Introduction

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Nicotinic acetylcholine receptors (nAChR) are modulators of neuronal function in the central nervous system (Role and Berg, 1996). One way in which nAChR can modify neuronal activity is by facilitating neurotransmitter release, and this may be accomplished by presynaptic or somatodendritic nAChR (Wonnacott, 1997). Dopamine release in rodent striatum may be modulated by presynaptic nAChR on dopamine terminals (Wonnacott, 1997; Zhou et al., 2001) as well as somatodendritic nAChR on dopamine neurons in the substantia nigra (Clarke et al., 1987; Reuben et al., 2000; Klink et al., 2001). The nigrostriatal system has merited attention because of its relevance to the motor stimulant and addictive properties of nicotine and because these nAChR represent potential therapeutic targets for the treatment of Parkinson's disease.

Neuronal nAChR are pentameric ligand-gated cation channels, comprised of one or more different subunits, from a portfolio of nine  (2-10) and three  (2-4) subunits expressed in the nervous system (Lukas et al., 1999; Elgoyhen et al., 2001). Determination of the subunit composition of native nAChR presents a major challenge. This endeavor is hampered by a lack of subtype-selective nicotinic ligands, the majority of those available are antagonists derived from natural products. The -conotoxins comprise a family of peptide toxins elaborated by Conus sp. and directed against particular nAChR subtypes (McIntosh et al., 1999). From studies of functional nAChR expressed in Xenopus oocytes, -conotoxin-MII (-CTx-MII) was originally defined as a specific antagonist of nAChR composed of 3 and 2 subunits (Cartier et al., 1996; Harvey et al., 1997). ¹²⁵I--CTx-MII labels a unique population of nicotinic binding sites in mouse and monkey brain (Whiteaker et al., 2000; Quik et al., 2001). This toxin partially blocks [³H]dopamine release from rat striatal synaptosomes stimulated with nicotine (Kulak et al., 1997) or anatoxin-a (Kaiser et al., 1998), providing evidence for the heterogeneity of presynaptic nAChR on dopamine terminals, with one population containing an 32 interface. Studies with the novel partial agonist UB-165 led us to propose an 42* nAChR subtype as a candidate for the other nAChR population (Sharples et al., 2000). The contribution of the 2 subunit to both nAChR subunits is consistent with the localization of 2 nAChR subunit immunoreactivity in most dopaminergic terminals in the dorsal striatum (Jones et al., 2001) and the absence of nicotine-evoked dopamine release from striatal synaptosomes or slices prepared from 2 null mutant mice (Grady et al., 2001; Zhou et al., 2001). However, the subunit composition of striatal presynaptic nAChR is likely to be more complex than pairwise combinations of subunits and this is denoted by the asterisk (Lukas et al., 1999).

Midbrain dopamine neurons express most nAChR subunits (Klink et al., 2001), including a particularly high expression of mRNA corresponding to the 6 and 3 nAChR subunits (Le Novère et al., 1996). Patch-clamp recording and single cell polymerase chain reaction analysis of rat midbrain dopamine neurons, and comparison with data from transgenic mice deficient in a particular nAChR subunit, led to the tentative subunit compositions 46/35(2)₂ and (4)₂5(2)₂ for two major nAChR subtypes found on cell bodies of these neurons in the substantia nigra and ventral tegmentum (Klink et al., 2001). Typical fast desensitizing 7-type nAChR currents are also observed in some midbrain dopamine neurons (Pidoplichko et al., 1997; Klink et al., 2001), but 7* nAChR do not seem to mediate [³H]dopamine release from striatal synaptosomes as the 7-selective antagonists -bungarotoxin (Bgt) and -conotoxin-ImI (-CTx-ImI) are without effect (Rapier et al., 1990; Kulak et al., 1997). However, we previously noted that 50 nM methyllycaconitine (MLA), a potent 7-selective antagonist, produced a partial inhibition of anatoxin-a-evoked [³H]dopamine release from striatal synaptosomes (Kaiser and Wonnacott, 2000), in agreement with the previous observation of Clarke and Reuben (1996). Klink et al. (2001) have also shown that low nanomolar concentrations of MLA inhibit a population of non-7 nAChR currents, correlated with the tentative subunit composition 465(2)₂, in the cell bodies of mesencephalic dopamine neurons.

