Introduction

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In the nervous system, nAChRs are ligand-gated cation channels composed of pentameric combinations of 11 subunits (2-9; 2-4) (Sargent, 1993; Lukas et al., 1999). The specific subunit composition yields receptors with pharmacological and electrophysiological properties that are unique to that subunit combination (Luetje and Patrick, 1991; Gerzanich et al., 1998). For example, antagonists can discriminate among nAChRs with different subunit compositions. -Methyllycaconitine and -bungarotoxin (-BGT) are potent and selective antagonists of 7 subunit-containing nAChRs (Couturier et al., 1990; Ward et al., 1990). Receptors composed of the 42 or 32 subunit combinations are more sensitive to block by dihydro--erythroidine (DHE) than receptors composed of other subunit pairs. Alternatively, mecamylamine has high affinity for nAChRs composed of 34 subunits (Albuquerque et al., 1997).

There are two main classes of neuron in the enteric nervous system: S neurons and AH neurons. S neurons are enteric interneurons or motor neurons (Kunze and Furness, 1999). S neurons receive fast excitatory synaptic input mediated partly by acetylcholine acting at nAChRs (Galligan and Bertrand, 1994). Studies of the pharmacological properties of nAChRs in the enteric nervous system would provide information about the mechanisms of synaptic excitation of enteric motor neurons and interneurons. AH neurons are enteric sensory neurons (Furness et al., 1998). The action potential in AH neurons is followed by a long-lasting afterhyperpolarization, and AH neurons contain the calcium binding protein calbindin (Furness et al., 1998). Although most AH neurons do not receive fast excitatory synaptic input, they express functional nAChRs because exogenously applied nAChR agonists depolarize AH neurons (Schneider and Galligan, 2000). Detailed studies of the properties of enteric nAChRs may help to identify the role of these receptors on enteric sensory neurons.

Recent immunohistochemical studies using antibodies that recognize 3, 5, 7, and 2 subunits have localized nAChR subunit immunoreactivity (ir) to nerve cell bodies, dendrites, and nerve terminals in enteric ganglia in acutely isolated myenteric plexus preparations (Kirchgessner and Liu, 1998). It is not known which of these subunits coassemble to form the functional receptors. The purpose of the present study was to characterize the pharmacological and electrophysiological properties of nAChRs in myenteric neurons in an effort to identify the nAChR subunit composition. Determining the subunit composition of nAChRs will help to understand the physiological role played by these receptors and to develop new drugs that may target particular nAChRs in the gut.

	Methods and Materials

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Tissue Culture. Myenteric neurons were cultured as described previously (Zhou and Galligan, 1996). Two newborn guinea pigs (1-2 days old) were sacrificed by severing the major neck blood vessels after halothane anesthesia. These procedures were approved by the All University Committee on Animal Use and Care at Michigan State University. The small intestine was placed in cold (4°C) sterile-filtered Krebs' solution of the following composition 117 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgCl₂, 1.2 mM NaH₂PO₄, 25 mM NaHCO₃, and 11 mM glucose. The longitudinal muscle with attached myenteric plexus was stripped free using a cotton swab and cut into 5-mm-long segments. Tissues were divided into four aliquots, and each aliquot was transferred to 1 ml of Krebs' solution containing 1600 U of trypsin for 25 to 30 min at 37°C. The tissues were triturated and then centrifuged at 900g for 5 min using a benchtop centrifuge. The supernatant was discarded, and the pellet was resuspended and incubated (30 min at 37°C) in Krebs' solution containing 2000 U of crab hepatopancreas collagenase. The suspension was triturated and then centrifuged for 5 min. The pellet was resuspended in Eagle's minimum essential medium containing 10% fetal bovine serum, gentamicin (10 µg ml¹), penicillin (100 units ml¹), and streptomycin (50 µg ml¹). Cells were plated on plastic dishes coated with (poly)L-lysine and maintained in an incubator at 37°C with an atmosphere containing 5% CO₂ for up to 2 weeks. After 2 days in culture, 10 µM cytosine arabinoside was added to the Eagle's minimum essential medium to limit smooth muscle and fibroblast proliferation. Medium was changed twice weekly.

