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J Neurophysiol 97: 2642-2650, 2007. First published January 31, 2007; doi:10.1152/jn.01234.2006
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Dorsal Unpaired Median Neurons of Locusta migratoria Express Ivermectin- and Fipronil-Sensitive Glutamate-Gated Chloride Channels

¹Centre of Environmental Sciences, Department of Physiology, and ²Biomedical Research Institute, Hasselt University and Transnationale Universiteit Limburg, Diepenbeek, Belgium; and ³Institute for Integrative Neuroanatomy, Berlin, Germany

Submitted 24 November 2006; accepted in final form 25 January 2007

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Together with type A GABA and strychnine-sensitive glycine receptors, glutamate-gated chloride channels (GluCl) are members of the Cys-loop family of ionotropic receptors, which mediate fast inhibitory neurotransmission. To date, GluCls are found in invertebrates only and therefore represent potential specific targets for insecticides, such as ivermectin and fipronil. In this study, we identified the functional expression of GluCls in dorsal unpaired median (DUM) neurons of the metathoracic ganglion of Locusta migratoria using electrophysiological and molecular biological techniques. In whole cell patch-clamped DUM neurons, glutamate-induced changes in both their membrane potentials (current-clamp) and currents (voltage-clamp) were dependent on the chloride equilibrium potential. On continuous application of glutamate, the glutamate-elicited current response became rapidly and completely desensitized. Application of glutamate in the presence of 10 µM fipronil or 100 µM picrotoxin reversibly decreased GluCl-mediated currents by 87 and 39%, respectively. Furthermore, 1 µM ivermectin induced a persistent chloride current, suggesting the expression of ivermectin-sensitive GluCl subunits. A degenerate PCR/RACE strategy was used to clone the full-length L. migratoria LmGlCl subunit. Finally, RT-PCR experiments demonstrated the presence of LmGluCl transcripts in locust DUM neurons. Our results provide the first direct evidence of a functional ivermectin-sensitive GluCl channel on the cell surface of DUM neurons of L. migratoria.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Glutamate is the main excitatory amino acid neurotransmitter in the CNS of vertebrates, where it interacts both with ionotropic receptors, i.e., ligand-gated ion channels, and metabotropic or G-protein–coupled receptors (Hogner et al. 2002). In vertebrates, ionotropic glutamate receptors are permeable to cations and can be subdivided into three groups according to their respective main agonist: N-methyl-D-aspartate (NMDA), -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate receptors.

In invertebrates, L-glutamate can also act as an excitatory transmitter at the neuromuscular junction (Ikeda et al. 2003; Wafford and Sattelle 1989). However, a biphasic response to L-glutamate is usually observed, consisting of a depolarization followed by a hyperpolarization. A pure hyperpolarizing effect can be obtained by applying ibotenate, a conformationally restricted structural analog of glutamate (Wafford and Sattelle 1989). These responses are mediated by another class of glutamate ionotropic receptors. These are involved in inhibitory synaptic transmission and consist of glutamate-gated chloride channels (GluCls). They were first identified in arthropods as extrajunctional glutamate receptors ("H" receptors) that cause hyperpolarization of the locust leg muscle (Cully et al. 1996; Dudel et al. 1989). The inhibitory ionotropic glutamate receptor gates a chloride-selective ion channel and belongs to the Cys-loop ligand-gated channel superfamily, together with the nicotinic acetylcholine, type A -aminobutyric acid (GABA), type C GABA, glycine, type 3 5-hydroxytryptamine (5-HT) receptors (Cleland 1996; Cull-Candy 1976) and the histamine-gated chloride channel (Zheng et al. 2002).

