Identification, localization and function of glutamate-gated chloride channel receptors in the honeybee brain.

Glutamate-gated chloride channels (GluCls) are members of the cys-loop ligand-gated ion channel superfamily whose presence has been reported in a variety of invertebrate tissues. In the honeybee, a single gene, amel_glucl, encoding a GluClα subunit, was found in the genome but both the pattern of expression of this gene in the bee brain and its functional role remained unknown. Here we localised the expression sites of the honeybee GluClα subunit at the mRNA and protein levels. To characterise the functional role of GluCls in the honeybee brain, we studied their implication in olfactory learning and memory by means of RNA interference (RNAi) against the GluClα subunit. We found that the GluClα subunit is expressed in the muscles, the antennae and the brain of honeybees. Expression of the GluClα protein was necessary for the retrieval of olfactory memories; more specifically, injection of dsRNA or siRNA resulted in a decrease in retention performances ∼24 h after injection. Knockdown of GluClα subunits impaired neither olfaction nor sucrose sensitivity, and did not affect the capacity to associate odor and sucrose. Our data provide the first evidence for the involvement of glutamate-gated chloride channels in olfactory memory in an invertebrate.

Inhibitory neurotransmission and olfactory memory in honeybees.

In insects, gamma-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.

Effects of sublethal doses of acetamiprid and thiamethoxam on the behavior of the honeybee (Apis mellifera).

Acetamiprid and thiamethoxam are insecticides introduced for pest control, but they can also affect non-target insects such as honeybees. In insects, these neonicotinoid insecticides are known to act on acetylcholine nicotinic receptors but the behavioral effects of low doses are not yet fully understood. The effects of acetamiprid and thiamethoxam were studied after acute sublethal treatment on the behavior of the honeybee (Apis mellifera) under controlled laboratory conditions. The drugs were either administered orally or applied topically on the thorax. After oral consumption acetamiprid increased sensitivity to antennal stimulation by sucrose solutions at doses of 1 microg/bee and impaired long-term retention of olfactory learning at the dose of 0.1 microg/bee. Acetamiprid thoracic application induced no effect in these behavioral assays but increased locomotor activity (0.1 and 0.5 microg/bee) and water-induced proboscis extension reflex (0.1, 0.5, and 1 microg/bee). Unlike acetamiprid, thiamethoxam had no effect on bees' behavior under the conditions used. Our results suggest a particular vulnerability of honeybee behavior to sublethal doses of acetamiprid.

Effects of sublethal doses of fipronil on the behavior of the honeybee (Apis mellifera).

Fipronil is a phenylpyrazole insecticide introduced for pest control, but it can also affect non-target insects such as honeybees. In insects, fipronil is known to block GABA receptors and to inhibit ionotropic glutamate-gated chloride channels, but the behavioral effects of low doses are not yet fully understood. We have studied the effect of sublethal doses of fipronil on the behavior of the honeybee (Apis mellifera) under controlled laboratory conditions. The drug was either administered orally or applied topically on the thorax. A significant reduction of sucrose sensitivity was observed for the dose of 1 ng/bee 1 h after a thoracic application. No significant effect on sucrose sensitivity was obtained with acute oral treatment. A lower dose of fipronil (0.5 ng/bee applied topically) impaired the olfactory learning of the honeybees. By contrast, locomotor activity was not affected. Our results suggest a particular vulnerability of the olfactory memory processes and sucrose perception to sublethal doses of fipronil in the honeybee.

Glutamatergic and GABAergic effects of fipronil on olfactory learning and memory in the honeybee.

We investigated here the role of transmissions mediated by GABA and glutamate-gated chloride channels (GluCls) in olfactory learning and memory in honeybees, both of these channels being a target for fipronil. To do so, we combined olfactory conditioning with injections of either the GABA- and glutamate-interfering fipronil alone, or in combination with the blocker of glutamate transporter L-trans-Pyrrolidine-2,4-Dicarboxylicacid (L-trans-PDC), or the GABA analog Trans-4-Aminocrotonic Acid (TACA). Our results show that a low dose of fipronil (0.1 ng/bee) impaired olfactory memory, while a higher dose (0.5 ng/bee) had no effect. The detrimental effect induced by the low dose of fipronil was rescued by the coinjection of L-trans-PDC but was rather increased by the coinjection of TACA. Moreover, using whole-cell patch-clamp recordings, we observed that L-trans-PDC reduced glutamate-induced chloride currents in antennal lobe cells. We interpret these results as reflecting the involvement of both GluCl and GABA receptors in the impairment of olfactory memory induced by fipronil.