curves allows an estimate of the EC₅₀ value of the removed subtype, assuming no large changes in expression in response to the gene deletion (see Discussion). By subtracting the 4-null mutant data from the wild-type data, EC₅₀ values for dopamine release mediated by the -CtxMII-sensitive subtype unique to the wild-type mouse could be estimated. Likewise, subtracting the data for the double-null mutation (4–/–)(3–/–) from the data for the 4-null mutant should isolate the contribution of the -CtxMII-sensitive subtype without the 4 subunit but with the 3 subunit (632-containing subtype). Table 1 presents EC₅₀ values for curves generated by subtraction as well as those from the curves from Figs. 4 and 5, and values for -CtxMII-resistant (42* nAChR) (Salminen et al., 2004) for comparison. This analysis indicated that the EC₅₀ values for the subtype unique to the wild-type mice (6432-nAChR) were significantly lower than those for the sum of all -CtxMII-sensitive subtypes, and the EC₅₀ values for 632-nAChR were lower than the values measured for the sum of subtypes in the 4-null mutant, but higher than that measured for the sum of subtypes in the wild-type.

Unlike the rather large differences seen for some of the agonists, effects of antagonists did not differ much for wild-type, 4-null mutant, and 43 double-null mutant genotypes. Inhibition of -CtxMII-sensitive ACh-stimulated dopamine release from striatal synaptosomes by DHE and MLA are shown in Fig. 6, a and b. IC₅₀ values are compared in Fig. 6c. IC₅₀ values for DHE did not differ significantly among genotypes and ranged from 2.83 to 3.89 µM, equivalent to a K_i value of 0.21 ± 0.08 µM. IC₅₀ values for MLA ranged from 0.83 to 1.92 µM. The small decrease in IC₅₀ value for MLA in mice with the double subunit deletion (–/–)(–/–) was significant compared with both wild-type mice and mice with the (4–/–)(+/+) genotype; however this difference was not evident when calculated as K_i values (0.04, 0.20, and 0.06 µM for the three genotypes, respectively).

View larger version (15K): [in this window] [in a new window]   Fig. 6. Inhibition curves for -CtxMII-sensitive dopamine release determined from synaptosomes prepared from ST of three genotypes of mice. Dopamine release was stimulated by an 18-s exposure to 10 µM ACh simultaneously with the indicated concentrations of DHE or MLA. For the (4+/+)(3+/+) genotype, release was determined with and without a 5-min exposure to -CtxMII (50 nM) immediately preceding the agonist (± antagonist) exposure. Data shown is determined by difference (total release – -CtxMII-resistant release). Single-site inhibition curve-fits to the data are shown. c shows a comparison of IC50 values. *, significantly different from wild-type, P < 0.05; +, significantly different from 4-null mutant, P < 0.05.

	Discussion

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The studies reported here comprise a first attempt to compare the pharmacology of the three native 62* nAChRs expressed in mouse striatum. In the past, pharmacological comparisons of nAChR subtypes have used transfected cell lines or X. laevis oocytes (see Papke and Heinemann, 1994; Parker et al., 2001; Wu et al., 2006, for a few of many examples). Unfortunately, for reasons that remain unknown, 6*-nAChRs have been difficult to express in X. laevis oocytes (Gerzanich et al., 1997). A chimera between the extracellular domain of 6 and the transmembrane domain of 3 has been successfully expressed (Kuryatov et al., 2000); however, its pharmacology may not reflect that of native subtypes (Nicke et al., 2003). Recently human 6 subunits were expressed in human embryonic kidney cells (Tumkosit et al., 2006), indicating that comparisons of 6* nAChRs may be possible in the future. We chose to study the pharmacological properties of native 6* nAChRs by using breeding strategies to limit the nAChR subtypes expressed in dopaminergic nerve terminals. This approach has several advantages: 1) functional receptors are formed in neurons where they are normally expressed, 2) receptors are composed of native subunits, and 3) receptors are assembled with normal processing. However, analysis does require differentiation among the native subtypes, and gene deletion may elicit compensatory changes in the remaining subtypes.

Compensatory changes in nAChR subunits expressed in dopaminergic nerve terminals, if they occur, would serve to complicate results obtained with the breeding strategy. However, there is little evidence for major compensatory changes in nAChR subunits. None of the known nAChR subunit mRNA levels are changed by either 4 (Ross et al., 2000) or 3 (Cui et al., 2003) gene deletion. Deletion of the 2 subunits eliminates all agonist-stimulated dopamine release; 4 subunits cannot replace 2 (Picciotto et al., 1998; Grady et al., 2001). Deletion of the 6 subunit eliminates all -CtxMII-sensitive agonist-stimulated dopamine release, as well as -CtxMII-sensitive ¹²⁵I-epibatidine binding, indicating that 3 subunits cannot substitute for 6 (Champtiaux et al., 2003). However, immunoprecipitation and binding experiments indicate that there is some increase in 4 subunits after 6 deletion (Champtiaux et al., 2003). With the 4 gene deletion, virtually all the agonist-stimulated -CtxMII-resistant response is eliminated, indicating that no subunit is capable of substituting for 4 in dopaminergic terminals (Champtiaux et al., 2003; Salminen et al., 2004). In addition, the 4 gene deletion eliminates approximately half of the -CtxMII-sensitive dopamine release, showing that additional 6 subunits do not take the place of 4 (Salminen et al., 2004). Some deletions of minor subunits do increase function of alternate subtypes of nAChR. For example, in 3 subunit-null mutant mice, an increase is seen in the agonist-stimulated dopamine release supported by the -CtxMII-resistant subtypes (Cui et al., 2003; Salminen et al., 2004), but immunoprecipitation experiments establish that levels of 4, 5, or 2 proteins do not accompany these functional changes (Gotti et al., 2005).

