Experimental Procedures

Materials.

The following were purchased from Sigma Chemical Co. (St. Louis, MO): (−)-nicotine tartrate, (+)-nicotine-(+)-di-p-toluoyltartrate, ACh, carbachol iodide, (±)-epibatidine hydrochloride, cytisine, (±)-nornicotine, (±)-anabasine, tetramethylammonium chloride (TMA),d-tubocurarine chloride (dTC), hexamethonium chloride, decamethonium chloride, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, atropine sulfate, tetrodotoxin, diisopropylflourophosphate (DFP), bovine serum albumin, and polyethylenimine. The following were purchased from Research Biochemicals International (Natick, MA): (+)-epibatidine hydrochloride, (−)-epibatidine hydrochloride, (+)-anatoxin-a, DHβE, MLA, epiboxidine and, 3-(2-(S)-azetidinylmethoxy)pyridine dihydrochloride (A-85380). Sucrose and HEPES were purchased from Boehinger-Mannheim (Indianapolis, IN). Dimethylphenylpiperazinium iodide (DMPP) was purchased from Aldrich Chemical Co. (Milwaukee, WI). Cesium chloride and Budget Solve Scintillation fluid were purchased from Research Products International (Mt. Prospect, IL). (S)-3-methyl-5-(1-methyl-2-pyrrodinyl)isoxazole (ABT-418) was a gift from Abbott Laboratories (Abbott Park, IL). [³H]Nicotine (81.5 Ci/mmol), [³H]epibatidine (33.8 Ci/mmol), and carrier-free ⁸⁶RbCl were purchased from DuPont-NEN (Boston, MA). α-Bungarotoxin (initial specific activity 210 Ci/mmol) was purchased from Amersham, Inc. (Arlington Heights, IL).

Mice.

Female C57BL/6J and β2 nicotinic acetylcholine receptor (nAChR)-null mutant mice (Picciotto et al., 1995) of either sex were bred at the Institute for Behavioral Genetics (University of Colorado, Boulder, CO). C57BL/6 mice were housed five per cage and β2 mutant mice were housed with like sex littermates (2–5 per cage). The mice were housed in a vivarium maintained at 22°C with a 12-h light/dark cycle (lights on from 7:00 AM to 7:00 PM). The mice were allowed free access to food (Harlan Teklad Rodent Diet) and water. Animals were 60- to 90-days old when used. Animal care and experimental procedures were performed in accordance with the guidelines and with the approval of the Animal Care and Utilization Committee of the University of Colorado, Boulder.

Synaptosome Preparation.

Crude synaptosomes were prepared from mouse forebrain or dissected brain regions. Samples were homogenized by hand (20 strokes with a Teflon-glass tissue grinder) in 10 volumes ice-cold 0.32 M sucrose buffered to pH 7.5 with 5 mM HEPES. The homogenate was centrifuged at 500g for 10 min. The resulting supernatant was subsequently centrifuged at 12,000g for 20 min to yield the crude synaptosomal pellet (P2).

⁸⁶Rb⁺ Uptake.

The uptake of⁸⁶Rb⁺ into the synaptosomal fractions was achieved by incubating the resuspended P2 for 30 min in uptake buffer (NaCl, 140 mM; KCl, 1.5 mM; CaCl₂, 2 mM; MgSO₄, 1 mM; HEPES hemisodium, 25 mM; glucose, 20 mM; pH, 7.5) containing 4 μCi of carrier-free⁸⁶RbCl. The final incubation volume was 35 μl. Uptake was terminated by filtration of the sample onto a 6-mm diameter glass fiber filter (Type AE; Gelman, Ann Arbor, MI) under gentle vacuum (−0.2 atmospheres) followed by two washes with 0.5 ml of uptake buffer. Samples to be tested for the effects of ACh were incubated with 10 μM diisopropyl fluorophosphate during the final 10 min of uptake to inhibit acetylcholinesterase.

Sample Perfusion.

After filtration and wash, the glass fiber filter containing the loaded synaptosomes was transferred to a polypropylene platform. Perfusion buffer (NaCl, 135 mM; CsCl, 5 mM; KCl, 1.5 mM; CaCl₂, 2 mM; MgSO₄, 1 mM; HEPES hemisodium, 25 mM; glucose, 20 mM; tetrodotoxin, 50 nM; atropine, 1 μM; bovine serum albumin (fraction V), 0.1%; pH, 7.5) was delivered to the filter at a rate of 2.5 ml/min using a Gilson Minipuls 3 peristaltic pump (Gilson, Middleton, WI). Atropine and tetrodotoxin were included in the buffer to prevent activation of muscarinic receptors and sodium channels, respectively. The 1 μM concentration of atropine is comparable to that used in studies of heterologously expressed receptors (Leutje and Patrick, 1991) and of nAChR in cell culture (Alkondon and Albuquerque, 1993, 1995). Preliminary experiments indicated that this concentration of atropine had no effect on nicotine-stimulated ⁸⁶Rb⁺efflux. TTX (50 nM) inhibits a fraction of nicotine-stimulated⁸⁶Rb⁺ efflux apparently resulting from secondary activation of Na⁺channels (Marks et al., 1995) and was included to block this secondary response. Buffer was actively removed using a second Gilson peristaltic pump set for a flow rate of 3.2 ml/min. The use of two pumps prevented the accumulation of perfusion buffer on the filter holding the sample. Sample effluent was pumped through a 200 μl volume flow-through Cherenkov cell in a β-RAM Radioactivity HPLC Detector (IN/US Systems, Inc., Tampa, FL) to achieve continuous monitoring of⁸⁶Rb efflux from the sample. Stimulation of the synaptosomes was accomplished by diverting perfusion buffer flow through a 200 μl loop containing the test solution by means of a 4-way rotary Teflon injection valve (Alltech Associates, Inc., Deerfield, IL). Thus, stimulation time was 5 s. The⁸⁶Rb⁺ efflux was monitored for 4 min and timing was adjusted so that any efflux resulting from stimulation was located in the middle of the sampling period. This timing permitted the definition of basal efflux rate measured before and after agonist application.

Agonist Application.

The stimulation of samples by agonists was achieved by filling the 200 μl sample loop with a solution of defined concentration of the agonist being evaluated. Each sample was stimulated once.

Antagonist Effects.

The effect of DHβE on⁸⁶Rb⁺ efflux stimulated by ACh, nicotine, or epibatidine was evaluated at two concentrations of each agonist (30 and 1000 μM, 10 and 300 μM, and 0.3 and 10 μM, respectively).

