Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

Molecular cloning approaches have revealed the expression of 10 nicotinic acetylcholine receptor (nAChR) subunits [2-7, 9 (to date 8 has been identified only in avian neurons), and 2-4] in mammalian neuronal tissue (Lindstrom et al., 1996). Each of the subunits' mRNA has a distinct pattern of expression, suggesting the possibility that they may mediate different processes. In the central nervous system, many of these neuronal nAChRs seem to be presynaptic, where they modulate the release of neurotransmitters such as dopamine, norepinephrine, acetylcholine, and -aminobutyric acid with differential pharmacologies (Wonnacott, 1997).

Heterologous expression of neuronal nAChR subunits has shown different subunit combinations (or expression of 7-9 alone) result in functional nAChR subtypes with differing pharmacological and biophysical properties (Lindstrom et al., 1996). The number of neuronal nAChR subunits known theoretically allows the possibility of vast numbers of potential subunit combinations and receptor subtypes. However, efforts to discover which nAChR subtypes exist in the central nervous system (and their locations) have been hindered by the lack of subtype-specific pharmacological probes to identify individual subtypes within the mixed native nAChR population. Indeed, only two subtypes of neuronal nAChRs, those which correspond to ¹²⁵I--Bgt and `high-affinity agonist binding' sites, have been thoroughly characterized so far. In mammalian neuronal systems, ¹²⁵I--Bgt is believed to bind largely (if not exclusively) to nAChRs containing the 7 subunit (Schoepfer et al., 1990; Seguela et al., 1992), whereas ()-[³H]nicotine, [³H]cytisine, [³H]acetylcholine, and [³H]methylcarbamylcholine binding is only detectable at the 42 combination of subunits (Whiting and Lindstrom, 1987; Flores et al., 1992). Neuronal bungarotoxin (Bgt), a minor component of the venom of the Taiwanese banded krait Bungarus multicinctus (also named -Bgt, toxin F, and Bgt 3.1) showed initial promise as a selective antagonist of 32 subtype nAChRs (Luetje et al., 1990). However, problems of availability (B. multicinctus is a protected species), complex kinetics of interaction at multiple nAChR subtypes (Papke et al., 1993), and the difficulty of ensuring the toxin's purity have severely restricted its usefulness. More recently, it has become apparent that the agonist ligand [³H]epibatidine binds with detectable affinity to other neuronal nAChRs in addition to the 42 subtype (Perry and Kellar, 1995; Marks et al., 1998; Parker et al., 1998), raising hopes of identifying further native nAChR populations.

-CtxMII was identified in the venom of Conus magus by sequential fractionation and testing of the isolates for inhibition of 32 nAChRs expressed in Xenopus laevis oocytes (Cartier et al., 1996). Experiments in native systems have demonstrated potent (nanomolar IC₅₀ values) and selective blockade of nAChR subpopulations in rodent striatal (Grady et al., 1997; Kulak et al., 1997; Kaiser et al., 1998) and avian ciliary ganglion (Ullian et al., 1997) preparations. These functional studies provided strong evidence that -CtxMII was a highly selective antagonist of a novel native receptor population. The high affinity and novel subtype selectivity of -CtxMII make it a potentially useful ligand, particularly in light of its proven ability to interact with native neuronal nAChRs and the present paucity of selective pharmacological tools.

In this study, a radiolabeled version of -CtxMII ([¹²⁵I]CtxMII) was used to identify, locate, and enumerate -CtxMII binding nAChRs in mouse brain. The distribution and pharmacology of these receptors differs from those previously characterized, indicating that they represent a novel population. Using unlabeled -CtxMII in combination with existing nicotinic ligands allowed identification of -CtxMII binding nAChRs as part of the set of high-affinity [³H]epibatidine binding nAChRs, but distinct from the traditionally recognized "high-affinity agonist binding" 42 subtype. The pharmacological characteristics and distribution of -CtxMII binding nAChRs indicate that they are likely to be composed of (at least) 3 and 2 subunits.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Animals. Male mice (C57BL/6J, 60-90 days old) were used throughout this study. Mice were bred at the Institute for Behavioral Genetics and housed five per cage. The vivarium was maintained on a 12-h/12-h light/dark cycle (lights on 7 AM to 7 PM), and mice were given free access to food and water. The Animal Care and Utilization Committee of the University of Colorado, Boulder, approved all procedures used in this study.

