Introduction

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The nicotinic AChR is known to be the target of various chemicals including nicotine, carbachol, d-tubocurarine, general anesthetics and several natural toxins (Albuquerque et al., 1989; Swanson and Albuquerque, 1992; Papke and Oswald, 1989; Rozental et al., 1989; Dilger et al., 1995, Kofuji et al., 1990; Ishihara et al., 1995; Castro and Albuquerque, 1993, 1995). Recently, nitromethylene heterocyclic insecticides have also been shown to act on the nicotinic AChR. Several studies using binding techniques have demonstrated that nitromethylene heterocycles compete with nicotine and -bungarotoxin (Bai et al., 1991; Sattelle et al., 1989) and that they bind with a high affinity to the AChR of various species of insects (Liu and Casida, 1993). A member of this group of insecticides, imidacloprid (1-(6-chloro-3-pyridylmetyl)-2-nitroimino-imidazolidine) has a biphasic effect initially evoking stimulation followed by block of the neuronal activity of the American cockroach (Sone et al., 1994). Benson (1989) has shown that nitromethylene heterocycles suppressed ACh-induced currents as antagonists. However, other studies have shown that these compounds themselves generate currents through activation of the nicotinic AChR and exert antagonistic effects depending on the concentration and chemical structures (Cheung, et al., 1992; Bai et al., 1991; Sattelle et al., 1989; Benson, 1992; Leech et al., 1991; Zwart et al., 1992, 1994). Thus, a question arises as to whether these conflicting observations result from multiple effects of nitromethylene heterocycles at the channel level, or from different subtypes of nicotinic AChR of preparations.

Recently, we have demonstrated that imidacloprid potently induced subconductance state single-channel currents mediated by nicotinic AChR channels (Nagata and Narahashi, 1995; Nagata et al., 1996a). We now report the results of whole-cell and single-channel patch clamp experiments that have unveiled more detailed mechanisms of imidacloprid action on neuronal nicotinic AChR in PC12 cells. Imidacloprid suppressed the carbachol-induced whole-cell currents and generated currents at nicotinic AChR. Contrary to the main conductance state currents induced by ACh, imidacloprid almost exclusively induced the subconductance state currents. When imidacloprid was co-applied with ACh, the mean open time and burst duration of main conductance state currents were decreased compared with the control in which ACh was applied alone. These results help to explain the multiple effects of imidacloprid on ACh-induced whole-cell currents and some of the results reported previously.

	Materials and Methods

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Culture of PC12 cell line. The PC12 cell line was kindly provided by Drs. Edson X. Albuquerque and Edna F. R. Pereira of the University of Maryland School of Medicine, Baltimore, MD. Cells were cultured in Dulbecco's modified Eagles' medium containing fetal bovine serum (0.1 mg/ml, Sigma Chemical Co., St. Louis, MO) at 36°C in an air + CO₂ (90 + 10%, by volume). For patch clamp experiments, cells were plated on glass cover-slips coated with poly-L-lysine and cultured for 2 to 7 days. PC12 cells without nerve growth factor treatment expressed the nicotinic AChR.

Whole-cell current recording. Membrane currents were recorded using the whole-cell patch clamp technique (Hamill et al., 1981) at room temperature (22°C). Pipette electrodes were made from 0.8 mm (I.D.) borosilicate glass capillary tubes and fire-polished before use. The electrodes had resistances of 2 to 3 M when filled with standard pipette solution. The membrane potential was clamped at -90 mV, and a 5- to 10-min period was allowed after rupture of the membrane to equilibrate the cell interior with pipette solution. Currents through the electrode were recorded by an Axopatch 200A amplifier (Axon Instruments, Foster City, CA), filtered at 10 kHz, and stored on an LSI 11/73 computer (Digital Equipment, Pittsburgh, PA). The data were transferred to a microcomputer (PowerBook 520c, Apple Computer, Cupertino, CA) for further analysis. Currents were continuously monitored by a chart recorder.

Cell-attached single-channel current recording. Single-channel currents were recorded using the cell-attached variation of the patch clamp technique (Hamill et al., 1981) at room temperature (22°C). Pipette electrodes were made by the same method as described above, coated by SigmaCote (Sigma) to minimize the background noise, and fire-polished. The electrodes had resistances of 10 to 12 M when filled with standard pipette solution. The membrane was hyperpolarized to various potentials from the resting potential.

Solutions. The external bath solution for both whole-cell and cell-attached patch clamp experiments contained (in mM): NaCl 165, KCl 5, CaCl₂ 2 and N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid 5. Tetrodotoxin (1 µM) was also added to eliminate sodium channel currents. The pH was adjusted to 7.3 with NaOH, and the osmolarity was adjusted to 330 mOsm by D-glucose. The internal pipette solution for the whole-cell recording contained (in mM): CsCl 80, CsF 80, ethyleneglycol bis(-aminoethylether)-N, N,N',N'-tetraacetic acid 10, and N-2-hydroxyethylpiperazine-N' acid 10. The pH was adjusted to 7.3 with CsOH, and the osmolarity was adjusted to 330 mOsm by D-glucose.