Results reported here are consistent with a lack of compensatory changes in other nAChR subtypes. The decreased -CtxMII binding that we found in ST (53%) is consistent with loss of the wild-type population of 6432-nAChR subtype estimated by immunoprecipitation (Gotti et al., 2005). Likewise, the decrease in ¹²⁵I--CtxMII binding of an additional 27% in ST in the double mutants is consistent with the immunoprecipitation estimate of the 632-nAChR population (Gotti et al., 2005). The agreement between binding and immunological results indicates that additional nAChR subtypes, or greater numbers of remaining subtypes, are not formed in these null mutant mice; i.e., expression of new or novel nAChRs does not easily occur in nAChR-null mutant mice.

Deletion of 4 and 4-plus-3 subunits profoundly affected maximal agonist-induced dopamine release. The amounts of decrease in dopamine release stimulated by ACh (Fig. 3) were similar to the decreases in ¹²⁵I--CtxMII binding. Qualitatively similar results were measured for R_max values for all five agonists, although decreases were somewhat less by this measurement. Although minor compensatory increases in 62* nAChR expression cannot be ruled out, these results could also indicate some differences in kinetic or other functional properties of the different subtypes.

Gene deletion of 4 had little effect on EC₅₀ value for A-85380, some effect for ACh and epibatidine, but elicited marked changes in EC₅₀ values for both cytisine- and nicotine-stimulated dopamine release. Subsequent deletion of the 3 gene elicited further increases for these agonists. These results build on the observation that the EC₅₀ value for ACh-induced -CtxMII-sensitive dopamine release is not changed significantly after deletion of the 4 gene (Salminen et al., 2004), whereas a significant increase (8-fold) in the EC₅₀ value for nicotine-stimulated dopamine release has been reported (Champtiaux et al., 2003). We measured a 31-fold increase in EC₅₀ value for cytisine activation of the -CtxMII-sensitive nAChR in (4–/–)(3+/+) mice over the wild-type nAChR and a 99-fold increase was seen with the (4–/–)(3–/–) mice over the wild type. Nicotine was also able to distinguish all three subtypes with 7- and 31-fold increases, respectively. Other compounds, including the agonists ACh and epibatidine and the antagonist MLA, did not differentiate as well among variations of 6*-nAChR subtypes. Affinity for A-85380, DHE, and -CtxMII did not differ with genotype. For those agonists showing large differences, subtraction of data allowed estimation of EC₅₀ values for individual subtypes (Table 1). The 6432 nAChR has the highest sensitivity for nicotine-stimulated activity measured to date in the mouse, with an EC₅₀ value for dopamine release of 0.2 µM, well within the range achieved by long-term treatment (Marks et al., 2004).

In summary, experiments reported here confirm the postulate that three 6* nAChR subtypes play important roles in modulating the -CtxMII-sensitive component of dopamine release (Salminen et al., 2004; Gotti et al., 2005). The 6432 subtype is unique in that it has an EC₅₀ value for nicotine-stimulated release that are 7-fold lower than the EC₅₀ value for nicotine-induced stimulation of the -CtxMII-resistant component of dopamine release (Salminen et al., 2004). Likewise, the EC₅₀ value for activating this receptor is 7-fold lower than the EC₅₀ value for nicotine-stimulated GABA release (Lu et al., 1998) and more than 200-fold lower than the EC₅₀ value for nicotine-stimulated-ACh release (Grady et al., 2001) from synaptosomes. Thus, the 6432 receptors may play a very important role in modulating behavioral effects of nicotine, particularly those associated with tobacco addiction. The results of the experiments reported here also indicate that interbreeding nAChR-null mutant mice may be used successfully to characterize pharmacological properties of native nAChRs normally expressed along with other, more abundant, nAChR subtypes. This strategy of sequential gene deletion revealed that large, and agonist selective, differences in sensitivity exist among 6*-nAChR subtypes. This approach may facilitate the identification of new compounds capable of selectively modulating nAChR subtypes in vivo that may be of therapeutic benefit and also may lead to a greater understanding of the natural role of these receptors.

	Acknowledgements

 
We thank Natalie M. Meinerz, Theresa delVecchio, and Tianna Purrington for technical assistance in genotyping. We also thank Megan Canon and Eric Laudenslager for assistance in dopamine release assays.

	Footnotes

 
This work was supported by National Institute on Drug Abuse grants DA12242 (to M.J.M.) and DA03194 (to A.C.C), National Institute of Mental Health grant MH53631 (to J.M.M.), Colorado Tobacco Research Grant 3F-034 (to O.S.), and an Emil Aaltonen Foundation grant (to O.S.). Production of the null mutant mice was supported by animal resources grant DA015663 (to A.C.C.).

A portion of this work has been presented as an abstract: Salminen OS, Marks MJ, McIntosh JM, Collins AC, and Grady SR. (2005) Characterization of a novel presynaptic nicotinic acetylcholine receptor subtype (62) on mouse brain dopaminergic terminals. Soc Neurosci Abstr 31:954.7

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

doi:10.1124/mol.106.031492.

ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; -CtxMII, -conotoxin MII; ACh, acetylcholine iodide; A-85380, 3-((2S)-azetidinylmethoxy)pyridine dihydrochloride; BSA, bovine serum albumin; DHE, dihydro--erythroidine; MLA, methyllycaconitine; ST, striatum; OT, olfactory tubercle; NAcc, nucleus accumbens; SC, superior colliculus.

¹ Current affiliation: Division of Pharmacology and Toxicology, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland.

Address correspondence to: Sharon R. Grady, Institute for Behavioral Genetics, University of Colorado, 447UCB, Boulder, CO 80309. E-mail: sharon.grady{at}colorado.edu

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