Whole-Cell Recording Technique. Patch-clamp recordings were carried out at room temperature with 3- to 5-M patch pipettes; seal resistances were greater than 5 G. The composition of the patch pipette solution was 160 mM CsCl, 2 mM MgCl₂, 1 mM EGTA, 10 mM HEPES, 1 mM ATP, and 0.25 mM GTP; the pH and osmolarity were adjusted to 7.4 (using CsOH) and 320 mOsM l¹ (using CsCl), respectively. Series resistance (up to 80%) and capacitative currents were electronically compensated. All recordings were made using an Axopatch 200A amplifier (Axon Instruments, Union City, CA). Data were acquired using Axotape 2.0 and pClamp 6.0 software (Axon Instruments). The currents were sampled at 2 kHz and were filtered at 1 kHz (four-pole Bessel filter; Warner Instruments, New Haven, CT) and stored on the computer hard drive.

Studies in Intact Myenteric Plexus. Longitudinal muscle myenteric plexus preparations were made from the ileum of adult guinea pigs (325-400 g; Bioport, Lansing, MI) or 1- to 2-day-old guinea pigs (70-90 g; Bioport). A segment of ileum taken 10 cm proximal to the ileal-cecal junction was removed and placed in oxygenated (95% O₂, 5% CO₂) Krebs' solution of the following composition: 117 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgCl₂, 25 mM NaHCO₃, 1.2 mM NaH₂PO₄, and 11 mM glucose. The Krebs' solution contained nifedipine (1 µM) and scopolamine (1 µM) to block contractions of the muscle layers during intracellular recordings. A segment of ileum (5 cm) was cut open along the mesenteric attachment and pinned flat in a silastic elastomer-lined Petri dish. The mucosa and submucosa were carefully peeled away and a 5-mm² section of longitudinal muscle myenteric plexus was cut out using fine scissors and forceps. The longitudinal muscle myenteric plexus preparation was transferred to a silastic elastomer-lined recording chamber and pinned flat. The chamber was then mounted on the stage of an inverted microscope (CK-2; Olympus, Tokyo, Japan), and the chamber was superfused continuously with warm (36°C) Krebs' solution at a flow rate of 3.5 ml/min. Neurons were impaled with 2 M KCl-containing electrodes (tip resistance 80-100 M), and membrane potential was recorded using an Axoclamp 2A amplifier (Axon Instruments).

Drug Application. Drugs were applied in two ways. When constructing steady-state concentration-response curves, external drug application was delivered by gravity flow from a linear array of quartz tubes (320-µm i.d. and 450-µm o.d.; Polymicron Technologies, Phoenix, AZ). The distance from the mouth of the tubes to the cells was ~200 µm with flow controlled manually using a micromanipulator. In experiments requiring precise timing of the onset and offset of drug application, computer-controlled solenoid valves (General Valve, Fairfield, NJ) were used to gate solution flow through the tubes. Our previous work showed that the rise time of drug concentration near the neurons is <50 ms (Zhou and Galligan, 1996). When using conventional intracellular methods to record from neurons in the intact plexus, nicotine or cytisine was applied from a fine-tipped (<10 µm) pipette positioned near the neuron. The concentration of drug in the pipette was 1 mM and was ejected from the pipette using a brief nitrogen pulse (10 psi) controlled by a Picospritzer (General Valve).