MLA is a hexacyclic norditerpenoid alkaloid, isolated from Delphinium sp. (Wonnacott et al., 1993). MLA is a competitive nicotinic antagonist with approximately 100-, 1,000-, and 10,000-fold higher affinity for 7 nAChR, compared with 32, 42, and muscle nAChR, respectively (Alkondon et al., 1992; Drasdo et al., 1992; Wonnacott et al., 1993). The pharmacology and distribution of [³H]MLA binding sites in rodent brain tissue corresponds well with that of ¹²⁵I-Bgt binding sites (Davies et al., 1999; Whiteaker et al., 1999), and sensitivity to low nanomolar concentrations of MLA has been interpreted as evidence for 7 nAChR. However, in avian neuronal preparations discrepancies between MLA- and Bgt-sensitive responses have been reported (Yum et al., 1996; Yu and Role, 1998), raising the possibility that minority populations of neuronal nAChR with particular subunit combinations may show differential sensitivity to these two toxins.

In this study, we have characterized pharmacologically the MLA-sensitive portion of nAChR-mediated [³H]dopamine release from striatal synaptosomes. We show that nanomolar concentrations of MLA inhibit the same nAChR population as -CTx-MII. Using ¹²⁵I--CTx-MII to label nAChR directly, we confirm that MLA potently interacts with this site. The relationship between presynaptic and somatodendritic nAChR on dopamine neurons, with respect to subunit composition, is discussed.

	Materials and Methods

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Sprague-Dawley rats were obtained from the University of Bath Animal House (Bath, UK) breeding colony and the Health Sciences Center (Denver, CO). [7,8-³H]Dopamine ([³H]dopamine, specific activity 1.78 TBq/mmol) and ¹²⁵I autoradiographic microscales were purchased from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK). -CTx-MII was provided by Tocris Cookson (Bristol, UK). -CTx-ImI, MLA, ()-nicotine, Bgt, mecamylamine, pargyline, and nomifensine were obtained from Sigma Chemical (Poole, Dorset, UK). (±)-UB-165 was synthesized by Professor T. Gallagher (University of Bristol, Bristol, UK) as described previously (Wright et al., 1997). ¹²⁵I--CTx-MII was synthesized by addition of an N-terminal tyrosine and subsequent iodination, as described by Whiteaker et al. (2000). All other chemicals used were of analytical grade and obtained from standard commercial sources.

Superfusion of Synaptosomes. [³H]Dopamine release from rat striatal synaptosomes was measured as described previously (Kaiser et al., 1998). In brief, male Sprague-Dawley rats (250-350 g) were killed by cervical dislocation and decapitated, and striata (including dorsal striatum and nucleus accumbens; 180-240 mg wet tissue/rat) were rapidly dissected. P2 synaptosomes were obtained by homogenization followed by differential centrifugation and were loaded with [³H]dopamine (0.1 µM, 0.132 MBq/ml) for 15 min at 37°C. Synaptosomes were deposited on GF/B filter discs (Whatman, Maidstone, UK) in open chambers of a superfusion apparatus (model SF-12; Brandel, Gaithersburg, MD), with striata from two rats providing enough tissue for 12 superfusion chambers. Synaptosomes were superfused with Krebs-bicarbonate buffer of the following composition: 118 mM NaCl, 2.4 mM KCl, 2.4 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, and 10 mM glucose, pH 7.4, saturated with 95% O₂, 5% CO₂ and supplemented with 1 mM ascorbic acid, 8 µM pargyline, and 0.5 µM nomifensine to prevent dopamine degradation and reuptake, respectively. The flow rate was 0.5 ml/min.

