Two full-length glutamate-gated chloride channel (GluCl) cDNAs, encoding GluCla3 and GluClb subunits, were cloned from ivermectin-susceptible (IVS) and -resistant (IVR) Cooperia oncophora adult worms. The IVS and IVR GluCla3 subunits differ at three amino acid positions, while the IVS and IVR GluClb subunits differ at two amino acid positions. The aim of this study was to determine whether mutations in the IVR subunits affect agonist sensitivity. The subunits were expressed singly and in combination in Xenopus laevis oocytes. Electrophysiological whole-cell voltage-clamp recordings showed that mutations in the IVR GluCla3 caused a modest but significant threefold loss of sensitivity to glutamate, the natural ligand for GluCl receptors. As well, a significant decrease in sensitivity to the anthelmintics ivermectin and moxidectin was observed in the IVR GluCla3 receptor.Mutations in the IVR GluClb subunit abolished glutamate sensitivity. Co-expressing the IVS GluCla3 and GluClb subunits resulted in heteromeric channels that were more sensitive to glutamate than the respective homomeric channels, demonstrating co-assembly of the subunits. In contrast, the heteromeric IVR channels were less sensitive to glutamate than the homomeric IVR GluCla3 channels. The heteromeric IVS channels were significantly more sensitive to glutamate than the heteromeric IVR channels. Of the three amino acids distinguishing the IVS and IVR GluCla3 subunits, only one of them, L256F, accounted for the differences in response between the IVS and IVR GluCla3 homomeric channels.

Two alleles of the HG1 gene, which encodes a putative GABA receptor a/c subunit, were isolated from Haemonchus contortus. These two alleles were shown previously to be associated with ivermectin susceptibility (HG1A) and resistance (HG1E), respectively. Sequence analysis indicates that they differ in four amino acids. To explore the functional properties of the two alleles, a full-length cDNA encoding the b subunit, a key functional component of the GABA receptor, was isolated from Caenorhabditis elegans (gab-1, corresponding to the GenBank locus ZC482.1) and coexpressed in Xenopus oocytes with the HG1 alleles. When gab-1 was coexpressed with either the HG1A allele or the HG1E allele in Xenopus oocytes, c-aminobutyric acid (GABA)-responsive channels with different sensitivity to the agonist were formed. The effects of ivermectin on the hetero-oligomeric receptors were determined. Application of ivermectin alone had no effect on the receptors. However, when coapplied with 10 lM GABA, ivermectin potentiated the GABA-evoked current of the GAB-1/ HG1A receptor, but attenuated the GABA response of the GAB-1/HG1E receptor. We demonstrated that the coexpressed HG1 and GAB-1 receptors are GABA-responsive, and provide evidence for the possible involvement of GABA receptors in the mechanism of ivermectin resistance.

To facilitate studies of the development and membrane biophysics of cells in the central olfactory pathway of the moth Manduca sexta, we have dissociated neurons and glial cells from the antennal lobes (ALs) and cultivated them in the controlled environment of tissue culture. Cultures produced from cells of the lateral group of AL neurons alone are enriched in local interneurons (LNs), while cultures made from cells of the median and anterior groups of AL neurons contain projection neurons (PNs) but lack LNs. Cultures containing only PNs, but not cultures with both PNs and LNs, require a conditioning factor derived from a conspecific cell line in order to ensure survival. Under these conditions, we identify 5 types of cells in the "PN-only" cultures that are consistently observed and distinguishable on the basis of their morphology and characteristic whole-cell current profiles. In cultures that contain both PNs and LNs, we find 3 additional types of cells with neuronlike appearance. Whereas the PNs have whole-cell currents that suggest a neuronal phenotype, we have not yet observed similar currents in the 3 other types of cells. We tentatively identify the latter 3 cell types as LNs whose development has been arrested. The conditioning factor also has a trophic effect on phase-dark, multipolar cells that are observed in freshly dispersed cultures. Their morphology and whole-cell currents lead us to suggest that they are glial cells.

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Neurons in the developing (antennal) olfactory lobe of the moth Manduca sexta undergo a period of extensive process outgrowth and branching that coincides temporally with both a rising titer of the steroid hormone 20-hydroxyecdysone and the ingrowth of sensory axons from receptors in the antenna. To evaluate the contribution of these two influences to the morphological development of antennal-lobe neurons, we placed the neurons in cell culture. Antennal-lobe neurons were dissociated from normal and chronically unafferented lobes at different stages of development and were exposed to different doses of hormone. Six neuronal cell types with distinctive and stable morphologies appeared in cultures from all stages of pupal development. Morphological changes in these neuronal types were examined quantitatively by comparison of the total length and number of branches. We found that 20-hydroxyecdysone had little direct effect on the morphological development of antennal-lobe neurons, but brief exposure to sensory axons in vivo prior to dissociation significantly enhanced subsequent outgrowth in culture.