Until now, GluCl channels have been found only in invertebrate nerve and muscle cells (Cleland 1996). GluCl receptors thus form a potentially important target for the development of insecticides having highly selective toxic activity against insects that have become resistant to other insecticides (Raymond and Satelle 2002; Zhao et al. 2004b). Chemical compounds that affect GluCl receptors include members of the avermectine/milbemycin (A/M) class of anthelmintics such as ivermectin, as well as the phenylpyrazole insecticides, including fipronil. These products are in common use and form effective prophylactic and curative treatments against endo- and/or ectoparasitic infestations (Bloomquist 2003; Horoszok 2001). Ivermectin, a macrocyclic lactone derived chemically from the parent macrocyclic lactone avermectin B_1A, appears directly to activate GluCl or, at lower concentrations, to potentiate the effect of glutamate (Yates and Wolstenholme 2004). However, because of their close homology with GluCl, it is likely that GABA-gated channels in invertebrates are also sites of ivermectin toxicity (Kane et al. 2000). Fipronil was designated as a new GABA-gated chloride channel blocker and introduced into pest control, for instance against the Colorado potato beetle and against some cotton pests (Moffat 1993; Smith and Lockwood 2003). The blocking action of fipronil on GluCl channels was demonstrated in oocytes transfected with GluCls (Horoszok et al. 2001) and a similar degree of block was observed on chloride currents induced by glutamate and ibotenate in dorsal unpaired median (DUM) neurons of the cockroach Periplaneta americana and the grasshopper Melanoplus sanguinipes (Ikeda et al. 2003; Raymond et al. 2000; Smith et al. 1999).

At the molecular and functional level, GluCls remain poorly characterized. Until now, cloning studies led to the identification of a small family of six GluCl genes in the nematode. In Hemonchus contortus, three genes encoding four subunits were identified. In Drosophila melanogaster, a cDNA clone (DmGluCl) was obtained by hybridization screening (Cully et al. 1996). Electrophysiological studies of D. melanogaster DmGluCl, expressed in oocytes, revealed direct activation by the acaricide nodulosporic acid and by ivermectin, as well as by the endogenous ligand glutamate (Kane et al. 2000).

The neurobiology of the locust has been extensively investigated and the action of L-glutamate on muscles of this insect has been well studied (Grolleau and Lapied 2000; Heidel and Pflüger 2006; Wicher et al. 2001). However, in the nervous system of L. migratoria, relatively little is known about the effect of glutamate on efferent DUM neurons. In the locust, efferent DUM neurons extend symmetrical pairs of axons to flight and leg (tibia) muscle, where they exert myo- and/or neuromodulatory effects. In other arthropod species, such as P. americana, GluCls seems to have a modulatory effect on the excitability of the DUM neurons and could play a substantial role in muscle and flight control (Dubas 1991; Washio 2002). It is thus tempting to postulate that GluCls fulfill the same role in DUM neurons of L. migratoria and that they might well therefore also represent a useful target for insecticide pest control in this species.

In the present study, we looked for the presence of a glutamate-gated chloride channel in an ex vivo preparation of DUM neurons of the metathoracic ganglion of L. migratoria using the whole cell patch-clamp technique. Under current-clamp conditions, glutamate-induced changes in membrane potential were measured, whereas under voltage-clamp conditions, glutamate-evoked currents were pharmacologically and kinetically characterized. Finally, using molecular biology techniques, we looked for the expression of GluCl transcripts that could account for the observed glutamate responses.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Isolation of metathoracic DUM neurons of the locust

Adult migratory locusts (L. migratoria) were used in all experiments. They were taken from a crowded laboratory colony maintained at about 32°C on a 14-h:10-h light/dark cycle and on a diet of grass and oatmeal. Isolated DUM neuronal cell bodies were prepared as described previously (Brône et al. 2003). Briefly, the dorsal median region containing the neuromeres T3, A1–A3 of the metathoraric ganglion were mechanically removed and subjected to collagenase/dispase (2 mg/ml) treatment. The cells were centrifuged and subsequently washed three times with culture medium consisting of equal parts of Basal Medium Eagle with Hank's salts (BME) and Grace's Insect Medium (GIM). In the first wash, a 1% penicillin/streptomycin mixture was added to the BME/GIM culture medium. The cells were dissociated by repetitive up and down aspirations through a pipette tip, then plated on Nunc petri dishes and incubated at 28°C in a 5% CO₂ atmosphere. All products were from GibcoBRL (Invitrogen, Merelbeke, Belgium), except for the Boehringer collagenase/dispase mixture (from Roche Diagnostics, Brussels, Belgium). Healthy efferent DUM neurons (diameter 40–60 µm) were recognized by their morphological characteristics under phase-contrast microscopy (Nikon Diaphot, Tokyo, Japan) (see, e.g., Brône et al. 2003).