Materials. [³H]Epibatidine (specific activity, 33.8 Ci/mmol), ()-[³H]nicotine (specific activity, 81.5 Ci/mmol), Na¹²⁵I (specific activity, 2200 Ci/mmol), and uridine triphosphate (-³⁵S; initial specific activity, 800 Ci/mmol) were obtained from DuPont NEN (Boston, MA). ¹²⁵I--Bgt (initial specific activity, 230 Ci/mmol), Hyperfilm -max, and Hyperfilm-³H were purchased from Amersham (Mt. Prospect, IL). NaCl, NaOH, KCl, MgCl₂, MgSO₄, CaCl₂, chloramine T, ammonium acetate, lysozyme, Tris · HCl, sodium carbonate, sodium bicarbonate, polyethylenimine (PEI; 50% w/v solution), yeast tRNA, triethanolamine, sodium citrate, dithiothreitol, Denhardt's solution, acetic anhydride, diethylpyrocarbonate, sodium phosphate, gelatin, poly-L-lysine, chromium aluminum sulfate, BSA (Fraction V), phenylmethylsulfonyl fluoride, EDTA, EGTA, aprotinin, leupeptin trifluoroacetate, and pepstatin A were obtained from Sigma Chemical Co. (St. Louis, MO). ()-nicotine bitartrate and DPX mountant were bought from BDH Chemicals (Poole, UK). Glass fiber filters Type A/E were obtained from Gelman Sciences (Ann Arbor, MI) and Type GB from MFS (Dublin, CA). Budget Solve scintillation fluid was purchased from RPI (Arlington Heights, IL). ATP, CTP, GTP, and RNase A were purchased from Boehringer-Mannheim (Indianapolis, IN). The enzymes SP6 RNA polymerase and HindIII, were obtained from Promega (Madison, WI). Dextran sulfate was purchased from Pharmacia (Uppsala, Sweden), and formamide from Fluka Chemical Corp. (Ronkonkoma, NY).

Preparation of -CtxMII and Y₀--CtxMII and Iodination of Y₀--CtxMII. Unmodified -CtxMII was synthesized as described previously (Cartier et al., 1996). To provide an iodination site, -CtxMII was synthesized with the addition of a tyrosine at the N terminus (Y₀--CtxMII). Although the two histidine residues present in native MII also provide potential iodination sites, structure-function studies suggested that modification of these sites would lead to unacceptable levels of decreased toxin potency (G. E. Cartier, unpublished observations). Synthesis of Y₀--CtxMII was achieved by methods described previously (Cartier et al., 1996).

Quantitative Autoradiography of ¹²⁵I--CtxMII and [³H]Epibatidine Binding. Quantitative autoradiography procedures were similar to those described previously (Pauly et al., 1989; Marks et al., 1998). Three C57BL/6J mice were sacrificed by cervical dislocation. The brains were removed from the skulls and rapidly frozen by immersion in isopentane (35°C, 10 s). The frozen brains were wrapped in aluminum foil, packed in ice, and stored at 70°C until sectioning. Tissue sections (14 µm thick) prepared using an IEC Minotome Cryostat refrigerated to 16°C were 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. Eight series of sections were collected from each mouse brain.

Membrane Preparation. Each C57BL/6J mouse was sacrificed by cervical dislocation. The brain was removed from the skull and placed on an ice-cold platform. Brains were either dissected into 12 regions [olfactory bulbs, cerebellum, hindbrain (pons-medulla), hypothalamus, hippocampus, striatum, cerebral cortex, thalamus, midbrain, interpeduncular nucleus, superior colliculus, and inferior colliculus] or the hindbrain, cerebellum, and olfactory bulbs were discarded without further dissection ("whole brain" preparation). Samples were homogenized in ice-cold hypotonic buffer (14.4 mM NaCl, 0.2 mM KCl, 0.2 mM CaCl₂, 0.1 mM MgSO₄, 2 mM HEPES, pH 7.5) using a glass-Teflon tissue grinder. The particulate fractions were obtained by centrifugation at 20,000g (15 min, 4°C; Sorval RC-2B centrifuge). The pellets were resuspended in fresh homogenization buffer, incubated at 37°C for 10 min, then harvested by centrifugation as before. Each pellet was washed twice more by resuspension/centrifugation, then stored (in pellet form under homogenization buffer) at 70°C until used. Protein concentrations in the membrane preparations were measured using the method of Lowry et al. (1951), using BSA as the standard.