Superfusion Data Analysis. Evoked tritium release above baseline was calculated as a percentage of the total radioactivity present in the synaptosomes immediately before stimulation. The baseline was derived, using SigmaPlot version 2.0 (Jandel Scientific, San Rafael, CA), by fitting the following double exponential decay equation to the data: y = ae^bx + ce^dx, where a and c are initial (at x = 0) release in each phase, b and d are the decay constants in each phase, and x is the fraction number.

Quantitative Autoradiography of ¹²⁵I--CTx-MII Binding. ¹²⁵I--CTx-MII binding was assessed by quantitative autoradiography. The methods used were similar to those detailed in Whiteaker et al. (2000). Six male Sprague-Dawley rats (300-350 g) were killed by cervical dislocation, and the brains were removed from the skull and rapidly frozen by immersion in isopentane (35°C, 30 s). The frozen brains were wrapped in aluminum foil, packed in ice, and stored at 70°C until sectioning. Tissue sections (20 µm in thickness) were prepared, using a Leica CM1850 cryostat/microtome refrigerated to 23°C, and thaw mounted onto subbed microscope slides (Richard Allen, Richland, MI). Slides were subbed by incubation with gelatin (1% w/v)/chromium aluminum sulfate (0.1% w/v) for 2 min at 37°C; drying overnight at 37°C, incubation at 37°C for 30 min in 0.1% (w/v) poly-L-lysine in 25 mM Tris, pH 8.0; and drying at 37°C overnight. Mounted sections were stored, desiccated, at 70°C until use. Twelve series of sections were collected from three brains for use in the saturation binding experiments, with 15 series being collected from the remaining brains for use in the inhibition binding experiments. Sections were collected from the front to the back of the striatum (approximately +2.2 mm to 1.8 mm relative to bregma).

Analysis of Autoradiography Experiments. Results for saturation binding experiments were calculated using the Hill equation B = B_max Lⁿ/(Lⁿ + K_dⁿ), where B is the binding at the free ligand concentration L, B_max is the maximum number of binding sites, K_d is the equilibrium binding constant, and n is the Hill coefficient. Values of B_max, K_d, and n were calculated using the nonlinear least-squares fitting algorithm of SigmaPlot version 5.0 (Jandel Scientific). Results for inhibition of ¹²⁵I--CTx-MII binding were calculated using a one-site fit: B = B₀/(1 + I/IC₅₀), where B is ligand bound at inhibitor concentration I, B_o is the binding in the absence of inhibitor, and IC₅₀ is the concentration of inhibitor required to reduce binding to 50% of B_o. Values for K_i (inhibition binding constant) were derived by the method of Cheng and Prusoff (1973): K_i = IC₅₀/(1 + L/K_d).

	Results

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MLA Partially Inhibits Nicotine-Evoked [³H]Dopamine Release from Striatal Synaptosomes. Nicotine-evoked [³H]dopamine release from rat striatal synaptosomes was monitored in the presence and absence of a variety of nicotinic antagonists. Nicotine (10 µM; delivered as a 40-s pulse) elicited a peak of radioactivity above basal release, and this was almost totally abolished in the presence of the general nAChR antagonist mecamylamine (10 µM; Fig. 1A). The residual release was equivalent to the nonspecific release observed when agonist was replaced with buffer. In the presence of MLA (50 nM), a small decrease in the amount of nicotine-evoked [³H]dopamine release was consistently observed (Fig. 1, A and B).