Immunohistochemistry. The medium in culture dishes was replaced with 3 ml of Zamboni's fixative [2% (v/v) formaldehyde and 0.2% (v/v) picric acid in 0.1 M sodium phosphate buffer, pH 7.0], and cells were fixed overnight at 4°C. The fixative was cleared using three washes of dimethyl sulfoxide at 10-min intervals. Cells were then washed three times with phosphate-buffered saline (PBS) (0.01 M; pH 7.2) at 10-min intervals and subsequently incubated overnight with primary antibody at room temperature. The primary antibodies, target antigens, host species, and working dilutions are listed in Table 1. In some experiments, antibodies raised against the 3 and 5 subunits were preincubated with the antigen peptide provided by the commercial supplier (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). For these experiments, diluted antibody was incubated with 10 µg of the antigen peptide for 2 h at room temperature. The antigen peptide preincubated antibody was then applied to preparations as described above. After primary antibody incubation, cells were washed three times at 10-min intervals with PBS. Cells were then incubated (1 h at 23°C) with sheep anti-mouse IgG (1:40 dilution in PBS; Jackson Immunoresearch Laboratories, West Grove, PA) conjugated to fluorescein isothiocyanate to reveal calbindin-ir, donkey anti-goat IgG (1:200 dilution in PBS; Jackson Immunoresearch Laboratories) conjugated to Cy3 to reveal 3 or 5 subunit-ir, goat anti-rat IgG (1:200 dilution in PBS; Jackson Immunoresearch Laboratories) conjugated to Cy3 to reveal mAb35-ir (3 and 5 subunits) and mAb270-ir (2 subunits) or goat anti-rabbit IgG (1:200 dilution in PBS; Jackson Immunoresearch Laboratories) conjugated to Cy3 to reveal 7 subunit-ir. Cells were then washed with three times with PBS and mounted in buffered glycerol for fluorescence microscopy using a LabroluxS upright microscope and a PL Fluotar 40 × 0.7 numerical aperture objective (Leica, Wetzlar, Germany). When using this microscope, digital images were obtained using a SPOT-2 cooled charge-coupled device color camera (Diagnostic Instruments, Sterling Heights, MI). Some images were obtained using Leica TCS-SL confocal scanning system (Leica Microsystems, Heidelberg, Germany) and a DMLFSA upright microscope with a HCX PL APO 63 × 1.32 numerical aperture oil immersion objective (Leica). Confocal image depth was 1.1 µm, and four images were averaged to obtain a single image for presentation. All images were then processed using Adobe Photoshop 5.5 (Adobe Systems, Mountain View, CA) and Powerpoint 7.0 (Microsoft, Redmond, WA) software.

Drugs. Crab hepatopancreas collagenase was obtained from Calbiochem-Novabiochem (La Jolla, CA). All other drugs and reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Statistics. Data are expressed as the mean ± S.E.M. Agonist concentration-response curves obtained from individual neurons were fit using the Hill equation as follows.
where E_max is the maximum response, EC₅₀ is the half-maximal effective concentration, n is the slope factor, and x is the ligand concentration. Mean values for each parameter obtained from a number of neurons in different treatment groups were compared using Student's t test. For electrophysiological studies, n values refer to the number of neurons from which data were obtained. For data from the immunohistochemical studies, n values refer to the number of separate batches of neuronal cultures on which observations were made; two guinea pigs provided neurons for each batch of cultures.

	Results

Top Abstract Introduction Methods and Materials Results Discussion References

Agonist Concentration-Response Relationships. ACh (1 mM) caused an inward current in every neuron tested (holding potential of 60 mV). The amplitude of currents activated by ACh remained stable during repeated ACh application for up to 60 min after whole-cell formation when recordings were obtained using an ATP/GTP-free pipette solution. At 2 min after establishing whole-cell recording conditions, the mean ACh current amplitude was 2.5 ± 0.6 nA, whereas at 60 min the mean current amplitude was 2.4 ± 0.6 nA (n = 4; P > 0.05).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Inward currents caused by nAChR agonists. A, representative traces of inward currents caused by maximum concentrations of ACh, nicotine, and DMPP. B, concentration-response curves for inward currents caused by four nAChR agonists. Data were expressed as a percentage of the current amplitude caused by ACh (1 mM) in each neuron. Points are mean ± S.E.M. response amplitude from the indicated number (n) of neurons for each curve. Curves were fitted to the data points using the Hill equation.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   DMPP is a partial agonist at myenteric nAChRs. Concentration-response curves for ACh-induced inward currents obtained in the absence and presence of a subthreshold concentration (10 µM) of DMPP. DMPP caused a rightward shift in the ACh concentration-response curve without decreasing the maximum response. Data are expressed as a percentage of the response caused by 1 mM ACh in each neuron tested. Points are the mean ± S.E.M. of the data obtained from the indicated number of neurons.

Effects of Antagonists. Because nAChR subtypes can be differentiated on the basis of their sensitivity to block by nAChR antagonists, inhibition curves for hexamethonium, mecamylamine, and DHE against responses caused by ACh (1 mM) were obtained. In these experiments, neurons were pretreated with each antagonist for 5 min, and responses to repeated applications of ACh in the presence of increasing concentration of each antagonist were obtained. The data summarized in Fig. 3 show antagonist inhibition curves where responses were normalized to the peak current induced by ACh alone. Mecamylamine was the most potent antagonist; the mecamylamine IC₅₀ value (micromolar) was 0.1 ± 0.04 (n = 6). The IC₅₀ (micromolar) value for hexamethonium was 1.6 ± 0.4 (n = 6). Full concentration-response curves for DHE were not obtained because concentrations higher than 300 µM were not tested. However, at 30 µM, DHE reduced the ACh response by 50 ± 4% (n = 5).