The effects of α-Bgt on ACh-stimulated⁸⁶Rb⁺ efflux were measured using 30 and 1000 μM ACh on samples that had been incubated with 100 nM α-Bgt for 60 min at 22°C before filtration. Perfusion buffer for these samples also contained 100 nM α-Bgt.

The effects of antagonists were examined for samples that had been perfused with the appropriate concentration of antagonist for 8 min before stimulation with either 10 μM nicotine or 10 μM epibatidine in the presence of 2 μM DHβE. Nicotine was used to stimulate in the absence of DHβE to provide data directly comparable to those obtained previously, whereas epibatidine was used in the presence of DHβE because of the large response obtained with this agonist. The agonist solutions contained the same concentration of antagonist that was used to perfuse the samples. Thus, antagonist was present before, during, and after agonist stimulation. The effect of DHβE on the response stimulated by 10 μM epibatidine was evaluated in samples that also contained either 2 μM DHβE or 2 μM MLA.

Agonist-Stimulated ⁸⁶Rb⁺ Efflux in Several Brain Regions.

The following fifteen brain regions were dissected and P2 fractions prepared: olfactory bulbs (OB), olfactory tubercles (OT), cerebral cortex (Cx), septum (Se), hippocampus (Hp), striatum (St), habenula (Hab), thalamus (Th), hypothalamus (HT), interpeduncular nucleus (IPN), midbrain (MB), superior colliculus (SC), inferior colliculus (IC), hindbrain (HB), and cerebellum (Cb). The P2 fractions were loaded with⁸⁶Rb⁺ and evaluated for the efflux stimulated by 10 μM nicotine and 10 μM epibatidine plus 2 μM DHβE. Nicotine was used to stimulate in the absence of DHβE to provide data directly comparable to those obtained previously, whereas epibatidine was used in the presence of DHβE because of the large response obtained with this agonist. To obtain adequate samples from the small brain areas (OT, Se, Hab, HT, IPN, SC, IC), tissue from several mice was pooled before homogenization.

Ligand Binding.

Particulate fractions were prepared from P2 after lysis of the crude synaptosomes by three cycles of suspension in hypotonic buffer (NaCl, 14 mM; KCl, 0.15 mM; CaCl₂, 0.2 mM; MgSO₄, 0.1 mM; HEPES, hemisodium salt, 2.5 mM; pH = 7.5) followed by centrifugation at 20,000g for 15 min.

Binding reactions were conducted in buffer of the following composition: NaCl, 140 mM; KCl, 1.5 mM; CaCl₂, 2 mM; MgSO₄, 1 mM; HEPES hemisodium, 25 mM; pH = 7.5. Incubations with α-Bgt also contained 0.1% bovine serum albumin.

The binding of [³H]nicotine and α-Bgt was conducted as described previously (Marks et al., 1986) as modified for 100 μl incubations in 96-well microtiter plates (Marks et al., 1996). For [³H]nicotine binding, samples were incubated with 17 nM [³H]nicotine (K _D = 2 nM) for 30 min at 22°C. Blanks were determined by including 10 μM unlabeled nicotine in the incubation. For α-Bgt binding, samples were incubated with 2.0 nM α-Bgt (K _D = 0.5 nM) for 4 h at 22°C. Blanks were determined by including 1 mM unlabeled nicotine in the incubation.

The binding of [³H]epibatidine at low concentrations (high-affinity binding) and the effect of cytisine on this binding was determined as described previously (Marks et al., 1998). Incubation volume was 500 μl. For total [³H]epibatidine binding, samples were incubated with 0.36 nM [³H]epibatidine (K _D = 6 pM) for 120 min at 22°C. Cytisine-resistant [³H]epibatidine binding was determined by including 50 nM cytisine in the incubation. Blanks were determined by including 100 μM unlabeled nicotine in the incubation.

The binding of [³H]epibatidine at high concentrations (low- plus high-affinity binding; Houghtling et al., 1995) was measured using a 100 μl incubation volume. For total [³H]epibatidine, samples were incubated with 10 nM [³H]epibatidine (low-affinityK _D = 6 nM) for 60 min at 22°C. Blanks were determined by including 1 mM unlabeled nicotine in the incubation. Low-affinity binding was estimated by subtracting the binding measured at 0.36 nM [³H]epibatidine from that measured at 10 nM [³H]epibatidine or as the amount of low-affinity binding inhibited by incubation with 300 μM dTC. The results obtained with these two calculations did not differ significantly.

At the completion of the incubations, all binding reactions were terminated by filtration using an 96-place manifold (Inotech Biosystems, Lansing, MI). Particulate fractions were collected with two glass fiber filters (top, Type GB100, Microfiltration Systems, Dublin, CA; bottom, Type A/E, Gelman Sciences, Ann Arbor, MI). Filters for collection of [³H]nicotine and [³H]epibatidine were treated with 0.5% polyethylenimine. GB100 filters for collection α-Bgt samples were treated with 5% nonfat skim milk dissolved in wash buffer, whereas A/E filters were treated with 0.5% polyethylenimine. Samples were washed six times. All filtrations and washes were conducted in a 4°C cold room using ice-cold buffer.

After the addition of 1 ml of Budget Solve Scintillation Fluid (Research Products International) to each sample, tritium was measured at 45% efficiency using a Packard 1600TR Liquid Scintillation Spectrometer. The ¹²⁵I was measured at 80% efficiency using a Packard Cobra Gamma Counter.

Protein.

Protein was measured using the method of Lowry et al. (1951) with bovine serum albumin as the standard.

Data Calculation.

The magnitude of agonist stimulated⁸⁶Rb⁺ efflux was determined as the counts exceeding basal efflux during time of exposure to agonist. Samples were normalized to the counts remaining in the sample at the end of the stimulation period such that the signal size represented the percent of total tissue⁸⁶Rb⁺ that was stimulated by agonist exposure.

Curve fitting was accomplished using the nonlinear curve fitting algorithm in Sigma Plot 5.0 (Jandel Scientific, San Rafael, CA). Concentration-effect curves were fit using either the Michaelis-Menten equation, the Hill equation, or two Michaelis-Menten equations. Relative potencies, efficacies, and regional distributions of various responses were compared using regression analysis.

Results

ACh-Stimulated ⁸⁶Rb⁺ Efflux.