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Inhibition by MLA of nicotine-evoked [3H]dopamine release from striatal synaptosomes. Synaptosomes were loaded with [3H]dopamine and superfused as described under Materials and Methods; 2-min (1-ml) fractions were collected and counted for radioactivity. Antagonist was introduced into the perfusing buffer 10 min before stimulation for 40 s with nicotine, except in the case of Bgt in which synaptosomes were incubated with it for 1 h before addition of agonist. A, release of [3H]dopamine over time, showing the response to 10 µM nicotine (arrow) in the absence () and presence () of 50 nM MLA, or 10 µM mecamylamine (). Evoked [3H]dopamine release is expressed as a percentage of the mean basal release; values are the mean ± S.E.M. of the triplicate chambers from a representative experiment. B, effect of increasing concentrations of MLA on nicotine-evoked dopamine release. Synaptosomes were stimulated with 10 µM nicotine, in the presence and absence of MLA. Data are expressed as a percentage of the response to 10 µM nicotine alone. Error bars indicate the S.E.M. from at least three separate experiments, each consisting of two or more replicate chambers (, p < 0.01, significantly different from control response; , p < 0.05, significantly different from response obtained in the presence of 30 and 50 nM MLA; one-way ANOVA, Tukey's post hoc test). C, effect of the 7-selective antagonists Bgt (40 nM), MLA (50 nM), -CTx ImI (1 µM), and the general nAChR antagonist mecamylamine (10 µM) was compared for their effect on [3H]dopamine release evoked by 10 µM nicotine () or 100 µM nicotine (). Values are expressed as a percentage of the response to 10 µM nicotine, in the absence of antagonist, determined in parallel. Inset, concentration-response relationship for nicotine-evoked [3H]dopamine release from striatal synaptosomes, expressed as a percentage of the response to 10 µM nicotine. Values are the mean ± S.E.M. from at least three separate experiments, each consisting of two or more replicate chambers (, p < 0.01, significantly different from control response; one-way ANOVA, Tukey's post hoc test)

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Inhibition of nicotine-evoked [3H]dopamine release from striatal synaptosomes by MLA and non-7 antagonists. Synaptosomes were loaded with [3H]dopamine and superfused as described under Materials and Methods. Antagonists (50 nM MLA, 100 nM -CTx-MII, and 1 µM DHE) were introduced into the perfusing buffer, either singly or in combination, 10 min before stimulation for 40 s with 10 µM nicotine. Evoked [3H]dopamine release in the presence of antagonist is expressed as a percentage of the response to 10 µM nicotine alone. Values are the mean ± S.E.M. from at least three independent experiments, each consisting of two or more replicate chambers (, p < 0.01, significantly different from control response; one-way ANOVA, Tukey's post hoc test; , p < 0.01, significantly different from responses obtained with nicotine alone and in combination with -CTx-MII, MLA, and -CTx-MII + MLA; one-way ANOVA, Tukey's post hoc test.)

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Sensitivity to MLA of [3H]dopamine release evoked by UB-165. Synaptosomes were loaded with [3H]dopamine and superfused as described under Materials and Methods. Antagonist (50 nM MLA, 100 nM -CTx-MII, and 10 µM mecamylamine) was added to the perfusing buffer 10 min before stimulation for 40 s with nicotine (10 µM) or UB-165 (0.2 or 1 µM). Results are expressed as a percentage of the response to 1 µM UB-165. Values are the mean ± S.E.M. from at least three independent experiments, each consisting of two or more replicate chambers (, p < 0.01, significantly different from responses obtained with 1 µM UB-165; one-way ANOVA, Tukey's post hoc test; , p < 0.01, , p < 0.05, significantly different from responses obtained with 0.2 µM UB-165; one-way ANOVA, Tukey's post hoc test).