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Antagonist inhibition of ACh-induced inward currents. Mecamylamine, hexamethonium, and DHE produce concentration-dependent inhibition of responses caused by 1 mM ACh. Data are expressed as a percentage of inhibition of the ACh response caused by each concentration of antagonist. Points are the mean ± S.E.M. of data obtained from the indicated number of neurons.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Inward currents caused by ACh or nicotine are not blocked by -bungarotoxin or MLA. A, ACh concentration-response curves obtained from untreated neurons and from neurons that had been preincubated 1 to 2 h with media containing -bungarotoxin. Data are peak current amplitudes and are the mean ± S.E.M. of the indicated number of neurons. There were no differences in these curves. B, nicotine concentration-response curves obtained from untreated neurons and from neurons that had been preincubated 1 to 2 h with media containing -bungarotoxin. Data are peak current amplitudes and are the mean ± S.E.M. of the indicated number of neurons. There were no differences in these curves. C, representative traces of currents caused by ACh before, during, and after treatment with MLA. D, pooled data from experiment shown in B. Data are the mean ± S.E.M. (n = 9) of the peak ACh current amplitude. MLA did not affect ACh responses.

Current-Voltage Relationship of ACh-Induced Current. Fig. 5 shows the inwardly rectifying I-V relationship for responses caused by ACh (300 µM). The slope conductances measured at 50 and 50 mV were 21 ± 3 and 0.7 ± 0.3 ns, respectively (G₅₀/G₊₅₀ = 66 ± 19; n = 10). The reversal potential of ACh-induced current was approximately 0 mV in the normal Krebs' solution (n = 18), and this was shifted by 20 mV when the external Na⁺ concentration was reduced by 50% (n = 3).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Current-voltage relationship for inward currents caused by ACh. A, ACh-induced currents recorded from one myenteric neuron at holding potentials ranging between 110 and 50 mV. B, mean current-voltage curve for ACh-induced currents. Current-voltage relationship exhibits marked inward rectification. Data points are the mean ± S.E.M. current amplitude recorded at each membrane potential. Data were obtained from 10 neurons.

Kinetic Properties of Currents Induced by Nicotinic Agonists. At 60 mV, the 10 to 90% rise times for ACh (1 mM)- and nicotine (1 mM)-induced currents were 79 ± 6 and 76 ± 7 ms, respectively. Currents caused by nicotine decayed more rapidly than those caused by ACh but more slowly than those activated by DMPP. The increase in the rate of current decay was accompanied by a rebound inward current after nicotine and DMPP washout (Fig. 6, arrow). To analyze the contributions of open-channel block, the decay rate of currents caused by ACh, nicotine, and DMPP were compared at different membrane potentials (Fig. 6). The 10 to 90% decay time for ACh-induced currents was voltage-independent (4 ± 0.4 s at 100 mV, 4 ± 0.3 s at 50 mV; n = 16; P > 0.05), whereas those for nicotine and DMPP were voltage-dependent (Fig. 6). The ratio of 10 to 90% decay times measured at 50 and 110 mV was 1.6 for nicotine (3.6/2.2 s; n = 10; P < 0.05) and 2.8 for DMPP (1.5/0.54 s; n = 11; P < 0.05).

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Voltage dependence of decay of nAChR-mediated inward currents. A, representative traces of inward currents caused by ACh, nicotine, and DMPP recorded at holding potentials of 110 mV (traces labeled 1) and at 50 mV (traces labeled 2). Recordings obtained at 50 mV were scaled to the same size as the traces obtained at 110 mV to compare decay rates. B, histograms show the mean 10 to 90% decay times for currents caused by ACh, nicotine, and DMPP at the indicated membrane potentials. The decay rates for nicotine and DMPP-induced currents significantly faster at 110 mV compared with 50 mV.