()-[³H]Nicotine Binding to Membranes. The binding of ()-[³H]nicotine was measured using the method of Marks et al. (1986), modified for use with a 96-well plate washer. Membrane samples (200 µg of whole brain preparation) were incubated in 96-well polystyrene plates with 20 nM ()-[³H]nicotine in 100 µl of binding buffer for 30 min at 22°C. Binding reactions were terminated by filtration of samples onto PEI-soaked (0.5% w/v in binding buffer) glass fiber filters (types GFA/E and GB) using an Inotech Cell Harvester (Inotech, East Lansing, MI). Samples were subsequently washed six times with ice-cold binding buffer. Total and nonspecific [in the presence of 1 mM ()-nicotine tartrate] binding were determined in triplicate. Where inhibition binding was being measured, various concentrations of competing ligands were included in triplicate wells.

¹²⁵I--Bgt Binding to Membranes. Binding of ¹²⁵I--Bgt to membrane preparations was performed using procedures similar to those used with ()-[³H]nicotine, except that incubation times were extended to 5 h and samples contained 1 nM ¹²⁵I--Bgt instead of 20 nM ()-[³H]nicotine. The GFA/E filters were treated with 0.5% PEI and the GFB filters were treated with 5% (w/v) nonfat dry milk before filtration.

[³H]Epibatidine Binding to Membranes. Binding of [³H]epibatidine was quantified as described previously (Marks et al., 1998). Incubations were performed in 1-ml polypropylene tubes in a 96-well format, using 50 to 200 µg of membrane protein per tube (depending on brain region). A 500-µl reaction volume was used to minimize problems of ligand depletion, and all incubations progressed for 2 h at 22°C. The concentration of [³H]epibatidine (500 pM) used in inhibition binding experiments was chosen to maintain binding of ligand to the tissue at 5% or less of total ligand added. Saturation binding experiments were performed for membrane preparations from each brain region, using ligand concentrations in the range 10 to 800 pM. At the lower concentrations, a significant proportion of ligand bound to the tissue. Free [³H]epibatidine concentrations were estimated by correcting for the amount of ligand bound to tissue, and these corrected concentrations were used to calculate K_d values for [³H]epibatidine binding in each brain region and, thus, the K_i values for each compound versus [³H]epibatidine binding.

¹²⁵I--CtxMII Binding to Membranes. Large amounts of nonspecific binding were seen when using ¹²⁵I--Ctx (0.2-32 nM) in filtration binding assays. Best assay performance was produced using the following modifications to the ()-[³H]nicotine binding procedure. Incubation times were extended to 2 h, and incubation buffer was supplemented with BSA [0.1% (w/v)], 5 mM EDTA, 5 mM EGTA, and 10 µg/ml each of aprotinin, leupeptin trifluoroacetate, and pepstatin A to protect the ligand from endogenous proteases. The glass fiber filters were treated with 5% (w/v) nonfat dry milk before filtration.

In Situ RNA Hybridization. The method used for in situ hybridization using riboprobes was identical with that used by Simmons et al. (1989) and Marks et al. (1992). Probes were prepared by in vitro transcription, using -³⁵S-UTP as the sole source of UTP. The 3 probe was prepared from clone pPCA48E(4) cloned in pSP65, linearized using HindIII, and synthesized using SP6 RNA polymerase. The synthesis was designed to yield full-length antisense transcript. Immediately before hybridization, the probe was subjected to alkaline hydrolysis using the method of Cox et al. (1984) to yield products with average sizes of 500 bases.

Calculations. 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 Sigma Plot version 5.0 (Jandel Scientific, San Rafael, CA). Results for inhibition of ()-[³H]nicotine and ¹²⁵I--Bgt binding were calculated using a one-site fit: B = B_o / [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. Results for inhibition of ligand binding were calculated using the formulae for either one (as above) or two binding sites: B = B₁ / [1 + (I / IC_50-1)] + B₂ / [1 + (I / IC_50-2)], where B is ligand bound at inhibitor concentration I, and B₁ and B₂ are binding sites sensitive to inhibition with IC_50-1 and IC_50-2, respectively. 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

Top Abstract Introduction Experimental Procedures Results Discussion References

¹²⁵I--CtxMII Autoradiography, Mouse Brain Sections. Preliminary experiments (using monoiodinated but nonradioactive Y₀--CtxMII) showed that it remained a potent inhibitor at 32 nAChRs expressed in X. laevis oocytes (K_d = 1.9 nM, compared with 0.35 nM for the native toxin; data not shown). In light of these data, attempts were made to identify specific, nicotinic -CtxMII binding using the monoradioiodinated version of this ligand (¹²⁵I--CtxMII) in mouse brain sections.