Inhibition by MLA of ¹²⁵I--CTx-MII Binding. The effects of MLA on nicotinic agonist-evoked [³H]dopamine release suggest that it acts competitively at the same nAChR subtype as -CTx-MII. To address this proposition, ¹²⁵I--CTx-MII was used to label nicotinic sites in rat striatum and nucleus accumbens, and the ability of MLA to displace this specific binding was investigated. ¹²⁵I--CTx-MII displayed saturable specific binding to striatum and nucleus accumbens (Fig. 4A), with B_max values of 9.8 ± 1.3 and 16.5 ± 4.6 fmol/mg of protein and K_d values of 0.63 ± 0.19 and 0.83 ± 0.55 nM, respectively. Competition assays with serial dilutions of MLA over the range 1 nM to 1 µM were carried out using 0.5 nM ¹²⁵I--CTx-MII, a concentration approximating its K_d. MLA was able to fully displace specific binding of the radioligand to both striatum and nucleus accumbens (Fig. 4B), with K_i values of 32.9 ± 12.9 and 34.6 ± 13.8 nM, respectively.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Inhibition by MLA of 125I--CTx-MII binding to rat striatum and nucleus accumbens. A, saturation binding of 125I--CTx-MII. Total and nonspecific binding of 125I--CTx-MII (0.05-2.5 nM) to serial sections of rat brain were determined by quantitative autoradiography as described under Materials and Methods. Nonspecific binding was measured in the presence of 10 mM nicotine in adjacent series of sections for each 125I--CTx-MII concentration. Binding to striatum and nucleus accumbens was determined from optical density measurements of labeling in those regions. Nonspecific binding was linear with concentration, reaching 18.5 ± 1.1 and 14.6 ± 2.1 fmol mg1 (protein) at 2.5 nM 125I--CTx-MII, for striatum and nucleus accumbens, respectively. Specific binding was calculated as the difference between total and nonspecific values for each radioligand concentration. Data indicate the mean ± S.E.M. of determinations from three animals and are fitted to the Hill equation. Bmax values of 9.8 ± 1.3 and 16.5 ± 4.6 fmol mg1 (protein), and Kd values of 0.63 ± 0.19 and 0.83 ± 0.55 nM were derived for striatum and nucleus accumbens, respectively. B, inhibition by MLA. Serial sections of rat brain were incubated with 0.5 nM 125I--CTx-MII in the presence of increasing concentrations of MLA (1-1000 nM), in the presence and absence of 10 mM nicotine to define nonspecific binding, as described under Materials and Methods. Data indicate the mean specific binding ± S.E.M. of determinations from three animals, and are fitted to single-site Hill inhibition equation. IC50 values for inhibition by MLA are 59.1 ± 23.2 and 55.5 ± 22.1 nM for striatum and nucleus accumbens, respectively.

	Discussion

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The present study demonstrates that the presumed 7-selective compound MLA may antagonize other nAChR subtypes found at rat striatal dopaminergic nerve terminals, at concentrations frequently used to selectively block 7 nAChR. The ability of 50 nM MLA to partially inhibit nicotinic agonist-evoked [³H]dopamine release was consistently observed at low agonist concentrations, but was surmountable at higher agonist concentrations, suggesting a competitive mode of action. Because none of the other 7-selective compounds examined (Bgt and -CTx-ImI) had any effect on nicotine-evoked [³H]dopamine release from striatal synaptosomes, this strongly argues that MLA is not acting through an 7 nAChR. Moreover, the lack of additivity with -CTx-MII suggests that both antagonists act at the -CTx-MII-sensitive nAChR subtype. Confirmation of this interaction was provided by the ability of low nanomolar concentrations of MLA to displace ¹²⁵I--CTx-MII binding to rat striatum and nucleus accumbens.

The absence of a discernible 7 nAChR-mediated component in nicotine-evoked [³H]dopamine release from striatal synaptosomes is well documented (Rapier et al., 1990; Kulak et al., 1997) and substantiated by the lack of effect of Bgt and -CTx-ImI in the present study (Fig. 1C). In contrast, we have shown previously that these agents, as well as MLA, antagonize nicotine-evoked [³H]dopamine release from striatal slices, interpreted as evidence for the involvement of an indirect 7 nAChR-mediated component in the slice (Kaiser and Wonnacott, 2000). In the present study, [³H]dopamine release from striatal synaptosomes was examined to focus on presynaptic nAChR localized on the dopamine terminals.