The⁸⁶Rb⁺ efflux stimulated by a 5-s exposure to several concentrations of ACh is illustrated in the left panel of Fig. 1. Each point represents datum collected for a 3-s time period using a continuous flow detector. The amount of⁸⁶Rb⁺ efflux increased with increasing concentrations of ACh between 1 and 1000 μM. The peak observed after stimulation with 1000 μM ACh was approximately 3-fold greater than the basal efflux. The concentration-response curve for ACh-stimulated ⁸⁶Rb⁺deviated markedly from simple Michaelis-Menten kinetics (Fig. 1, right). The Hill coefficient for this curve was 0.44 ± 0.08. The results were consistent with those of a two-component process displaying EC₅₀ values of 0.80 ± 0.57 μM and 82.6 ± 40.2 μM with maximal efflux representing 0.079 ± 0.019% and 0.160 ± 0.018% of total tissue⁸⁶Rb⁺ during the 5-s stimulation, respectively. These results suggest that more than one process mediates ACh-stimulated⁸⁶Rb⁺ efflux from mouse brain synaptosomes.

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Figure 1

Stimulation of ⁸⁶Rb⁺efflux by ACh. Crude whole brain synaptosomes stimulated for 5 s with ACh. The traces (left) are actual data obtained at the indicated concentrations of ACh. Each point represents the counts measured during the 3-s sampling period. Basal efflux for each condition was approximately 50 counts per fraction. The ACh concentration-effect curve is shown in the right panel. Each point represents the mean ± S.E.M. of six determinations from three separate experiments. The curve is that obtained for a two-site fit of the data.

Effects of DHβE and α-Bgt.

The effects of the nicotinic antagonists DHβE and α-Bgt on⁸⁶Rb⁺ efflux stimulated by either 30 or 1000 μM ACh were evaluated in an attempt to pharmacologically differentiate the putative multiple processes underlying ACh-stimulated⁸⁶Rb⁺ efflux from mouse brain synaptosomes (Fig. 2). Samples were treated with DHβE, an antagonist that is somewhat selective for α4/β2 nicotinic receptors, for 8 min or with α-Bgt, an antagonist that is selective for neuronal α7 nicotinic receptors, for 70 min before stimulation with ACh. The main panel on the left side of Fig. 2presents data from representative perfusion profiles obtained with a 5-s stimulation with 30 μM ACh without antagonist treatment, and after treatment with 2 μM DHβE, 100 nM α-Bgt, or both antagonists. DHβE treatment inhibited ACh-stimulated⁸⁶Rb⁺ efflux approximately 75%, whereas α-Bgt treatment had no significant effect. α-Bgt also had no additional effect in the presence of DHβE (Fig. 2, left, inset). The main panel on the right side of Fig. 2 presents data from representative perfusion profiles obtained with a 5-s stimulation with 1000 μM ACh. The response was inhibited approximately 40% by 2 μM DHβE, but was unaffected by 100 nM α-Bgt alone or in combination with DHβE. These results indicate that a significant fraction of ACh-stimulated ⁸⁶Rb⁺ efflux is sensitive to inhibition by DHβE, but not by α-Bgt. Furthermore, the DHβE-sensitive component was the same (0.8% of tissue content) whether the samples were stimulated with 30 or 1000 μM ACh.

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Figure 2

Effect of 2 μM DHβE and 100 nM α-Bgt on⁸⁶Rb⁺ efflux stimulated by 30 or 1000 μM ACh. The main panel of each figure illustrates primary data collected from samples of crude whole-brain synaptosomes stimulated with 30 μM ACh (left) or 1000 μM ACh (right) in the presence of 2 μM DHβE, 100 nM α-Bgt, or both agents, as indicated. Samples were stimulated with ACh for 5 s. Samples were treated with DHβE for 8 min before stimulation and with α-Bgt for 70 min before stimulation. DHβE and α-Bgt were present before, during, and after exposure to ACh. The insets summarize the responses (mean ± S.E.M.,n = 6) obtained under each experimental condition: C, control; D, 2 μM DHβE; B, 100 nM α-Bgt; D+B, 2 μM DHβE plus 100 nM α-Bgt.

DHβE Inhibition.

The partial inhibition of ACh-stimulated⁸⁶Rb⁺ efflux by 2 μM DHβE and the difference in the magnitude of this effect at different ACh concentrations suggests that this antagonist may differentially inhibit subsets of responses. Thus, the inhibitory effects of DHβE were examined by constructing concentration-response curves for responses stimulated by 30 or 1000 μM ACh (Fig.3). Samples were exposed to DHβE for 8 min before stimulation and DHβE was present during and after the 5-s stimulation with ACh. DHβE produced a concentration-dependent decrease in ⁸⁶Rb⁺ efflux at both concentrations of ACh. An apparently monophasic inhibition was observed for samples stimulated with 30 μM ACh with an estimated IC₅₀ value of 0.17 ± 0.02 μM. Higher concentrations of DHβE were required to inhibit⁸⁶Rb⁺ efflux stimulated by 1000 μM ACh. Inasmuch as DHβE is a competitive antagonist, the shift in the concentration-effect curve could have arisen merely because of the increased ACh concentration. Inhibition by DHβE was reversible (k = 0.021/s,T _1/2 = 33 s), however with this dissociation rate, reversal of inhibition was approximately 5% during the 5-s stimulation.

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Figure 3

Inhibition of ACh-stimulated⁸⁶Rb⁺ efflux by DHβE. Crude whole-brain synaptosomes were stimulated with 30 μM ACh (○) or 1000 μM ACh (●) for 5 s after an 8-min exposure to the indicated concentrations of DHβE. The inset displays the difference in response at 1000 μM and 30 μM ACh. Each data point represents the mean ± S.E.M. of eight determinations from four separate experiments.

When the results obtained with 30 μM ACh were subtracted from those obtained with 1000 μM ACh, the curve displayed in the inset of Fig. 3was generated. The IC₅₀ value estimated for this component was 2.6 ± 0.6 μM.

Differential DHβE inhibition curves were also generated using (−)-nicotine (10 and 300 μM) and (±)-epibatidine (0.3 and 10 μM) as agonists. Apparent IC₅₀ values of 0.27 ± 0.03 and 0.28 ± 0.04 μM at the lower concentrations and 7.2 ± 3.2 and 11.8 ± 3.0 μM for the difference between the higher and lower concentrations were calculated for nicotine and epibatidine, respectively. These results suggest the existence of a relatively high-affinity, DHβE-sensitive nicotinic response and a relatively low-affinity, DHβE-resistant nicotinic response for ACh, nicotine, and epibatidine.