View larger version (90K): [in this window] [in a new window]   Fig. 1.   Autoradiographic representation of 125I--CtxMII binding in mouse brain [total, with cytisine (20 nM), and nonspecific]. Sections (14 µm) were incubated with 0.5 nM 125I--CtxMII alone (left column), in the presence of 20 nM cytisine (center column), and with 1 µM epibatidine (nonspecific 125I--CtxMII; right column) as described under Experimental Procedures. The sections are adjacent in each row, and the panels are digital images of autoradiograms. The abbreviations used to identify brain regions are: AC, anterior commissure; DLGN, dorsolateral geniculate nucleus; FR, fasciculus retroflexus; IPN, interpeduncular nucleus; LH, lateral habenula; LSZ, lambdoid septal zone; MH, medial habenula; MVN, medial vestibular nucleus; NAC, nucleus accumbens core; NAS, nucleus accumbens shell; OMN, oculomotor nerve (or root); OPN, olivary pretectal nucleus; Opt, optic tract; OT, olfactory tubercle; PHN, prepositus hypoglossal nucleus; SCO, superior colliculus, optic nerve layer; SCS, superior colliculus, superficial gray; SCZ, superior colliculus, zonal layer; SN, substantia nigra; SOD, supraoptic decussation; Str, striatum; VLGN, ventrolateral geniculate nucleus.

View this table: [in this window] [in a new window]   TABLE 1 Regional distribution of specific mouse brain 125I--CtxMII binding in the presence and absence of 20 nM cytisine. Mouse brain sections (14 µm) were incubated in the presence of 125I--CtxMII (0.5 nM) with or without 20 nM cytisine. Binding of 125I--CtxMII was detected by exposing max film to the sections, followed by digital densitometry of the autoradiographic images. Amounts of ligand bound were calculated by reference to 125I-containing tissue paste standards. Because levels of 125I--CtxMII nonspecific binding varied noticeably between brain regions, nonspecific binding was determined in each region, and was used to calculate specific binding (presented in terms of femtomoles of 125I--CtxMII bound per milligram of tissue weight). Measurements were made six times in each region, in each mouse, where possible. Values are the mean ± S.E. of binding measured in three different mice.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Competition by cytisine, epibatidine, ()-nicotine, and -Bgt for 125I--CtxMII binding sites in mouse brain. Sections (14 µm) were incubated with 0.5 nM 125I--CtxMII in the presence of varying unlabeled ligand concentrations as described in the Methods section. Left, competition for specific 125I--CtxMII binding in the superficial gray of the superior colliculus by epibatidine (, IC50 = 103 ± 41 pM, nH = 0.87 ± 0.09), cytisine (, IC50 = 18 ± 8 nM, nH = 0.94 ± 0.12), and ()-nicotine (, IC50 = 481 ± 54 nM, nH = 1.47 ± 0.20). Right, competition for specific 125I--CtxMII binding in the striatum by epibatidine (, IC50 = 112 ± 38 pM, nH = 0.80 ± 0.23), cytisine (, IC50 = 23 ± 9 nM, nH = 1.12 ± 0.17), and ()-nicotine (, IC50 = 348 ± 84 nM, nH = 1.05 ± 0.24). -Bgt (1 µM) did not displace 125I--CtxMII binding in either region. Nonspecific binding was determined in the presence of 1 µM unlabeled epibatidine. Each point and value is the mean ± S.E.M. of four or five separate determinations. Data were fitted to a one-site Hill inhibition equation (see Experimental Procedures).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Regional comparison of mouse brain-specific 125I--CtxMII binding in the presence and absence of cytisine (20 nM). Levels of specific 125I--CtxMII (0.5 nM) binding with and without the addition of 20 nM cytisine were determined by quantitative autoradiography and compared in 23 different brain regions. Each point is the mean ± S.E.M. of data gathered from three different animals. Linear regression showed r = 0.96, slope = 0.600.