The inhibition by 50 nM MLA of [³H]dopamine release evoked by either nicotine or UB-165 was surmountable (Figs. 1C and 3), consistent with a competitive interaction. Similarly, Clarke and Reuben (1996) observed a surmountable inhibition by MLA of nicotine-evoked [³H]dopamine release from striatal synaptosomes. These authors demonstrated complete inhibition by MLA with an IC₅₀ value of 38 nM, whereas nicotine-evoked [³H]noradrenaline release was notably less sensitive to MLA (IC₅₀ = 1 µM). Full dose-response curves were not determined in the present study because the current appreciation of the heterogeneity of nAChR subtypes governing nicotine-evoked [³H]dopamine release compromises the analysis of such profiles. MLA (50 nM) also partially inhibited (by 37%) [³H]dopamine release from striatal synaptosomes stimulated with 1 µM anatoxin-a, but had no effect when the agonist concentration was increased to 25 µM (Kaiser and Wonnacott, 2000). Thus, MLA potently and competitively inhibits a portion of striatal [³H]dopamine release elicited by a number of nicotinic agonists acting at presynaptic nAChR.

MLA seems to interact with -CTx-MII-sensitive nAChR because inhibition by the two toxins of nicotine- or UB-165-evoked [³H]dopamine release is not additive when they are applied together (Figs. 2 and 3). This inference is supported by the ability of MLA to potently displace ¹²⁵I--CTx-MII binding. ¹²⁵I--CTx-MII labeled a small population of specific sites in rat striatum and nucleus accumbens, with a subnanomolar binding affinity. The density of sites (10 and 16 fmol/mg of protein, respectively) agrees well with numbers of specific binding sites reported for the corresponding brain regions in mouse (Whiteaker et al., 2000) and monkey (Quik et al., 2001), assuming 1 mg of protein corresponds to 10 mg of tissue. In the monkey caudate putamen, more than 95% of specific ¹²⁵I--CTx-MII binding sites were lost in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned animals. Together with the correlation with dopamine transporters, this strongly supports the localization of ¹²⁵I--CTx-MII binding sites to dopaminergic terminals in these brain regions (Quik et al., 2001). Bgt (1 µM) failed to inhibit ¹²⁵I--CTx-MII binding to mouse striatum, and conversely -CTx-MII, at concentrations up to 1 µM, did not compete for ¹²⁵I-Bgt binding to mouse brain membranes (Whiteaker et al., 2000). Thus, the ability of MLA to fully displace ¹²⁵I--CTx-MII binding (Fig. 4) with a K_i of ~33 nM reflects a non-7 nAChR activity for this ligand.

The observations in the present study parallel the findings of Klink et al. (2001) for nAChR responses recorded electrophysiologically from dopamine cell bodies in the rat midbrain. These authors showed that in most (75%) dopamine neurons, acetylcholine or nicotine evoked slow whole cell currents that were partially blocked by low nanomolar concentrations of MLA and -CTx-MII in a nonadditive manner.