Stimulation of DHβE-Sensitive and DHβE-Resistant⁸⁶Rb⁺ Efflux by Nicotinic Agonists.

The existence of ACh-stimulated responses that are differentially sensitive to inhibition by DHβE was used to evaluate the concentration dependence of nicotinic agonist-stimulated⁸⁶Rb⁺ efflux. Concentration-effect curves for ACh activation of⁸⁶Rb⁺ efflux from mouse forebrain were constructed with either 0 or 2 μM DHβE present in the perfusion buffer and are shown in Fig.4. Similar to the results presented in Fig. 1, ⁸⁶Rb⁺ efflux stimulated by ACh displayed a biphasic concentration-effect curve with estimated EC₅₀ values of 5.4 ± 4.7 and 244 ± 328 μM and efflux of 0.12 ± 0.05 and 0.16 ± 0.05%, respectively. However, at concentrations higher than 1 mM the responses decreased. In the presence of 2 μM DHβE, a monophasic concentration-effect curve with an EC₅₀ value of 540 ± 60 μM and maximal efflux of 0.16 ± 0.01% was obtained. These parameters are comparable to the low-affinity component of the full concentration-effect curve in the absence of DHβE. The difference in response between total ACh-stimulated efflux and that observed in the presence of 2 μM DHβE is shown in the inset to Fig.4. At ACh concentrations at or below 1000 μM, the ACh-stimulated⁸⁶Rb⁺ efflux appeared to be a monophasic process with an EC₅₀ of 7.5 ± 2.6 μM and a maximal efflux of 0.15 ± 0.01%. These parameters are comparable to the high-affinity component of the full dose-response curve in the absence of DHβE. Also note that a substantial decrease in efflux occurred at concentrations above 1 mM. These results indicate that differential inhibition by DHβE is a useful tool to study agonist stimulation of⁸⁶Rb⁺ efflux.

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Figure 4

Concentration-response curves for ACh in the presence of 0 or 2 μM DHβE. Crude whole-brain synaptosomes were treated with 0 μM DHβE (○) or 2 μM DHβE (●) for 8 min before a 5-s exposure to the indicated concentrations of ACh. Each point represents the mean ± S.E.M. for six determinations from three separate experiments. The inset displays the difference between the results in the absence or presence of DHβE. Curves shown for the response in the presence of DHβE and the inset are theoretical fits of the data to the Michaelis-Menten equation. The curve for the response in the absence of DHβE is a two-site fit of these data.

Differential DHβE inhibition was used to evaluate the stimulation of⁸⁶Rb⁺ efflux by 17 nicotinic agonists (Fig. 5). Most agonists elicited an increase in both DHβE-sensitive and DHβE-resistant ⁸⁶Rb⁺efflux, but substantial differences in both potency and efficacy were observed (Table 1).

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Figure 5

Agonist simulation of DHβE-sensitive and DHβE-resistant ⁸⁶Rb⁺ efflux. Crude whole brain synaptosomes were stimulated with the indicated concentrations of each agonist in the presence or absence of 2 μM DHβE. Data points represent results obtained in the presence of 2 μM DHβE (●; DHβE-resistant) or the difference between the response measured in the absence or presence of 2 μM DHβE (○; DHβE-sensitive). Each point represents the mean ± S.E.M. of six to eight determinations from three to four separate experiments. Curves are theoretical fits of the data to the Michaelis-Menten equation.

The EC₅₀ values for activation of the DHβE-sensitive ⁸⁶Rb⁺efflux were all substantially lower than the EC₅₀values for activation of DHβE-resistant⁸⁶Rb⁺ efflux. However, the relative potency observed for the DHβE-sensitive and DHβE-resistant responses varied among the agonists. Many of the agonists (ACh,l-nicotine, (±)-epibatidine, (+)-epibatidine, (−)-epibatidine, methylcarbachol, and epiboxidine) were 40- to 150-fold more potent for the DHβE-sensitive response. However, DMPP, TMA, carbachol, nornicotine, and anatoxin-a exhibited less selectivity between the two responses with potency ratios of about 20. Two weak agonists, cytisine and d-nicotine, displayed large differences in potency. A-85380 was unique in that this compound stimulated substantial⁸⁶Rb⁺ efflux and also displayed a large difference in potency (about 2500-fold) between the DHβE-sensitive and DHβE-resistant responses.

The agonists tested also differed considerably in the maximal responses that were measured. Furthermore, the maximal efflux observed for a given agonist for the DHβE-sensitive response could be larger than, similar to, or smaller than the maximal efflux observed for the DHβE-resistant response. Maximal⁸⁶Rb⁺ efflux measured for the endogenous transmitter, ACh, was very similar for the DHβE-sensitive and DHβE-resistant responses, as was the maximal efflux for l-nicotine. Epibatidine, epiboxidine, and A-85380 elicited considerably more DHβE-resistant efflux, as did cytisine. However, cytisine was not particularly efficacious for either component. Three quaternary agonists, carbachol, DMPP, and TMA, elicited 1.6- to 5-fold greater DHβE-sensitive response than DHβE-resistant response. No detectable DHβE-resistant⁸⁶Rb⁺ efflux was elicited by anabasine.

The relative potency and efficacy of the seventeen nicotinic agonists for the DHβE-sensitive and DHβE-resistant responses were compared by regression analysis (Fig. 6). The correlations between both potency and efficacy of the agonists were statistically significant (r = 0.75 andr = 0.74, respectively; p < .05), indicating a general correspondence between these two parameters for the DHβE-sensitive and DHβE-resistant responses. However, these relationships are not particularly strong. For example, the six agonists with EC₅₀ values of about 100 μM for stimulation of DHβE-resistant⁸⁶Rb⁺ efflux have EC₅₀ values that differ by approximately 1000-fold for the DHβE-sensitive efflux. Furthermore, the five agonists with EC₅₀ values around 10 μM for DHβE-sensitive ⁸⁶Rb⁺efflux have EC₅₀ values that differ approximately 100-fold for the DHβE-resistant response. Similar incongruities exist for maximal efflux (E_max), as well.