Electrophysiology

Changes in the membrane potential of and current in DUM neurons of L. migratoria were recorded at room temperature using the whole cell configuration of the patch-clamp technique both in current-clamp and voltage-clamp conditions. Locust DUM neurons were transferred onto the stage of an inverted phase-contrast microscope (Nikon Diaphot) in a recording chamber that was continuously perfused with solutions further described in Table 1. Electrodes made from 1.5 mm (OD) borosilicate glass capillary tubes, having a resistance between 800 k and 1.5 M, were also appropriately filled with the solutions listed in Table 1. To reduce contaminating Na⁺ and K⁺ currents through the voltage-gated ion channels, some experiments were conducted using modified intracellular and extracellular solutions (Table 1). The osmolality of these solutions (about 380 mOsm/kg), corresponding to that of L. migratoria hemolymph (Brône et al. 2003), was attained by addition of glucose. Liquid junction (LJ) potentials were calibrated using the Junction Potential Calculator for Windows (JPCalcW, Peter H. Barry, Dept. of Physiology and Pharmacology, Australia and Axon Instruments, Union City, CA) and were taken into account at the start of each experiment. The LJ potential between the standard intracellular solution (SIS) and standard extracellular solution (SES) was –18 mV, between high Cl^––IS and SES was –21.8 mV, between low Na⁺–ES and low K⁺–high Cl^––IS was –6.7 mV and between low Na⁺–ES and low K⁺–low Cl^––IS was –19.2 mV. The experimental protocols and data acquisition were carried out using an EPC-10 patch-clamp amplifier (HEKA Elektronik, Lambrecht, Germany) controlled by the program Pulse (HEKA Elektronik) running on a personal computer (equipped with a Pentium III processor). Capacitive and leak currents were compensated and residual capacitances and leak currents were eliminated, by means of a P/6 protocol (Moyer and Brown 2002). The series resistance was 1.5 M and was compensated 53%. The recording of whole cell current was started 5 min after rupture of the cell membrane to allow adequate equilibration of the cell interior with the pipette solution.

Data were filtered at 2.9 kHz and sampled at 20 kHz. They were stored on a computer hard disk and analyzed with the program PulseFit 8.77 (HEKA Instruments). Further analyses, including nonlinear regression, were carried out using Origin 6.0 Professional (Microcal Software, Northampton, MA).

Current–voltage relationships were fitted with the Goldman–Hodgkin–Katz (GHK) equation

Desensitization kinetics of individual glutamate-evoked currents was assessed by fitting the desensitizing phase with the following equation

Except where stated otherwise, results are expressed as means ± SE, where n is the number of experiments. Statistical comparisons were made using the Student's t-test or one-way ANOVA followed by Dunnett's posttests. Differences were considered significant for P 0.05.

Chemicals

Monosodium glutamate (Sigma–Aldrich Chemie, Steinheim, Germany) and avermectin B1A (Sigma–Aldrich) were dissolved in triply distilled water. Stock solutions of fipronil (PESTANAL) and picrotoxin (PTX), both from Sigma–Aldrich (Seelze, Germany), were made in ethanol (EtOH) and dimethylsulfoxide (DMSO), respectively. These stock solutions were diluted into the extracellular solutions immediately before the start of each experiment. The final concentrations of EtOH and DMSO in the solutions were 0.1%, at which they had no effect on glutamate-induced currents (data not shown). L-Glutamate and ivermectin were applied topically through a valve-controlled tubing system positioned laterally above the cell at a distance of about 50–100 µm. The antagonists fipronil and PTX were applied to the bath.

Degenerate PCR and RACE

To obtain the full-length sequence of the L. migratoria GluCl channel, a Marathon RACE library was constructed using a mixture of several tissues (including muscle and neuronal tissues) as described by the manufacturer (Clontech, Hamburg, Germany). Degenerated oligonucleotides were designed and synthesized based on the predicted amino acid sequences of the conserved regions of the glutamate-activated chloride alpha channel (GluCl) after alignment of the GluCls from Drosophila melanogaster and Caenorhabditis elegans: DEG-F:5'-GT(CG)TCATGGGT(TA)TCATT(CT)TGG-3' DEG-R:5'-TC(CG)AG(CA)AG(TG)GC(CT)CCGAA(CT)AC(AG)AA(CT)GT-3'.

The thermocycler was programmed for 35 cycles at 94°C for 30 s, 41.5°C for 45 s, and 68°C for 45 s using the L. migratoria RACE library as a template and the Advantage Taq II polymerase mixture (Clontech) as polymerase. The resulting band of 193 bp was directly sequenced and the following primers were designed for rapid amplification of cDNA ends (RACE): GSP-F1:5'-GGCTCGATCAGGGTGCCGTTC-3' GSP-F2:5'-CCGTTCCCGCACGAGTGTCC-3' GSP-R1:5'-TCGAGAAGTGCCCCGAATACGA-3' GSP-R2:5'-ATGTTAGGCAGACGCCGGTCCA-3'.