This raises the question of the subunit composition of nAChR subtype(s) with which MLA potently interacts. The majority of rat midbrain dopamine neurons express the 3, 4, 5, 6, 2, and 3 subunits (Klink et al., 2001). -CTx-MII was originally defined as a selective antagonist of nAChR composed of 3 and 2 subunits, with >100-fold lower potency at other pairwise nAChR subunit combinations or 7 nAChR expressed in Xenopus oocytes (Cartier et al., 1996; Harvey et al., 1997; Kaiser et al., 1998). Because binding of CTx-MII to one 32 interface would be sufficient for functional inhibition, the toxin specificity was extended to include 32-containing (or 32*) nAChR (Kulak et al., 1997; Kaiser et al., 1998). However, the 3 subunit shares high sequence identity with the 6 subunit (Le Novère and Changeux, 1995) that is highly expressed in dopamine neurons (Le Novère et al., 1996; Goldner et al., 1997; Klink et al., 2001). Deletion of 6 subunit expression by in vivo administration of antisense oligonucleotides decreased nicotine-induced effects on locomotor activity (Le Novère et al., 1999), consistent with a role in motor functions executed by the striatum. The 6 subunit is reluctant to form functional nAChRs in pairwise combination with a  subunit, supporting its participation in more complex subunit assemblies, and the efficient expression of 6 in combination with a variety of mammalian subunits has been demonstrated in heterologous systems (Kuryatov et al., 2000). Immunoisolated 6-containing nAChR from chick retina display moderately high affinity for -CTx-MII (K_i = 66 nM), but relatively low affinity for MLA (1.3 µM; Vailati et al., 1999).

Expression of the 3 subunit is also limited to a few brain regions, notably catecholaminergic areas where it colocalizes with 6 nAChR subunit expression in the rat brain (Le Novère et al., 1996). Functional nAChRs comprised of rat 3, 2, and 3 subunits and expressed in Xenopus oocytes retain high sensitivity to -CTx-MII (Luo et al., 2000), whereas transgenic mice lacking expression of the 3 nAChR subunit are deficient in specific binding sites for ¹²⁵I--CTx-MII in the striatum (Booker et al., 1999). However, chicken immunoisolated 3-containing nAChR that are devoid of 6 subunits do not exhibit high affinity for -CTx-MII or MLA (Vailati et al., 2000). Thus, the 3 subunit may not confer sensitivity to -CTx-MII or MLA to nAChR but is crucial for the formation, targeting or stability of -CTx-MII-sensitive nAChR in basal ganglia in vivo. Klink et al. (2001) propose that the 3 subunit is targeted to terminal regions of dopaminergic neurons and does not participate in somatodendritic nAChR; thus, nAChR at these two locations could exhibit pharmacological (Reuben et al., 2000) and biophysical differences. Indeed, Klink et al. (2001) reported that 1 nM MLA was maximally effective in blocking cell body responses, which is more potent than predicted from the neurochemical measurements of [³H]dopamine release summarized above, or its K_i for interaction with ¹²⁵I--CTx-MII binding sites (Fig. 4). Although this discrepancy may reflect methodological differences, it could also arise from distinct nAChR subtypes at cell body and terminal locations.

These considerations lead to the proposition that -CTx-MII- and MLA-sensitive presynaptic nAChRs mediating striatal [³H]dopamine release are comprised of 3 and/or 6 subunits together with 2 and 3 subunits. The finding that ¹²⁵I--CTx-MII binding to basal ganglia is absent in 6 null mutant mice (Champtiaux et al., 2002), but persists in 3 null mutant mice (Whiteaker et al., 2002), argues for the involvement of the 6 subunit, rather than the 3 subunit, in this nAChR subtype. Klink et al. (2001) proposed the subunit composition 465(2)₂ for -CTx-MII- and MLA-sensitive somatodendritic nAChRs. Inclusion of the 4 subunit was based on the absence of responses in midbrain slices from 4 null mutant mice. The ability of nicotinic agonists to elicit dopamine release from striatal synaptosomes prepared from these transgenic animals has not been reported, so no evidence is available for the participation of the 4 subunit in the -CTx-MII- and MLA-sensitive presynaptic nAChR.

One implication of this and related studies is that sensitivity to nanomolar concentrations of MLA should not be considered diagnostic of 7 nAChR, at least in studies of rodent basal ganglia. This is particularly pertinent for in vivo studies, where drug concentrations reaching the nAChR are not known. However, the caveat that must be placed on the nAChR subtype selectivity of MLA may also be exploited for the differentiation of minority subtypes of nAChR, by comparison of sensitivities to MLA and -CTx-MII.