Identification of neuron-specific ivermectin binding sites in Drosophila melanogaster and Schistocerca americana

doi:10.1016/0965-1748(94)00047-L

Insect Biochemistry and Molecular Biology

Volume 25, Issue 1, 1995, Pages 11-17

https://doi.org/10.1016/0965-1748(94)00047-L Get rights and content

Abstract

High affinity avermectin binding sites have been identified and partially characterized in membranes from two insect species, Drosophila melanogaster and the locust Schistocerca americana. There is a 10-fold increase in the density of ivermectin binding sites associated with membranes isolated from Drosophila heads (a neuronally enriched tissue source) compared to the bodies (B_max values were 3.5 and 0.22 pmol/mg, respectively) with only a small difference in the apparent dissociation constant (K_d values of 0.20 and 0.34 nM for heads and bodies, respectively). Membranes prepared from metathoracic ganglia of the locust, Schistocerca americana, were highly enriched in high affinity avermectin binding sites (K_d = 0.2 nM and B_max = 42 pmol/mg). Using an [¹²⁵I]arylazido-avermectin analog as a photoaffinity probe, a 45 kDa protein was identified in both the Drosophila head and body tissue preparations. A 45 kDa protein was also specifically labeled with [¹²⁵I]azido-avermectin in the locust neuronal membranes.

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In insects, γ-aminobutyric acid (GABA) and glutamate mediate fast inhibitory neurotransmission through ligand-gated chloride channel receptors. Both GABA and glutamate have been identified in the olfactory circuit of the honeybee. Here we investigated the role of inhibitory transmission mediated by GABA and glutamate-gated chloride channels (GluCls) in olfactory learning and memory in honeybees. We combined olfactory conditioning with injection of ivermectin, an agonist of GluCl receptors. We also injected a blocker of glutamate transporters (l-trans-PDC) or a GABA analog (TACA). We measured acquisition and retention 1, 24 and 48 h after the last acquisition trial. A low dose of ivermectin (0.01 ng/bee) impaired long-term olfactory memory (48 h) while a higher dose (0.05 ng/bee) had no effect. Double injections of ivermectin and l-trans-PDC or TACA had different effects on memory retention, depending on the doses and agents combined. When the low dose of ivermectin was injected after Ringer, long-term memory was again impaired (48 h). Such an effect was rescued by injection of both TACA and l-trans-PDC. A combination of the higher dose of ivermectin and TACA decreased retention at 48 h. We interpret these results as reflecting the involvement of both GluCl and GABA receptors in the impairment of olfactory long-term memory induced by ivermectin. These results illustrate the diversity of inhibitory transmission and its implication in long-term olfactory memory in honeybees.
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The avermectins are a group of pesticides whose main mode of action is to block synaptic transmission between neurons or between neurons and muscle cells. A number of closely related genes encoding avermectin-binding proteins have been cloned from a variety of invertebrates, including Drosophila melanogaster. Here we present the results of our study on the responses to avermectins of the D. melanogaster ort (ora transientless; 3-66.4) mutants. Mutations in this gene are known to affect synaptic transmission in the visual system. By the use of bioassays that we developed, three ort mutations were examined for resistance to two avermectins: ivermectin and abamectin. The results demonstrated an increased susceptibility to both toxins in all the mutant strains analyzed, compared to the wild-type flies. The levels of the susceptibility were allele specific in either the homo- or the hemizygous state of the mutations and in interstrain hybrids between different ort mutants. These observations allowed us to eliminate the genetic background of the mutants as a possible reason for their changed resistance. At the third instar larval stage, all the mutants were also more susceptible than the wild type, though a specific response to ivermectin was only shown by one mutant and there were no mutant-specific larval responses to abamectin. Double ort heterozygotes displayed a level of susceptibility inherited from the more resistant parent. No increased susceptibility of the ort mutants was found to treatment with dieldrin, which specifically blocks γ-aminobutyric acid receptors. Together, these data provide evidence that ort is implicated in the genetic determination of avermectin sensitivity and provide the first example of mutations establishing hypersensitivity to these insecticides in Drosophila.

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Identification of neuron-specific ivermectin binding sites in Drosophila melanogaster and Schistocerca americana

Abstract

Molec. Brain Res.

Parasitol. Today

Pesticide Biochem. Physiol.

Pesticide Biochem. Physiol.

Biochem. biophys. Res. Commun.

Brain Res.

Neurochem. Int.

Comp. Biochem. Physiol.

Biochem. Pharmac.

Neurosci. Lett.

Neurochem. Int.

Avermectin-sensitive chloride currents induced by Caenorhabditis elegans RNA in Xenopus oocytes

Molec. Pharmac.

Expression of Caenorhabditis elegans mRNA in Xenopus oocytes: A model system to study the mechanism of action of avermectins

Parasitol. Today

Avermectin potentiates an l-glutamate dependent current in insect neurones

Actions of insecticides on the insect GABA receptor complex

J. Receptor Res.