Chemicals. Acetylcholine and carbachol were first dissolved in distilled water to make stock solutions. Imidacloprid was dissolved in dimethylsulfoxide. These stock solutions were then diluted with the internal pipette solution for the cell-attached and with the standard external solution for the whole-cell patch clamp experiments. The final concentrations of dimethylsulfoxide in test solutions were .3% (v/v) or less which had no effect on the activity of ACh- and carbacohol-induced currents.

Drug application. For whole-cell experiments, test solutions were applied to the cell using a locally developed application system (Nagata and Narahashi, 1994). The application was controlled by a computer-operated magnetic valve. Using this application system, the external solution surrounding the cell could be completely changed within 100 msec.

	Results

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Effects of imidacloprid on carbachol-induced currents. Imidacloprid suppressed currents evoked by carbachol. When carbachol was applied for 25 sec at a concentration of 100 µM, a transient inward current was generated and decayed to a very low level (fig. 1Aa). This concentration was somewhat less than the ED₅₀ value (fig. 2), and was chosen to be able to observe the effect of a test compound clearly. Imidacloprid (30 µM) coapplied with 100 µM carbachol suppressed the peak current amplitude (fig.1Ab) to 70% of the control, and the effect was completely reversible after washing with imidacloprid-free solution. Further experiments showed that imidacloprid suppressed the ACh-induced current in a dose-dependent manner (fig. 1B).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Effects of imidacloprid on the nicotinic ACh receptor in PC12 cells. A, Currents induced by 25-sec application of (a) 100 µM carbachol (solid bar), (b) 100 µM carbachol and 30 µM imidacloprid (broken bar) and (c) 30 µM imidacloprid. B, Currents induced by applications of 100 µM carbachol (control), co-application of 100 µM carbachol and various concentrations of imidacloprid and 100 µM carbachol after washing out imidacloprid. C. The dose-response relationship for imidacloprid suppression of currents induced by 100 µM carbachol. D, Structure of imidacloprid.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Carbachol and imidacloprid activation of currents in PC12 cells. A, Carbachol-induced currents. Currents were induced by 3-sec applications of carbachol to avoid desensitization. B, Dose-response relationships for carbachol-induced (closed circle) and imidacloprid-induced (open circle) currents. Current amplitudes were normalized to the current induced by 1 mM carbachol. Each point represents the mean ± S.D. (n = 4). The EC50 value of carbachol was estimated to be 116 µM and the Hill coefficient 1.0. Because the desensitization rate of current induced by carbachol was fast, the peak amplitude of current might have been attenuated somewhat resulting in inaccurate values for the EC50 and the Hill cofficient. For imidacloprid, currents were too small to estimate EC50 value and Hill coefficient accurately.

Imidacloprid-generated whole-cell currents. Imidacloprid generated currents when applied alone (fig. 1Ac). At a concentration of 30 µM, imidacloprid induced a small transient inward current that reached approximately 10% of the current produced by 100 µM carbachol. The peak amplitude of currents generated by 30 µM imidacloprid was 51.9 ± 8.4 pA (n = 5, mean ± S.D.). The dose-response relationship of imidacloprid-generated current is shown in figure 2B. The minimum effective concentration to induce currents was 1 µM and the currents reached maximum at 30 µM.

Single-channel currents induced by acetylcholine. Single-channel currents were recorded by the cell-attached patch clamp technique with the recording electrode containing 10 µM ACh (fig. 3A). The membrane was hyperpolarized by 40 mV from the resting potential which was close to -90 mV. The inward single-channel currents were generated by activation of the nicotinic AChR by ACh (Nagata et al., 1996a). In addition to currents that represented the main conductance state, there were small currents of a subconductance state (fig. 3A) and large currents of the supraconductance state (data not shown). The subconductance state currents occurred much less frequently than the main conductance state currents. The supraconductance state currents had brief open times (<1 msec), and have been observed in other preparations (Hamill and Sakmann, 1981; Brehm et al., 1984; Morris and Montpetit, 1986). However, this type of current was not analyzed in our study as brief open times prevented accurate measurements from being made. Main and subconductance state currents are clearly discernible. The amplitude of the main conductance state currents was 3.0 ± .25 pA (n = 6, mean ± S.D.) and that of the subconductance state currents was .85 ± .18 pA (n = 6).