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Figure 6

Correlations between EC₅₀ values and maximal efflux for DHβE-sensitive and DHβE-resistant⁸⁶Rb⁺ efflux. EC₅₀ values (left) and maximal efflux (right) determined for the agonist curves of Fig. 5for the DHβE-sensitive response are compared to the corresponding values for the DHβE-resistant response.

Effects of Nicotinic Antagonists.

Inhibition of DHβE-sensitive (measured with 10 μM nicotine) and DHβE-resistant (measured with 10 μM epibatidine plus 2 μM DHβE) nicotinic responses by six antagonists was studied. Concentration-effect curves for these antagonists are shown in Fig. 7and IC₅₀ values are summarized in Table2. In contrast to the agonist effects, where every agonist was more potent in stimulating DHβE-sensitive⁸⁶Rb⁺ efflux, the antagonists were not uniformly more potent inhibitors of DHβE-sensitive response. Results obtained for DHβE under these conditions confirm the differential effect of DHβE. An IC₅₀ value of 0.15 ± 0.05 μM was calculated for DHβE-sensitive efflux and an IC₅₀ value of 8.3 ± 1.7 μM or 13.7 ± 3.3 was calculated for the response stimulated by 10 μM epibatidine in the presence of 2 μM DHβE or 2 μM MLA, respectively. MLA is also a more potent inhibitor of DHβE-sensitive efflux (IC₅₀ = 0.20 ± 0.09 μM) than it is of the DHβE-resistant response (IC₅₀ = 4.4 ± 1.6 μM). Decamethonium inhibited both responses with equal potency (IC₅₀ = 4.1 μM). Mecamylamine and dTC were slightly more potent inhibitors of the DHβE-resistant efflux (IC₅₀ = .16 ± 0.03 μM and 1.1 ± 0.2 μM, respectively) than of the DHβE-resistant efflux (IC₅₀ = .59 ± 0.09 and 2.8 ± 0.7 μM, respectively). Hexamethonium was a significantly more potent inhibitor of the DHβE-resistant response (IC₅₀= .89 ± 0.17 μM) than the DHβE-sensitive (IC₅₀ = 17.6 ± 6.1 μM). Thus, all six nicotinic antagonists inhibited both DHβE-sensitive and DHβE-resistant ⁸⁶Rb⁺efflux, but the relative potency of the compounds as inhibitors of the two processes varied markedly.

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Figure 7

Inhibition of DHβE-sensitive and DHβE-resistant⁸⁶Rb⁺ efflux by nicotinic antagonists. Crude whole-brain synaptosomes were exposed to the indicated concentration of antagonists for 8 min before a 5-s exposure to 10 μMl-nicotine (○) or 10 μM (±)-epibatidine plus 2 μM DHβE (●). Those samples to be stimulated with (±)-epibatidine were treated with 2 μM DHβE as well as the test antagonist for 8 min before stimulation. In a separate set of of experiments, samples for which DHβE inhibition of the DHβE-resistant response were measured were treated with 2 μM MLA (▪) instead of DHβE to allow analysis of DHβE inhibition in the absence of this antagonist. Each point represents the mean ± S.E.M. of six to eight individual measurements from three to four separate experiments. The curves are theoretical one-site fits using the equation: A_I = 100/(1+(I/IC₅₀)), where A_I is the percentage of control activity at antagonist concentration, I, and IC₅₀is the antagonist concentration that gives 50% inhibition.

Responses in β2 Mutant Mice.

Analysis of responses of mice expressing mutant nicotinic receptors provides one means by which the molecular basis of nicotinic responses can be investigated. The effect of null mutation of the β2 nAChR subunit on DHβE-sensitive and DHβE-resistant ⁸⁶Rb⁺efflux was determined. Perfusion profiles for⁸⁶Rb⁺ efflux stimulated by a 5-s exposure to 10 μM nicotine for β2 homozygote wild type, heterozygote, and homozygote mutant mice are shown in Fig.8 (top left). Although exposure to nicotine produced substantial increases in⁸⁶Rb⁺ efflux from whole brain synaptosomes of wild type and heterozygote mice, little response was observed in the homozygote mutants. The effect of β2 genotype is summarized in Fig. 8 (bottom left). A small decrease (12%) in response was observed for the heterozygotes, but⁸⁶Rb⁺ efflux stimulated by 10 μM nicotine decreased 96% in the β2 null mutants. The efflux remaining in the homozygote mutants was not significantly different from zero.

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Figure 8

Effect of β2 genotype on DHβE-sensitive and DHβE-resistant ⁸⁶Rb⁺ efflux. Crude synaptosomes were prepared from whole brains of mice with the following genotype for the β2 nAChR subunit: +/+, homozygote wild type; ±, heterozygote; −/−, homozygote mutant. The top panels illustrate primary data for mice of each genotype receiving a 5-s stimulation with 10 μM l-nicotine (left) or 10 μM (±)-epibatidine plus 2 μM DHβE (right). DHβE was present before, during, and after the stimulation with epibatidine. The bottom panels summarize data (mean ± S.E.M.) obtained for four wild type, seven heterozygote, and five mutant mice.

Perfusion profiles for⁸⁶Rb⁺ efflux stimulated by 10 μM epibatidine in the presence of 2 μM DHβE for β2 homozygote wild type, heterozygote, and homozygote mutant mice are shown in Fig. 8 (top right). Although exposure to epibatidine in the presence of DHβE resulted in marked stimulation of⁸⁶Rb⁺ efflux for both wild type and heterozygote mice, the response observed for homozygote mutants was substantially reduced. The effect of genotype on the DHβE-resistant response is summarized in Fig. 8 (bottom right). The average signal observed for β2 heterozygote mice was 29% lower than that measured for wild type mice, a significant decrease in response. DHβE-resistant ⁸⁶Rb⁺efflux measured in the homozygote mutant mice was 4% of that measured for the homozygote wild type mice and was not significantly different from zero. Therefore, the β2 subunit is required for most, if not all, of the receptors that mediate both DHβE-sensitive and DHβE-resistant components of nAChR-mediated⁸⁶Rb⁺ efflux.

Distribution of DHβE-Sensitive and DHβE-Resistant⁸⁶Rb⁺ Efflux in Mouse Brain.

The experiments described above were performed using crude synaptosomes prepared from whole mouse forebrain. To determine the distribution of DHβE-sensitive and DHβE-resistant nicotinic responses throughout the brain, ⁸⁶Rb⁺ efflux stimulated by 10 μM nicotine or 10 μM epibatidine in the presence of 2 μM DHβE was measured in crude synaptosomes prepared from 15 brain areas. The results of these experiments are shown in Fig.9.