The first RACE PCR was performed for 35 cycles at 94°C for 30 s and 68°C for 4 min using Advantage Taq II polymerase mixture and GSP-F1 (3'-RACE) or GSP-R1 (5'-RACE) combined with adaptor primer 1 (AP1). A second nested PCR was performed with GSP-F2 or GSP-R2 and AP2 primer. The PCR fragments (1,000–2,000 bp) obtained were cloned into the pGEM-T vector (Promega, Karlsruhe, Germany) and sequenced until start and stop codons were identified.

RNA isolation from DUM neurons and RT-PCR

DUM neurons of L. migratoria were isolated as described earlier with slight modifications. After the collagenase/dispase treatment, the cells were plated on a NUNC petri dish and incubated for 15 min at 28°C. The cells were picked up using a borosilicate patch pipette, then washed with ice-cold PBS (pH 7.4) and transferred into an RNase-free microtube.

Total RNA was prepared from the material obtained from about 40 DUM neurons with the PicoPure RNA Isolation kit (Arcturus Bioscience, Westburg, Leusden, The Netherlands) with an additional DNase treatment (Rnase-free DNase set, Qiagen) following the instructions of the suppliers. In addition, RNA from a mixture of several neuronal tissues (abdominal and thoracic ganglia, brain) was prepared. First-strand cDNA was synthesized using the Promega RT system (Promega Benelux, Leiden, The Netherlands).

The cDNA of isolated locust DUM neurons and locust neuronal tissues was used as a template for a PCR using Taq DNA polymerase (Applied Biosystems). A fruitfly cDNA was used as a positive control. PCR primers DEG-F and DEG-R were used for amplification during 40 cycles at 94°C for 30 s, 41.5°C for 45 s, and 72°C for 45 s. PCR products were than separated on a 2% high-resolution agarose gel. Several distinctive bands were observed. The band of interest (about 190–200 bp) was extracted from the gel using the Qiaquick gel extraction kit (Qiagen), purified, and, in the case of isolated DUM neurons, subjected to a second PCR amplification using the same primers and incubation conditions. The amplified PCR fragments were either directly sequenced or cloned into pGEM-T for sequencing.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Glutamate elicits changes in the membrane properties of locust DUM neurons

When recorded with solutions close to physiological conditions (SIS–SES; see METHODS and Table 1), whole cell patch- and current-clamp locust DUM neurons had a resting membrane potential of –34.4 ± 3.8 mV (n = 6). As shown in Fig. 1A, application of 1 mM glutamate hyperpolarized the membrane to –62.6 ± 1.81 mV (n = 6). When Cl^– concentrations were identical in intracellular and extracellular solutions, DUM neurons had a resting membrane potential of –58.8 ± 1.4 mV (n = 5) and, in that case, glutamate induced a depolarization of the membrane to –7.9 ± 3.4 mV (n = 5; Fig. 1B).

View larger version (14K): [in this window] [in a new window]   FIG. 1. Glutamate-induced membrane potential changes in locust metathoracic ganglion dorsal unpaired median (DUM) neurons, recorded with the whole cell patch-clamp technique in current-clamp mode. A: in the presence of standard intracellular and extracellular solutions (SIS–SES; see inset), application of 1 mM glutamate for 1 s hyperpolarized the DUM neuron membrane potential from –35 to –61 mV. B: when using almost identical chloride concentrations in the intracellular and extracellular solutions (high Cl––IS–SES; see inset), glutamate generated a depolarization from –55 to –19 mV.

View larger version (20K): [in this window] [in a new window]   FIG. 2. Glutamate-evoked currents in locust metathoracic ganglion DUM neurons, recorded with the whole cell patch-clamp technique in voltage-clamp conditions. This figure shows a typical experiment. In the presence of almost identical chloride concentrations in the intracellular and extracellular solutions (High Cl––IS–SES; see inset) and at a holding potential (VH) of –70 mV), the application of 1 mM glutamate evoked an inward current that reached a peak amplitude of 5.6 nA. Current production desensitized completely during glutamate administration, with a monoexponential time course ( = 246 ms).