Immunohistochemical Localization of nAChR Subunits in Myenteric Neurons. The monoclonal antibody mAb35 recognizes neuronal 3 and 5 nAChR subunits. mAb35-ir was found in nearly every myenteric neuron maintained in primary culture (n = 5) (Fig. 7A). Calbindin-ir was also localized to a subset of mAb35-ir neurons (n = 2) (Fig. 7B). Because mAb35 recognizes 3 and 5 subunits, antibodies that recognize 3 or 5 subunits were used to determine whether one or both of these subunits was expressed by myenteric neurons. These antibodies revealed that most neurons were immunoreactive for the 3 (n = 5) and 5 (n = 4) subunits (Fig. 7, B and C). Labeling revealed by these two antibodies was blocked when the antibodies were preincubated with the antigen peptide (n = 3) (data not shown). These data indicate that myenteric neurons express nAChRs that contain 3 and 5 subunits. A monoclonal antibody (mAb270) that recognizes the 2 subunit revealed that myenteric neurons also expressed nAChRs containing this subunit (n = 3) (Fig. 7D). Finally, an antibody that recognizes the 7 subunit revealed only a few faintly stained neurons and nerve fibers (n = 2) (Fig. 7E).

View larger version (103K): [in this window] [in a new window]   Fig. 7.   Immunohistochemical localization of ir for nAChR subunits and calbindin in myenteric neurons maintained primary culture. A and B, photomicrographs showing colocalization of mAb35-ir (A) and calbindin-ir (B). Most mAb35-ir neurons did not contain calbindin-ir. mAb35 localizes 3 and 5 nAChR subunits. Arrows indicate a single neuron labeled by both antibodies. C, localization of 5 subunit-ir in myenteric neurons. D, confocal image showing localization of 3 subunit-ir in myenteric neurons. E, photomicrograph showing localization of mAb270-ir in myenteric neurons. mAb270 localizes 2 subunits. F, confocal image showing faint labeling of two myenteric neurons with 7 subunit-ir. Scale bars, 35 µm.

Studies in Acutely Isolated Myenteric Plexus. The data described above for agonist effects on myenteric neurons maintained in primary culture show that cytisine was a weak agonist at nAChRs expressed by these neurons. However, previous work in the acutely isolated myenteric plexus from adult guinea pigs showed that cytisine was equieffective with nicotine at causing depolarizations of a subset of myenteric neurons (Schneider and Galligan, 2000). This apparent difference in results could be due to the use of tissue culture or whole-cell recording conditions or to developmental differences because the neurons used for primary culture studies were obtained from 1- to 2-day-old guinea pigs. This issue was addressed by directly comparing responses to cytisine and nicotine in neurons in acutely isolated myenteric plexus preparations from adult and newborn guinea pigs. Nicotine and cytisine (each at 1 mM) were applied by pressure ejection from a pipette positioned near the impaled neuron. Both agonists caused a rapid depolarization associated with a fall in input resistance in neurons from the adult and newborn myenteric plexus (Fig. 8A). Furthermore, response amplitudes were also similar for nicotine and cytisine in adult and newborn neurons (Fig. 8B). These data indicate that nAChRs expressed by adult and newborn guinea pig myenteric neurons are similar in their sensitivity to cytisine.

View larger version (37K): [in this window] [in a new window]   Fig. 8.   Cytisine and nicotine depolarize myenteric neurons in the acutely isolated myenteric plexus from newborn (1-2-day-old) and adult guinea pig small intestine. A, representative traces of depolarizations caused by nicotine or cytisine applied from a pipette positioned near the impaled neuron. Recordings were obtained using conventional intracellular microelectrodes. The pipette drug concentration was 1 mM. Downward deflections are voltage responses to 200-pA, 20-ms current pulses passed through the intracellular recording electrode. B, mean data from experiments illustrated in A. Data are mean + S.E.M. depolarization amplitude caused by nicotine or cytisine in myenteric neurons from newborn or adult small intestine; n is the number of neurons from which the data were obtained.

View larger version (24K): [in this window] [in a new window]   Fig. 9.   Antagonist concentration-response curves for inhibition of depolarizations caused by ionophoretically applied ACh (1 M) onto myenteric neurons in the myenteric plexus preparation from adult guinea pig ileum. Points are the mean ± S.E.M. inhibition of the ACh response caused by each concentration of antagonist. Data are percentage of inhibition of ACh responses obtained in each neuron before antagonist application; n values are the number of neurons from which data were obtained.