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Figure 9

DHβE-sensitive and DHβE-resistant⁸⁶Rb⁺ efflux in 15 brain regions. Crude synaptosomes were prepared from the following brain regions: OB, OT, Cx, Se, Hp, St, Hab, Th, HT, IPN, MB, SC, IC, HB, Cb.⁸⁶Rb⁺ efflux was stimulated by a 5-s exposure to 10 μM nicotine (top left) or 10 μM epibatidine plus 2 μM DHβE (bottom left). DHβE was present before, during, and after stimulation with DHβE. Values represent mean ± S.E.M. of four separate experiments. The magnitude of the DHβE-resistant response is compared to the magnitude of the DHβE-sensitive response in the right panel.

The amount of ⁸⁶Rb⁺ efflux stimulated by 10 μM nicotine (DHβE-sensitive) varied substantially among brain regions (Fig. 9, top left). The least efflux was observed in Cb. Low levels were also observed in OB and Se. The largest nicotine-stimulated efflux was observed in Th. High levels were also measured for IPN and Hab.

The amount of ⁸⁶Rb⁺ efflux stimulated by 10 μM epibatidine in the presence 2 μM DHβE (DHβE-resistant) also varied substantially among brain regions (Fig.9, bottom left). The lowest DHβE-resistant efflux was measured in Cb and Se, whereas the greatest DHβE-resistant response was measured in SC and Th.

The regional distribution of the DHβE-sensitive⁸⁶Rb⁺ efflux was compared to that for the DHβE-resistant response as shown in Fig. 9 (right). The efflux stimulated by 10 μM nicotine is significantly correlated to that stimulated by 10 μM epibatidine plus 2 μM DHβE (r = 0.86, df = 13, p < .05) indicating that regional distribution of these two responses is similar. However, the relative amount of DHβE-resistant efflux measured in SC, IC, MB, and HB is substantially greater than that predicted from the amount of DHβE-sensitive efflux observed in these regions.

Comparison of nAChR-Mediated ⁸⁶Rb⁺ Efflux to Nicotinic Binding Sites.

Because the pharmacological properties, and to a lesser extent the regional distribution, of the DHβE-sensitive response differ from those of the DHβE-resistant response, a comparison of the distribution of the functional responses to that of nicotinic binding sites may indicate a relationship between binding and function. Several nicotinic binding sites can be measured using radioligand binding assays. High-affinity [³H]nicotine binding and α-Bgt binding have been used to measure two distinct binding sites (Marks et al., 1986). Recently the binding of [³H]epibatidine has been characterized (Houghtling et al., 1995). This ligand measures several nicotinic binding sites: a high-affinity site sensitive to inhibition by cytisine (identical with the high-affinity agonist site), a high-affinity site relatively resistant to inhibition by cytisine and that may include more than one site, and a low-affinity site (Houghtling et al., 1995; Marks et al., 1998; Zoli et al., 1998).

The properties of [³H]epibatidine binding to whole mouse brain are illustrated in Fig.10. The saturation curve for [³H]epibatidine binding is shown in Fig. 10(top). The binding isotherm deviates from that of a single site, as illustrated by the biphasic Scatchard plot shown in the inset to this panel. The binding constants for the two components as estimated from nonlinear least-squares fitting of the primary data wereK _D of 0.020 ± 0.004 nM andB _max of 98 ± 4 fmol/mg protein for the high-affinity site and K _D of 6.4 ± 2.8 nM and B _max of 65 ± 7 fmol/mg protein for the low-affinity site. The inhibition of [³H]epibatidine binding at two ligand concentrations by cytisine and dTC was used to further evaluate the properties of the sites. Fig. 10 (bottom left) displays the results for cytisine inhibition. At a [³H]epibatidine concentration of 0.44 nM, when the high-affinity site is fully saturated with little binding at the low-affinity site, inhibition by cytisine is biphasic. Approximately 85% of the binding was sensitive to cytisine (IC₅₀ = 6.7 nM, estimatedK _I = .26 nM) with the remaining 15% of the binding relatively resistant to cytisine (IC₅₀ = 340 nM, estimatedK _I = 13 nM). At a [³H]epibatidine concentration of 12.5 nM, where both high- and low-affinity binding occurs, the pattern of cytisine inhibition was not markedly different from that observed at 0.44 nM. Inhibition of the low-affinity site was estimated and is illustrated in the inset to the bottom left panel. The binding parameters estimated were an IC₅₀ value of 1.8 ± 0.3 (estimatedK _I = .59 μM) with a initial binding of 49 ± 2 fmol/mg protein. The similarity of the ratios of cytisine K _I values for the high- and low-affinity sites (∼15- and ∼95-fold for the high- and low-affinity [³H]epibatidine binding sites, respectively) underlies the inability of cytisine inhibition to distinguish these sites. Fig. 10 (bottom right) demonstrates the inhibition of [³H]epibatidine binding by dTC. With a [³H]epibatidine concentration of 0.44 nM, inhibition by dTC is biphasic, similar to the biphasic inhibition obtained with cytisine. Approximately 87% of the sites were sensitive to inhibition by dTC (IC₅₀ = 49 ± 10 μM, estimated K _I = 2.2 μM), whereas the remaining 13% of the sites were less sensitive (IC₅₀ = 1500 ± 1400 μM, estimatedK _I = 64 μM). With 12.5 nM [³H]epibatidine, the inhibition by dTC was substantially different than that by cytisine in that approximately one-third of the binding was inhibited by 100 μM dTC. This fraction of the inhibition corresponded to the low-affinity binding as illustrated in the inset to the bottom right panel. The binding site parameters estimated were an IC₅₀ of 12.2 ± 2.4 μM (estimated K _I = 4.1 μM) with a binding site density of 53 ± 2 fmol/mg. This low-affinity [³H]epibatidine binding component was similar to that calculated with cytisine inhibition. The ability of dTC to selectively inhibit low-affinity [³H]epibatidine binding was made possible because theK _I/K _Dratio was about 100,000 for the high-affinity site compared with about 650 for the low-affinity site, and theK _I values were comparable (2.1 and 4.1 μM, respectively). Thus, low-affinity [³H]epibatidine binding sites were estimated by selective dTC inhibition at a high ligand concentration (about 10 nM), and cytisine-resistant sites were estimated by selective cytisine inhibition at a low ligand concentration (about 0.5 nM).