To assess whether the main ionic species carrying the glutamate-induced currents was chloride, the I–V relationships were obtained using two different intracellular solutions: one containing nearly identical intracellular and extracellular Cl^– concentrations (low K⁺–high Cl^––IS) and a second one containing a lower Cl^– concentration in the intracellular solution (low K⁺–low Cl^––IS). These solutions were also modified (see Table 1) to reduce contaminating currents through the voltage-dependent channels (e.g., K_v, Na_v, Ca_v). Currents were evoked by repeatedly applying 1 mM glutamate for 3 s and at the same time applying a voltage step ranging from –60 to +60 mV. In between applications, V_H was returned to –70 mV and glutamate was washed out (Fig. 3A).

View larger version (19K): [in this window] [in a new window]   FIG. 3. Current–voltage (I–V) relationship of glutamate-induced currents in locust DUM neurons recorded with the whole cell patch-clamp technique. A: currents (A2) were evoked by 1 mM glutamate applications at holding potentials ranging from –60 to +60 mV (A1), in the presence of almost identical Cl– concentrations and intracellular (low K+–high Cl––IS) and extracellular solutions (low Na+–ES) modified to reduce contaminating voltage-dependent currents. Between voltage steps, VH was returned to –70 mV and glutamate was washed out. B: I–V relationship, obtained from glutamate induced currents described in A ([high Cl–]I; closed circles). Maximum peak currents were normalized for the cell capacitance and are plotted against the holding potentials. Relation was best-fitted with the GHK equation, which yielded a reversal potential of +2.65 mV. This was close to the calculated equilibrium potential (–0.9 mV). When the intracellular chloride concentration was lowered to 83 mM ([low Cl–]I; open circles), the reversal potential shifted to –13.6 mV in agreement with the theoretically expected shift (–20 mV).

These results clearly demonstrate that the glutamate-induced currents in locust DUM neurons are mainly carried by chloride ions.

Pharmacology of glutamate-induced currents

Glutamate-induced currents in locust DUM neurons were then tested for their sensitivity to two known blockers of GluCls: fipronil and picrotoxin (PTX). As shown in Fig. 4, A and B, both drugs reversibly inhibited glutamate-gated currents. Ten micromolar fipronil inhibited to 13.1 ± 2.5% of controls (n = 5; Fig. 4A₂). After washout, glutamate currents recovered to 74.4 ± 4.4% (n = 5) of the control. In the presence of PTX, glutamate-evoked currents were reduced to 38.8 ± 4.2% (n = 4; Fig. 4B₂), whereas recovery was to 87.8 ± 8.2% (n = 4) after washout.

View larger version (22K): [in this window] [in a new window]   FIG. 4. Pharmacological properties of the glutamate-gated chloride channel in DUM neurons of L. migratoria. 1 mM glutamate was applied for 3 s at a VH of –70 mV in high Cl––IS and SES conditions (see METHODS), alone or in combination with 10 µM fipronil (A1) or 100 µM picrotoxin (PTX, B1). Recovery from inhibition (washout) was also checked. Histograms in A2 and B2 summarize fipronil- and PTX-induced inhibition of glutamate-activated currents (means ± SE; n = 5 for fipronil, 4 for PTX; **P < 0.001 and ***P < 0.0001: one-way ANOVA followed by Dunnett's posttests). C: a 1-mM glutamate application was followed by perfusion for 3 s with 1 µM ivermectin (IVM), which elicited an inward current that persisted on washout of IVM.

These pharmacological data further support the presence of a glutamate-gated chloride channel in efferent DUM neurons of L. migratoria.

Cloning of full-length LmGluCl

Because functional GluCls usually require the expression of at least GluCl subunits (Cully et al. 1996; Semenov and Pak 1999), we developed a degenerate PCR and RACE strategy in an attempt to clone the full-length sequence of L. migratoria GluCl (LmGluCl). Within the isolated cDNA, prepared as described in METHODS, a 1,362-bp open reading frame was identified encoding for a 453 amino acid protein. Typical features of GluCl subunits as in other members of the Cys-loop ligand-gated ion channel superfamily were found: a large extracellular N-terminal domain containing the ligand-binding site, a conserved Cys-loop and four transmembrane regions (Fig. 5). Moreover, the LmGluCl subunit showed strong homology to orthologous sequences from Drosophila melanogaster (CAA05260 [GenBank] ; 82.2% identity, 87.6% similarity), Tribolium castaneum (XP_973383 [GenBank] ; 84.8% identity, 91.4% similarity), Anopheles gambiae (XP_321697 [GenBank] ; 83.8% identity, 88.6% similarity), Apis melifera (ABG75738 [GenBank] ; Jones and Sattelle 2006; 78.3% identity, 87.1% similarity), Musca domestica (BAD16657 [GenBank] ; 81.3% identity, 86.7% similarity), and Lucilia cuprina (AAC31949 [GenBank] ; 83.7% identity, 89.1% similarity). The cDNA sequence of the LmGluCl subunit was deposited in GenBank under Accession Number DQ643254.