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Single-channel currents activated by 10 µM ACh, 10 µM imidacloprid and co-application of 10 µM ACh and 10 µM imidacloprid to cell-attached membrane patches clamped at a membrane potential 40 mV more positive than the resting potential. A, Currents induced by 10 µM ACh occurred during brief isolated openings or longer openings interrupted by a few short closures or gaps. Main conductance state currents were observed more frequently than subconductance state currents. B, Currents induced by 10 µM imidacloprid. Subconductance state currents were more frequently observed than main conductance state currents. C, Co-application of 10 µM ACh and 10 µM imidacloprid. Main conductance and subconductance state currents were induced, and channel openings were shortened.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Single-channel current-voltage relationships of main conductance (closed circles) and subconductance states (open circles) induced by 10 µM ACh (A) or 10 µM imidacloprid (B). The reversal potential of currents was extrapolated to be 0 mV. The main conductances were estimated to be 25 pS for ACh-induced currents and 25.4 pS for imidacloprid-induced currents. The subconductances are estimated to be 10.2 pS for ACh-induced and 9.8 pS for imidacloprid-induced currents. There is no significant difference in the slope conductances between ACh-induced and imidacloprid-induced currents for each conductance state.

Single-channel current induced by imidacloprid. Single-channel currents were induced by 10 µM imidacloprid alone (fig. 3B). Currents evoked by imidacloprid showed multiple conductance states. The two most frequently observed conductance states were further analyzed. The amplitudes of these two conductance state currents were 2.81 ± 0.47 pA (n = 6) and 0.88 ± 0.16 pA (n = 6). Imidacloprid-induced single-channel currents were blocked by 30 µM d-tubocurarine indicating that they were the result of activation of the nicotinic AChR (Nagata et al., 1996a). Imidacloprid first induced currents at a high frequency and with relatively short closings within a long opening forming a burst. This pattern repeated as a cluster of bursts for several tens of seconds followed by quiescence for a few minutes before starting new cluster of bursts. The current-voltage relationships of 10 µM imidacloprid-induced currents are plotted in figure 4B. Two conductances were estimated to be 25.4 and 9.8 pS, and are virtually identical to the main conductance and subconductance of ACh-induced currents (fig. 4A). Thus, imidacloprid opens the main conductance and subconductance channels which are the same as those opened by ACh in addition to at least two other conductance levels which were not analyzed.

Comparison of single-channel current parameters evoked by ACh and Imidacloprid. The open time distributions for ACh- and imidacloprid-induced main conductance currents are shown in figure 5A and B, respectively. The time axis is drawn on a logarithmic scale so that the effective bin width increases exponentially from left to right. This displays a multiexponential distribution as a series of skewed bells whose peaks overlie the time constants of several exponential components (Sigworth and Sine, 1987).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Open time distributions for main conductance currents induced by 10 µM ACh (A), 10 µM imidacloprid (B), and co-application of 10 µM ACh and 10 µM imidacloprid (C) to cell-attached membrane patches clamped at a membrane potential 40 mV more positive than the resting potential. The distributions are shown on a logarithmic time axis. The best fit of three exponential functions is shown. A, Time constants were estimated to be 0.7 msec (23% of total observations), 9.9 msec (56%) and 42.3 msec (21%). B, Time constants were estimated to be 0.7 msec (35.1% of total observation), 2.7 msec (52.7%) and 11.5 msec (12.2%). C, Time constants were estimated to be 0.9 msec (28.5%), 4.2 msec(37.8%) and 12.1 msec (33.7%).

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Closed time distributions for main conductance currents induced by 10 µM ACh (A), 10 µM imidacloprid (B) and co-application of 10 µM ACh and 10 µM imidacloprid (C) to cell-attached membrane patches clamped at a membrane potential 40 mV more positive than the resting potential. The distributions are shown on a logarithmic time axis. The best fit of exponential functions is shown. A, Time constants were estimated to be 1.6 msec (5.3% of total observations), 22.5 msec (12.4%), 295 msec (82.3%). B, Time constants were estimated to be 0.7 msec (3.2% of total observations), 2.2 msec (6.4%), 440 msec (58.4%) and 5050 msec (3.2%). C, Time constants were estimated to be 0.7 msec (0.1%), 3.4 msec (5.4%), 273 msec (83.5%) and 1950 msec (10.9%).

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Distributions of burst duration for main conductance currents induced by 10 µM ACh (A), 10 µM imidacloprid (B) and co-application of 10 µM ACh and 10 µM imidacloprid (C) to cell-attached membrane patches clamped at a membrane potential 40 mV more positive than the resting potential. The distributions are shown on a logarithmic time axis. The best fit of three exponential functions is shown. A, Time constants were estimated to be 0.6 msec (13.6% of total observations), 11.1 msec (64.5%) and 71.9 msec (21.9%). B, Time constants were estimated to be 0.9 msec (2.8% of total observations), 3.8 msec (57.9%) and 12.2 msec (13.7%). C, Time constants were estimated to be 0.8 msec (26.5%), 3.0 msec (27.9%) and 12.5 msec (45.5%).