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Figure 10

[³H]Epibatidine binding and inhibition by cytisine and dTC. The main top panel illustrates a representative saturation curve for the binding of [³H]epibatidine to whole mouse brain particulate fraction. Actual data are represented by the points (●). The solid curve is the least-squares fit of the data to a two-site model and the dotted curve is the theoretical binding to the high-affinity site. The inset to this panel is the Scatchard plot of these data. The main panel at the bottom left displays the inhibition of [³H]epibatidine binding by cytisine and the main panel at the bottom right displays the inhibition of binding by dTC at 0.44 nM (○) or 12.5 nM (●) [³H]epibatidine. The solid curve at 0.44 nM [³H]epibatidine is the least-squares two-site fit of the data, whereas the dotted curve presents the theoretical low-affinity binding site. The dotted curve at 12.5 nM is the least-squares two-site fit of these data. The inset to this panel is the calculated inhibition profile for the low-affinity binding site calculated as described in Experimental Procedures. The curve is the theoretical one-site fit of these data.

[³H]Nicotine, α-Bgt, and three [³H]epibatidine binding sites were measured in P2s prepared from fifteen regions of mouse brain for comparison to the amount of DHβE-sensitive and DHβE-resistant⁸⁶Rb⁺ efflux measured in these regions (Fig. 11). Correlational analyses indicate that neither cytisine-resistant high-affinity epibatidine binding nor α-Bgt binding have regional distributions similar to those for the two functional responses. However, significant correlations between functional response and high-affinity nicotine binding as well as between functional response and low-affinity epibatidine binding were observed. The two highest correlation coefficients were observed for nicotine binding and DHβE-sensitive⁸⁶Rb⁺ efflux (r = 0.94) and for low-affinity epibatidine binding and DHβE-resistant ⁸⁶Rb⁺efflux (r = 0.94). The correlations between nicotine binding and DHβE-resistant⁸⁶Rb⁺ efflux and low-affinity epibatidine binding and DHβE-sensitive⁸⁶Rb⁺ efflux were also significant (r = 0.87 and r = 0.85, respectively).

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Figure 11

Comparison of regional distribution of DHβE-sensitive or DHβE-resistant ⁸⁶Rb⁺efflux to the regional distribution of nicotinic binding sites.⁸⁶Rb⁺ efflux stimulated by 10 μM nicotine (top) or by 10 μM epibatidine plus 2 μM DHβE (bottom) in 15 brain regions is compared to the density of nicotinic binding sites measured with 17 nM [³H]nicotine (Nicotine Binding), 0.36 nM [³H]epibatidine binding in the presence of 50 nM cytisine (Cytisine-Resistant High-Affinity Epibatidine Binding), as the difference between binding of 10 nM [³H]epibatidine in the absence or presence of 300 μM dTC (Low-Affinity Epibatidine Binding), or with 2 nM α-Bgt (α-Bungarotoxin Binding). Each point represents the mean ± S.E.M. of the efflux or binding in each of 15 brain regions. Correlation coefficients are displayed in each panel.

Discussion

Two pharmacologically distinct components of nicotinic agonist-stimulated ⁸⁶Rb⁺efflux from mouse brain synaptosomes were analyzed using an online continuous flow radioactivity monitor. Differential sensitivity to inhibition by the nicotinic antagonist DHβE was exploited to study the pharmacology and regional distribution of these responses. The efflux inhibited by low concentrations of DHβE displayed a higher affinity for nicotinic agonists than did the response that was less sensitive to DHβE inhibition. Both the DHβE-sensitive and DHβE-resistant components were reduced more than 95% in β2 null mutant mice, revealing an absolute requirement for this subunit. The DHβE-sensitive and DHβE-resistant responses were widely distributed throughout the brain with similar, but not identical, regional distribution. Thus, a novel DHβE-resistant functional nicotinic response is widely distributed in mouse brain. This response is robust and pharmacologically distinct from those characterized previously. Whether this response is mediated by a single receptor subtype remains to be determined.

Differential inhibition by DHβE was used to measure the two pharmacologically distinct nicotinic responses. Because DHβE is a competitive antagonist, the DHβE-resistant response may have occurred because inhibition was overcome at high agonist concentrations. To reduce agonist-dependent changes in IC₅₀ values and subsequent changes in the amount of inhibition, samples were treated with DHβE before stimulation. Therefore, reversal of blockade requires dissociation of bound DHβE. Although dissociation is relatively rapid (k = 0.02 sec^-1), little reversal (about 5%) of inhibition would occur during the 5-s stimulation. Thus, under the conditions used in these experiments, differential inhibition by DHβE appears to be a valid experimental approach to resolve different nicotinic responses.

Differential sensitivity of nicotinic agonist-stimulated responses to inhibition by DHβE is not a unique observation. Pharmacologically distinct nicotinic responses in hippocampal cell cultures (Alkondon and Albuquerque, 1993) and in neurons isolated from the IPN compared to neurons isolated from medial habenula (Mulle et al., 1991) displayed differential responses to DHβE inhibition. Indeed, differential DHβE sensitivity was used to investigate nAChR subtypes in hippocampal cultures (Alkondon and Albuquerque, 1995) in a manner analogous to that used in the current study. Biochemical assays of nicotinic receptor function also exhibit differential sensitivity to inhibition by DHβE. For example, nicotine-stimulated dopamine release is relatively more sensitive to inhibition by DHβE (Grady et al., 1992; Sacaan et al., 1995; Clarke and Reuben, 1996), than is nicotine-stimulated norepinephrine release (Sacaan et al., 1995; Clarke and Reuben, 1996). Defined nicotinic receptor subtypes expressed in Xenopus oocytes are differentially affected by DHβE (Harvey et al., 1996; Chavez-Noriega et al., 1997). Structural determinants for differential DHβE sensitivity are present in both α (Harvey et al., 1996) and β subunits (Harvey and Leutje, 1996).

In addition to DHβE, two antagonists, MLA and hexamethonium, differed markedly in their inhibitory potency. The selectivity demonstrated by MLA was comparable to that of DHβE. In contrast, hexamethonium was a more potent inhibitor of the DHβE-resistant response. Three other antagonists displayed similar inhibition of the DHβE-sensitive and DHβE-resistant components. Potency and efficacy for both DHβE-sensitive and DHβE-resistant⁸⁶Rb⁺ efflux differed among nicotinic agonists, but every agonist tested was more potent for the DHβE-sensitive component. Differential effects of antagonists and agonists on DHβE-sensitive and DHβE-resistant⁸⁶Rb⁺ efflux indicate the existence of at least two receptors.