View larger version (42K): [in this window] [in a new window]   FIG. 5. cDNA and protein sequence of L. migratoria glutamate-activated chloride alpha channel (LmGluCl). Start and stop codons are shown in bold/italics. Transmembrane regions (gray) and conserved cysteine residues of the Cys-loop (in box) are indicated. In addition, the position of the primers used for initial degenerate PCR are shown (underlined).

DUM neurons of L. migratoria express LmGluCl messengers

The expression of LmGluCl in locust DUM neurons was assessed as follows. mRNAs of isolated DUM neurons were reverse transcribed and used as template for a two-step PCR procedure. After the first PCR, a faint band was visible (Fig. 6A). This PCR band was excised, purified, and reamplified (Fig. 6B). Direct DNA sequencing confirmed the LmGluCl sequence.

View larger version (65K): [in this window] [in a new window]   FIG. 6. Expression of the GluCl channel in DUM neurons of L. migratoria. A: agarose electrophoresis of the first PCR using locust DUM neuron cDNA as a template (lane a2; marker = 100-bp ladder shown in lane a1). B: reamplification of the corresponding 193-bp PCR product for DNA sequencing (lane b3; marker = 50-bp ladder shown in lane b1). Positive control used was Drosophila melanogaster glutamate-activated chloride alpha channel (DmGluCl, lane b2).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

In invertebrates, glutamate can interact with two classes of ionotropic receptors: excitatory channels, mainly permeable to cations as in vertebrates, and inhibitory anionic channels that are found only in invertebrates. When using intra- and extracellular solutions with an ionic composition close to that of the hemolymph, we observed a hyperpolarizing response on glutamate application, suggesting that locust DUM neurons express only inhibitory glutamate-activated channels (Ikeda et al. 2003; Wafford and Sattelle 1989). The chloride dependency of glutamate-induced responses was further confirmed by the shift to depolarization when E_Cl^– was changed from –79.6 to –4.1 mV and by the concordance of reversal potentials of glutamate-induced currents with chloride equilibrium potentials. Thus it appears that L. migratoria DUM neurons express inhibitory ionotropic glutamate receptors only and not their excitatory counterparts. This was also observed for DUM neurons of other insect species such as the cockroach Periplaneta americana (Washio et al. 2002) and for the stomatogastric ganglion of the lobster Panulus interruptus (Cleland and Selverston 1998). This is in contrast to the expression of both inhibitory and excitatory glutamate receptors by muscle cells in the locust or the crayfish (Dudel and Franke 1987; Kerry et al. 1987, 1988) or by motor neurons such as the fast coxal depressor motor neuron of the cockroach thoracic ganglion (Wafford and Satelle 1989).

In our hands, both glutamate-induced voltage and current changes were monophasic. Furthermore, under voltage-clamp conditions, glutamate-induced currents completely desensitized in about 1 s in the continuous presence of glutamate. These properties are similar to those reported for Caenorhabditis elegans GluCls expressed in Xenopus oocytes (Forrester et al. 2003). In cockroach DUM neurons, on the other hand, biphasic glutamate responses were observed, consisting of an initial transient and a subsequent prolonged phase (Heckman and Dudel 1995; Raymond et al. 2000; Zhao et al. 2004a). The desensitizing and nondesensitizing components of the glutamate-induced responses seem to be attributable to the presence of pharmacologically distinct GluCls: the former sensitive and the latter insensitive to PTX or BIDN. The desensitizing glutamate-evoked current we report in locust DUM neurons is also PTX sensitive. It therefore probably corresponds to the transient current described in cockroach CNS neurons.