()-[³H]Nicotine, ¹²⁵I--Bgt and [³H]Epibatidine Binding, Mouse Brain Membrane Preparations. To expand on the data provided by ¹²⁵I--CtxMII competition binding experiments, the ability of -CtxMII to displace ()-[³H]nicotine, ¹²⁵I--Bgt, and [³H]epibatidine binding to mouse brain membrane preparations was assessed. For comparison, the ability of the nicotinic agonist cytisine to inhibit binding of the same ligands was also measured.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Competition by cytisine and -CtxMII for ()-[3H]nicotine, 125I--Bgt and [3H]epibatidine binding sites in mouse brain membranes. Left, mouse whole brain particulate fractions were incubated at 22°C with ()-[3H]nicotine (20 nM, 30 min; squares) or 125I--Bgt (1 nM, 5 h; triangles) in the presence of unlabeled cytisine (1 nM - 10 µM; filled symbols) or -CtxMII (1 nM - 10 µM; open symbols). Right, mouse superior colliculus particulate fractions were incubated at 22°C with [3H]epibatidine (500 pM, 2 h; circles) in the presence of unlabeled cytisine (1 nM - 30 µM; filled symbols) or -CtxMII (30 pM-3 µM; open symbols). Nonspecific binding was determined in the presence of 1 mM unlabeled ()-nicotine. Each point represents the mean ± S.E.M. of three separate determinations. Data were fitted to either one- or two-site (for cytisine inhibition of [3H]epibatidine binding) Hill inhibition equations (see Experimental Procedures). Both cytisine-sensitive and cytisine-resistant components of [3H]epibatidine binding are illustrated on the right (dotted lines).

View this table: [in this window] [in a new window]   TABLE 2 [3H]Epibatidine binding: cytisine-resistant and -CtxMII-sensitive populations in 12 brain regions Particulate fractions were prepared from the indicated brain regions of C57BL/6 mice. The Kd and Bmax values for each region were calculated from saturation binding experiments, as described under Experimental Procedures. Inhibition of [3H]epibatidine (500 pM) binding by cytisine and -CtxMII was determined as illustrated in Figure 4. Cytisine inhibition was fitted to a two-site model, with IC50 and Bmax values for the less-sensitive binding site being used to calculate the inhibition binding constant (Ki) and size of the cytisine-resistant [3H]epibatidine binding site population in each region. A one-site model was used to determine the Ki values and numbers of -CtxMII sensitive binding sites in each region. The Ki value of -CtxMII inhibition of [3H]epibatidine binding did not vary significantly between regions (one-way ANOVA; F(6,13) = 2.64, P > .05). All values are the mean ± S.E. of values derived from three to four independent experiments.

[³H]Epibatidine Autoradiography, Mouse Brain Sections. Competition binding experiments demonstrated that -CtxMII was able to potently displace a fraction of [³H]epibatidine binding to mouse brain membrane preparations. In addition, the same data showed that the density of -CtxMII-sensitive [³H]epibatidine binding sites varied widely among regions. Because the proportion of -CtxMII-sensitive [³H]epibatidine binding never exceeded that of the cytisine-resistant population (and in many regions was lower), it seemed possible that -CtxMII was selectively interacting with a subpopulation of cytisine-resistant [³H]epibatidine binding nAChRs, as suggested by the earlier ¹²⁵I--CtxMII autoradiography experiments. Crude regional dissection of the mouse brain provided limited anatomical resolution, so the relationship between cytisine-resistant and -CtxMII-sensitive [³H]epibatidine binding sites was explored using an autoradiographic approach. Inhibition binding experiments conducted using filtration binding indicated that 100 nM cytisine would essentially eliminate [³H]epibatidine binding to cytisine-sensitive sites (at a [³H]epibatidine concentration of 500 pM) but would leave the cytisine-resistant [³H]epibatidine binding largely unaffected (Fig. 4, right). For this reason, cytisine-resistant [³H]epibatidine binding was visualized using 500 pM [³H]epibatidine, in combination with 100 nM cytisine. The same series of experiments also suggested that 50 nM -CtxMII would be sufficient to displace -CtxMII-sensitive [³H]epibatidine binding, without affecting binding to other types of [³H]epibatidine binding sites.