Determining the molecular compositions of the nAChR subtypes that mediate the DHβE-sensitive and DHβE-resistant responses is important. Both DHβE-sensitive and DHβE-resistant⁸⁶Rb⁺ efflux were reduced more than 95% in β2-null mutant mice (Picciotto et al., 1995). Thus, both responses are nicotinic and the β2 subunit is an essential component of the nAChR mediating these responses. The identity of the α subunits remains to be unequivocally established.

In the absence of definitive assignment of subunit composition beyond the absolute requirement for β2, pharmacological and physiological data provide information about the potential subunit identity. Differences in sensitivity to agonist stimulation have been observed for other nicotinic receptors and these differences have been used to investigate potential molecular composition of the receptors. Four classical nicotinic agonists (ACh, (−)-nicotine, cytisine, and DMPP) show distinct differences in the activation of heterologously expressed receptors of defined composition (Leutje and Patrick, 1991;Chavez-Noriega et al., 1997). DHβE-sensitive⁸⁶Rb⁺ efflux stimulated by these agonists most closely resembles the responses observed for the α4β2 nAChR. Several other agonists that were evaluated in the current study have also been measured in systems of defined receptor composition. The relative potency and efficacy of epibatidine (Gerzanich et al., 1995; Gopalakrishnan et al., 1996), ABT-418 (Gopalakrishnan et al., 1996), and A-85380 (Sullivan et al., 1996) measured for α4β2 nAChR are comparable to those determined for DHβE-sensitive ⁸⁶Rb⁺efflux. Furthermore, the potencies and relative efficacies of ACh, anatoxin-a, cytisine, (+)-epibatidine, (−)-epibatidine, (−)-nicotine, and DMPP, for which both the Type II responses of hippocampal cells (Alkondon and Albuquerque, 1995; Zoli et al., 1998) and DHβE-sensitive ⁸⁶Rb⁺efflux were determined, are comparable. The Type II response most probably is mediated by α4β2 the nAChR (Alkondon et al., 1994). Finally, the close correlation between the regional distribution of high-affinity nicotine binding and DHβE-sensitive⁸⁶Rb⁺ efflux reported here and previously (Marks et al., 1993, 1996) suggests that this component is an α4β2 nAChR because high-affinity agonist binding measures primarily, if not exclusively, this subtype (Whiting and Lindstrom, 1988; Flores et al., 1992). Thus, the receptor mediating DHβE-sensitive ⁸⁶Rb⁺efflux in the majority of brain regions is likely to be the α4β2 subtype, perhaps with other subunits contributing in certain brain regions.

The possible molecular structure(s) of the DHβE-resistant component is less clear. Results with null mutant mice demonstrate that the β2 subunit is required. The broad distribution of the DHβE-resistant response makes it unlikely to be mediated by receptors containing either α2 or α3 nAChR subunits because the mRNA encoding these subunits has restricted expression in rat and mouse brain (Wada et al., 1989; Marks et al., 1992). Furthermore, the poor correlation between DHβE-resistant ⁸⁶Rb⁺efflux and cytisine-resistant high-affinity epibatidine binding, which may measure α3 nAChR (Marks et al., 1998; Zoli et al., 1998), suggests no relationship between the functional response and this binding component. The failure of α-Bgt or low concentrations of MLA to measurably inhibit either the DHβE-sensitive or DHβE-resistant responses indicates that α7-containing nAChRs are not involved. Considering the rapid desensitization and rundown that is displayed by α7 nAChRs, the experimental conditions used in these studies may not be well suited to measure these receptors.

The DHβE-resistant response may be mediated by a receptor with an as yet unidentified α subunit. Because subunit composition profoundly affects nAChR pharmacology and physiology (Luetje and Patrick, 1991;Chavez-Noriega et al., 1997), expression of an nAChR with an α subunit other than α4 could produce the DHβE-resistant response. If such a subunit exists, it must be widely distributed, inasmuch as the DHβE-resistant response is found throughout the brain. Receptors assembled from more than two different subunits also can display distinct pharmacology. For example, when the α5 subunit coassembles with other nAChR subunits, properties of the resulting receptor are altered (Ramirez-Latorre et al., 1996; Wang et al., 1996; Conroy and Berg, 1998). Although receptors containing α5 subunits are known, this subunit may be too sparsely expressed in rat and mouse brain to account for all of the DHβE-resistant response (Wada et al., 1990; Marks et al., 1992). The DHβE-resistant response may also be mediated by an α4β2 receptor with a subunit stoichiometry other than (α4)₂(β2)₃, a receptor that exists in an alternative conformational state, or a receptor that has undergone differential post-translational processing. The response might also be mediated by receptor subunits containing alternatively spliced subunits. Splice variants at the C terminus of rat α4 subunit are known (Goldman et al., 1987; Yu et al., 1996), but the properties of the subsequent nAChR formed from these variants do not differ. However, changes in the amino acids in the ligand binding domain can substantially change agonist (Corringer et al., 1998) or antagonist (Harvey et al., 1996) affinity. Splice variants with changes at or near the ligand binding sites could conceivably alter both agonist and antagonist affinity to produce the DHβE-resistant receptors.

Whatever the molecular identity of the DHβE-resistant response, it is likely to serve an important functional role. This response is expressed throughout the brain, and, if the in vitro measurements are any indication, would produce rapid, large responses. Although higher concentrations of ACh or nicotine are required to activate the DHβE-resistant response than the DHβE-sensitive response, the potency of these agonists for the DHβE-resistant response is comparable to that observed for many nAChR subtypes (Couturier et al., 1990; Gross et al., 1991; Seguela et al., 1993; Alkondon and Albuquerque, 1993; Gerzanich et al., 1995; Chavez-Noriega et al., 1997).

In summary, the experiments presented in this paper describe two components of nicotinic receptor-mediated⁸⁶Rb⁺ efflux that are of similar magnitude but are differentially sensitive to inhibition to DHβE, and are predominantly, if not exclusively, composed of β2 containing nAChR. The DHβE-sensitive component appears to be identical with one described previously. The DHβE-resistant component represents a previously undescribed, but ubiquitous and robust nAChR-mediated response that may be of significant functional consequence.