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Distributions of open time (A), closed time (B) and burst duration (C) for subconductance state currents induced by 10 µM imidacloprid to cell-attached membrane patches clamped at a membrane potential 40 mV more positive than the resting potential. The distributions are shown on a logarithmic time axis. The best fit of exponential functions is shown. A, Time constants were estimated to be 0.6 msec (51.5% of total observations), 3.0 msec (9.5%) and 29.7 msec (38.9%). B, Time constants were estimated to be 0.5 msec (49.3%), 1.1 msec (25.7%) and 406 msec (25%). C, Time constants were estimated to be 0.5 msec (39.1%), 2.6 msec (17.3%) and 136 msec (43.5%).

Coapplication of acetylcholine and imidacloprid. Coapplication of 10 µM ACh and 10 µM imidacloprid opened the channels exhibiting both the main conductance state and subconductance state (fig. 3C). The current amplitudes were almost the same as those induced by 10 µM ACh or 10 µM imidacloprid, being 2.92 ± 0.36 pA (n = 6, mean ± S.D.) and 1.11 ± 0.43 pA (n = 6) for the main conductance and subconductance states, respectively. The time constants of open time distribution of the main conductance state currents were, 0.9 msec (28.5%), 4.2 msec (37.8%) and 12.1 msec (33.7%) (fig. 5C). The slowest value was closer to that of imidacloprid than ACh. The two faster values were between the values of ACh and imidacloprid.

	Discussion

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Effects of imidacloprid on whole-cell currents of ACh receptors. Imidacloprid suppressed the whole-cell currents induced by carbachol and generated small whole-cell currents by itself (figs. 1 and 2). Both types of currents were blocked by d-tubocurarine indicating that they were generated at the nicotinic AChR. The inhibitory effects of nitromethylene heterocyclic compounds on the nicotinic AChR were reported by several investigators using different preparations (Bai et al., 1991; Benson, 1989; Cheung et al., 1992; Zwart et al., 1994). Our results are consistent with the previous reports using insect species and are the first with the neuronal AChR of mammals. Most previous reports failed to show the agonistic effect of nitromethylene heterocycles on the mammalian nicotinic AChRs (Solowey et al., 1978; Liu and Casida, 1993; Zwart et al., 1992, 1994). Zwart et al. (1994) have shown that imidacloprid induced currents in locust thoracic ganglion neurons, but not in N1E-115 and BC3H1 neuroblastoma cells. Because the maximum current induced by imidacloprid in PC12 cells was much smaller than that induced by carbachol, imidacloprid can be considered as a partial agonist.

Blocking effect of imidacloprid on carbachol-induced currents. When imidacloprid was co-applied with carbachol, whole-cell currents were suppressed in a dose-dependent manner (figs. 1 and 2). Zwart et al. (1994) proposed that the mechanism of suppression by nitrometylene heterocyclic compounds is due to the acceleration of desensitization of nicotinic AChR. However, channel openings occurring as bursts, which are characteristic of desensitization of channels (Colquhoun and Ogden, 1988; Nagata et al., 1996b), were not observed in the present co-application study (fig. 3C). Thus, it is uncertain whether imidacloprid suppression of carbachol-induced currents is due to receptor desensitization.

Imidacloprid-induced single-channel currents. As we have reported previously (Nagata et al., 1996a), imidacloprid generated single-channel currents of multi-conductance states in PC12 cells. The main conductance and subconductance of currents induced by imidacloprid were identical to those induced by ACh. Cheung et al. (1992) have reported that one of the nitromethylene heterocyclic compounds generated currents of two conductance states in cultured brain cells isolated from American cockroaches. However, they did not perform detailed analysis of the two conductance states. Therefore, it is not clear whether the two conductance states they observed in insects are the same as those observed in PC12 cells.

Possible mechanisms of the two conductance states. There are three possible mechanisms of subconductance state generated by chemicals. First, the receptor conformation may be modified allosterically through the binding of chemicals to the agonist binding site resulting in reduced conductance (Hamill and Sakmann, 1981; Morris and Montpetit, 1986; Morris et al., 1983,1989). A second hypothesis is that the conductance of fully opened channel is reduced by chemicals through binding to a site within the channel pore thereby reducing ion permeation (Takeda and Trautmann, 1984; Trautmann, 1982; Strecker and Jackson, 1989). Third, since the neuronal nicotinic ACh receptor comprises several subunits (Lindstrom, 1996; McGehee and Role, 1995), chemicals may activate the ACh receptors with different subunits to give rise to different channel conductance levels.