View larger version (94K): [in this window] [in a new window]   Fig. 5.   Autoradiographic comparison of total [3H]epibatidine (500 pM) binding with that in the presence of cytisine (100 nM) or cytisine (100 nM) + -CtxMII (50 nM) and 3 mRNA expression in mouse brain. Sections (14 µm) were incubated in the presence of 500 pM [3H]epibatidine (left column), 500 pM [3H]epibatidine + 100 nM cytisine (center-left column), 500 pM [3H]epibatidine + 100 nM cytisine + 50 nM -CtxMII (center-right column), or were subjected to 3 mRNA in situ hybridization (3 mRNA; right column) as described under Experimental Procedures. Nonspecific labeling was indistinguishable from film background. Panels are digital images of autoradiograms. The sections in each row are adjacent. Abbreviations for the indicated brain regions are: 4, somatosensory cortex layer four; AC, anterior commissure; AOBG, accessory olfactory bulb glomerular layer AOBM, accessory olfactory bulb mitral layer; CC, corpus callosum; CgCx, cingulate cortex; DG, dentate gyrus; DLGN, dorsolateral geniculate nucleus; FR, fasciculus retroflexus; FC, frontal cortex; ICDC, inferior colliculus, dorsal cortex; IPN, interpeduncular nucleus; LH, lateral habenula; MGN, medial geniculate nucleus; MH, medial habenula; MVN, medial vestibular nucleus; NAC, nucleus accumbens core; NAS, nucleus accumbens shell; OBG, olfactory bulb glomerular layer; OBP, olfactory bulb internal plexiform layer; OMN, oculomotor nerve (or root); OPN, olivary pretectal nucleus; Opt, optic tract; OT, olfactory tubercle; PHN, prepositus hypoglossal nucleus; RN, reticular nuclei; SCO, superior colliculus, optic nerve layer; SCS, superior colliculus, superficial gray; SCZ, superior colliculus, zonal layer; SN, substantia nigra; SOD, supraoptic decussation; SpCN suprachiasmatic nucleus; Str, striatum; Sub, subiculum; VLGN, ventrolateral geniculate nucleus; VTA, ventral tegmental area.

View this table: [in this window] [in a new window]   TABLE 3 Regional distribution of specific [3H]epibatidine binding [total, + cytisine (100 nM), and + cytisine (100 nM) + -CtxMII (50 nM)] Sections (14 µm) were incubated in the presence of 500 pM [3H]epibatidine (total [3H]epibatidine), 500 pM [3H]epibatidine + 100 nM cytisine or 500 pM [3H]epibatidine + 100 nM cytisine + 50 nM -CtxMII. Binding of [3H]epibatidine was detected by exposing max 3H-sensitive film to the sections, followed by digital densitometry of the resultant autoradiograms. Specific binding was calculated by reference to 3H-containing tissue paste standards, and is presented in terms of femtomoles of [3H]epibatidine bound per milligram of tissue weight. The amount of -CtxMII-sensitive [3H]epibatidine binding in each region was calculated as the difference between the [3H]epibatidine binding in the presence of cytisine (100 nM) alone and that with both cytisine (100 nM) and -CtxMII (50 nM) present. Six individual measurements were made from each region, under each condition in each mouse where possible. Each value represents the mean ± S.E. of binding measured in three different mice.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Comparison of specific 125I--CtxMII binding and [3H]epibatidine binding sensitive to -CtxMII (50 nM) in mouse brain. Amounts of specific 125I--CtxMII (0.5 nM) binding were determined in 23 different brain regions by quantitative autoradiography. The amount of -CtxMII-sensitive [3H]epibatidine binding was quantified in the same regions as the difference between [3H]epibatidine binding in the presence of cytisine (100 nM) and cytisine (100 nM) + -CtxMII (50 nM). Each point is the mean ± S.E.M. of data gathered from three different animals. In regions with small or zero cytisine- + -Ctx-MII-resistant [3H]epibatidine binding populations (filled symbols), linear regression showed r = 0.96, slope = 2.6. Those regions containing large amounts of -CtxMII-resistant [3H]epibatidine binding (open symbols) deviated markedly above the regression line. Abbreviations for the indicated brain regions are: FR, fasciculus retroflexus; IPN, interpeduncular nucleus; LH, lateral habenula; MH, medial habenula; OMN, oculomotor nerve (or root); SCZ, superior colliculus zonal layer.