In locust DUM neurons, fipronil seems to be a more powerful antagonist of GluCl-mediated responses than does PTX. At 10 µM, it blocked 86% of glutamate-gated currents. This is in good agreement with the 75% block by 10 µM fipronil of the transient component of glutamate responses in cockroach DUM neurons reported by Raymond et al. (2000). To the best of our knowledge, our work is the first report of an effect of fipronil on isolated DUM neurons of L. migratoria. The blocking effect of fipronil on GluCls was previously proposed to explain part of the toxicity of this insecticide, which is widely used in pest control to eradicate dieldrin-resistant insects (Horoszok et al. 2001; Smith and Lockwood 2003; Tingle et al. 2003; Zhao et al. 2004b).

Ivermectin (IVM) at 1 µM generated an irreversible inward current under identical chloride concentrations in DUM neurons of L. migratoria. At concentrations in the micromolar range, avermectin derivatives are known activators of ligand-gated chloride channels, i.e., strychnine-sensitive glycine receptors (Shan et al. 2001), GABA receptors (Adelsberger et al. 2000; Robertson 1989), and GluCls (Cleland 1996). Because glycine receptors are not found in invertebrates, the IVM-induced current observed in DUM neurons could be the consequence of the activation of either GABA receptors or GluCls—or even both, as already demonstrated in the fruit fly (Ludmerer et al. 2002). Accordingly, the amplitude of the current induced by 1 µM IVM was significantly higher than that induced by 1 mM glutamate, a potentially saturating concentration for GluCls. This could suggest that IVM activates both GABA receptors and GluCls in DUM neurons of the locust. On the other hand, avermectin-sensitive chloride currents in Xenopus oocytes injected with C. elegans mRNA were GABA insensitive, but sensitive to glutamate (Yates et al. 2003). Further characterization of IVM-induced currents is needed to solve this issue, but that is beyond the scope of this work. With respect to GluCls, it is usually accepted that the ability to irreversibly bind IVM is typically diagnostic for -like subunits of GluCls (Cully et al. 1994; Li et al. 2002). This was previously demonstrated in several invertebrate species: locust (Schistocerca americana; Rohrer et al. 1994), barber pole worm (Hemonthus contortus; Forrester et al. 2001; Portillo et al. 2003), heartworm (Dirofilaria immitis; Yates et al. 2004), elegant worm (Caenorhabditis elegans; Dent et al. 1997, 2000; Horoszok et al. 2001), and strongylid worm (Cooperia oncophora; Njue et al. 2004). In contrast to GluCl subunits, GluCl subunits were never shown to respond to IVM (Cully et al. 1994; Li et al. 2002). Note, however, in some species, splice variants of GluCl subunits were shown to be IVM resistant, e.g., DiGluCl3A from Dirofilaria immitis (Yates et al. 2004) or CoGluCl3 from Cooperia oncophora (Njue et al. 2004).

The functional expression of -subunit–containing GluCls by locust DUM neurons suggested by their IVM sensitivity was demonstrated at the molecular level. A full-length LmGluCl channel was cloned, using a PCR/RACE strategy, and its messenger RNA in isolated DUM neurons was detected by RT-PCR. This does not rule out the expression of other LmGluCl subunits and thus does not allow any conclusion about the homo- or heteromeric nature of locust GluCls. Transfection of the cloned LmGluCl in heterologous expression systems might help to solve this question.

The functional implication of glutamate-gated chloride channels in DUM neurons of L. migratoria certainly deserves further attention. Because metathoracic efferent DUM neurons innervate flight and leg muscles, their modulation by inhibitory GluCls is likely to influence flight control (Groleau and Lapied 2000). Furthermore, and more generally, because GluCls have been described until now in invertebrates only, a better insight into their functioning would undoubtedly contribute to the development of novel insecticides both more selective and more potent (Raymond and Satelle 2002; Zhao et al. 2004b).

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
We thank J. Janssen and R. Beenaerts for excellent technical support, Dr. Robert Dale for careful reading of the manuscript, and Dr. Inge Mertens, Laboratory of Developmental Physiology, Genomics and Proteomics of the Katholieke Universiteit Leuven for supplying the D. melanogaster.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Address for reprint requests and other correspondence: D. Janssen, Centre of Environmental Sciences, Department of Physiology, Hasselt University and Transnationale Universiteit Limburg, Agoralaan, Diepenbeek, 3590, Belgium (E-mail: daniel.janssen{at}uhasselt.be)

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
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