	Discussion

Top Abstract Introduction Methods and Materials Results Discussion References

Immuonohistochemical Studies. Previous studies of nAChR subunits in whole mounts of guinea pig gut showed that most enteric neurons were labeled by mAb35 (Kirchgessner and Liu, 1998; Obaid et al., 1999), an antibody that recognizes 3 and 5 subunits (Conroy et al., 1992; Conroy and Berg, 1995). Similarly, most myenteric neurons maintained in culture were labeled by mAb35 and some of these neurons contained calbindin. Calbindin is a marker for intrinsic sensory/AH type neurons (Furness et al., 1998) so most mAb35-labeled neurons were likely to be S-type neurons.

Electrophysiological Properties of Myenteric nAChRs. Whole-cell currents evoked by ACh were associated with a conductance increase, a reversal potential of 0 mV, and inward rectification. These properties are similar to those of nAChRs in other autonomic neurons (Mathie et al., 1990; Aibara and Akaike, 1991; Fieber and Adams, 1991). Although currents caused by nicotinic agonists desensitized, ACh produced stable responses if the period of agonist application was 2 s and the application interval was 2 min. Therefore, decay of whole-cell currents during agonist application is not time-dependent rundown. In addition, desensitization is the main mechanism for decay of ACh-induced currents, whereas desensitization and channel blockade contribute to the decay of currents activated by nicotine and DMPP. This latter conclusion is based on the voltage independence of the decay of ACh currents, whereas decay of nicotine and DMPP currents was faster at hyperpolarized potentials. Nicotine and DMPP are open channel blockers of nAChRs in myenteric neurons as in other neurons (Mathie et al., 1991; Maconochie and Knight, 1992).

Agonist Responses. The agonist rank order potency for myenteric neuronal nAChRs is DMPP > ACh = nicotine > cytisine. Because the agonist rank order potency for activation of homomeric 7 subunit nAChRs is nicotine = DMPP > cytisine > ACh (Couturier et al., 1990; Zhang et al., 1994), it is unlikely that myenteric neurons express these receptors on the cell body. Although DMPP was the most potent agonist, the Hill coefficient and E_max for DMPP were lower than those for ACh and nicotine. However, DMPP is a partial agonist at myenteric neuronal nAChRs and blockade of nAChRs is due partly to voltage-dependent channel block by DMPP. At high DMPP concentrations, the channel blocking effect would be most prominent accounting for the shallow slope of the concentration-response curve and decreased E_max. Therefore, the potency and efficacy of DMPP as an agonist at myenteric neuronal nAChRs was underestimated. Because DMPP is the most potent agonist at nAChRs composed of 32 or 34 subunits (Chavez-Noriega et al., 1997; Gerzanich et al., 1998), the data presented herein indicate that myenteric neurons express nAChRs composed of one or both of these subunit compositions.

Studies with Antagonists. -BGT and MLA block 7 subunit-containing AChRs (Couturier et al., 1990; Zhang et al., 1994), but nAChR-mediated responses in myenteric neurons were insensitive to these antagonists. Therefore, it is unlikely that guinea pig myenteric neurons express homomeric 7 subunit-containing nAChRs on the soma. Similar conclusions about the absence of somatodendritic 7 subunit-containing nAChRs in myenteric neurons have been reached by others (Töröscik et al., 1991; Barajas-Lopez et al., 2001).

Summary and Conclusions. These studies used immunohistochemical and electrophysiological approaches to identify the subunit composition of myenteric neuronal nAChRs. Immunohistochemical studies revealed the predominant presence of 3, 5, and 2 subunits. Although cytisine was a weak agonist at nAChRs expressed by myenteric neurons in culture, it was equieffective with nicotine at depolarizing neurons in the myenteric plexus. Furthermore, EC₅₀ values for ACh, nicotine, and DMPP obtained in neurons maintained in culture are similar to those for heterologously expressed 34 subunit-containing receptors. In addition, necamylamine was the most potent antagonist of myenteric neuronal nAChRs. These data indicate that myenteric neurons predominately express nAChRs composed of 3, 5, 2, and 4 subunits as occurs in a subset of nAChRs in chick ciliary ganglia (Conroy and Berg, 1995). These subunits may be in a homogenous population of receptors with unique pharmacological properties, or multiple receptors of different subunit composition may be expressed by individual neurons.