3 Subunit Expression: In Situ Hybridization. A previous investigation (Marks et al., 1998) suggested that cytisine-resistant [³H]epibatidine binding is found in regions that express 3 nAChR subunit mRNA or in regions innervated by those that do. To explore this link more thoroughly, the distribution of 3 mRNA was mapped by in situ hybridization, and compared with that of cytisine-resistant [³H]epibatidine binding. Hybridization was performed in brain slices prepared from the same animals used for autoradiographic investigations of ligand binding in this study, allowing direct comparisons to be made.

View this table: [in this window] [in a new window]   TABLE 4 Regional quantification of 3 mRNA Data show in situ hybridization of 35S-cRNA for 3 in mouse brain regions. Hybridization was performed as described under Experimental Procedures. Hybridized probe was detected by exposing max film to the sections, followed by digital densitometry of the autoradiographic images. Specific hybridization was quantified by reference to dot-blotted samples of 35S-cRNA probe of known activity and is presented in terms of pCi/mm2. Where possible, measurements were made six times in each region, in each mouse. Values are the mean ± S.E. of binding measured in three different mice.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

-CtxMII, originally isolated from the venom of the predatory cone snail, C. magus (Cartier et al., 1996), has been used to investigate the diversity of nicotinic receptor binding sites in mouse brain. The data presented here demonstrate that mouse brain high-affinity epibatidine binding has three components: 1) a component that corresponds to the ()-[³H]nicotine- and [³H]cytisine-binding sites ["cytisine-sensitive" sites, probably the 42 subtype (Whiting and Lindstrom, 1987; Flores et al., 1992)] and two sites with lower cytisine affinity ("cytisine-resistant" sites); 2) a cytisine-resistant component that displays high affinity for -CtxMII and that has been visualized here using ¹²⁵I--CtxMII; 3) a cytisine-resistant component that displays lower affinity for -CtxMII. This subdivision of mouse brain [³H]epibatidine-binding nAChRs is illustrated in Fig. 7.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Summary of [3H]epibatidine binding sites in mouse brain. Using [3H]epibatidine in combination with cytisine reveals that [3H]epibatidine binds specifically and with high affinity in mouse brain to both the well-established ()-[3H]nicotine and [3H]cytisine binding site of 42 composition (Whiting and Lindstrom, 1987; Flores et al., 1992), and a smaller population of nAChRs that are less cytisine sensitive (Marks et al., 1998). This allows the use of cytisine to selectively remove [3H]epibatidine binding at the ()-[3H]nicotine binding site, revealing sites less sensitive to cytisine binding. The [3H]epibatidine sites that are less cytisine-sensitive are likely to contain the 3 nAChR subunit, and may be divided into two groups according to their -CtxMII sensitivity. 125I--CtxMII may be used to identify and quantify the population that is more -CtxMII-sensitive and was used in this study to map the distribution of mouse brain high-affinity -CtxMII binding nAChRs. The population that is more -CtxMII-sensitive is likely to contain (minimally) 3 and 2 nAChR subunits, whereas the subpopulation that is less -CtxMII-sensitive can tentatively be assigned a minimal 3 and 4 nAChR subunit composition.

¹²⁵I--CtxMII Binds to a Novel nAChR Population. Competition binding experiments demonstrated that -CtxMII has a low affinity (K_i >10 µM) at mouse brain ()-[³H]nicotine and ¹²⁵I--Bgt binding sites (Fig. 4). This shows that it binds to a novel neuronal nAChR population, distinct from the well- characterized ()-[³H]nicotine- and ¹²⁵I--Bgt-binding sites (corresponding to 42 and 7-containing subtypes in mammalian neurons; Whiting and Lindstrom, 1987; Schoepfer et al., 1990; Flores et al., 1992; Seguela et al., 1992).

-CtxMII-Binding nAChRs are a Subset of Cytisine-Resistant [³H]Epibatidine Binding Sites. Confirming the data provided by the ¹²⁵I--CtxMII autoradiography experiments, -CtxMII was a potent inhibitor (mean K_i = 2.7 nM) of a fraction of [³H]epibatidine binding sites in mouse brain regional homogenates (Fig. 4; Table 2). Densities of these -CtxMII-sensitive [³H]epibatidine-binding sites varied among brain regions, in many cases having a lower density than cytisine-resistant [³H]epibatidine binding sites. Indeed, quantitative autoradiography (Fig. 5) showed that -CtxMII-sensitive sites are a subset of the cytisine-resistant [³H]epibatidine-binding sites describe