Homomeric RDL and heteromeric RDL/LCCH3 GABA receptors in the honeybee antennal lobes: two candidates for inhibitory transmission in olfactory processing.

gamma-Aminobutyric acid (GABA)-gated chloride channel receptors are abundant in the CNS, where their physiological role is to mediate fast inhibitory neurotransmission. In insects, this inhibitory transmission plays a crucial role in olfactory information processing. In an effort to understand the nature and properties of the ionotropic receptors involved in these processes in the honeybee Apis mellifera, we performed a pharmacological and molecular characterization of GABA-gated channels in the primary olfactory neuropile of the honeybee brain-the antennal lobe (AL)-using whole cell patch-clamp recordings coupled with single-cell RT-PCR. Application of GABA onto AL cells at -110 mV elicited fast inward currents, demonstrating the existence of ionotropic GABA-gated chloride channels. Molecular analysis of the GABA-responding cells revealed that both subunits RDL and LCCH3 were expressed out of the three orthologs of Drosophila melanogaster GABA-receptor subunits encoded within the honeybee genome (RDL, resistant to dieldrin; GRD, GABA/glycine-like receptor of Drosophila; LCCH3, ligand-gated chloride channel homologue 3), opening the door to possible homo- and/or heteromeric associations. The resulting receptors were activated by insect GABA-receptor agonists muscimol and CACA and blocked by antagonists fipronil, dieldrin, and picrotoxin, but not bicuculline, displaying a typical RDL-like pharmacology. Interestingly, increasing the intracellular calcium concentration potentiated GABA-elicited currents, suggesting a modulating effect of calcium on GABA receptors possibly through phosphorylation processes that remain to be determined. These results indicate that adult honeybee AL cells express typical RDL-like GABA receptors whose properties support a major role in synaptic inhibitory transmission during olfactory information processing.

Study of nicotinic acetylcholine receptors on cultured antennal lobe neurones from adult honeybee brains.

In insects, acetylcholine (ACh) is the main neurotransmitter, and nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission. In the honeybee, nAChRs are expressed in diverse structures including the primary olfactory centres of the brain, the antennal lobes (AL) and the mushroom bodies. Whole-cell, voltage-clamp recordings were used to characterize the nAChRs present on cultured AL cells from adult honeybee, Apis mellifera. In 90% of the cells, applications of ACh induced fast inward currents that desensitized slowly. The classical nicotinic agonists nicotine and imidacloprid elicited respectively 45 and 43% of the maximum ACh-induced currents. The ACh-elicited currents were blocked by nicotinic antagonists methyllycaconitine, dihydroxy-beta-erythroidine and alpha-bungarotoxin. The nAChRs on adult AL cells are cation permeable channels. Our data indicate the existence of functional nAChRs on adult AL cells that differ from nAChRs on pupal Kenyon cells from mushroom bodies by their pharmacological profile and ionic permeability, suggesting that these receptors could be implicated in different functions.

Acetylcholine, GABA and glutamate induce ionic currents in cultured antennal lobe neurons of the honeybee, Apis mellifera.

The honeybee, Apis mellifera, is a valuable model system for the study of olfactory coding and its learning and memory capabilities. In order to understand the synaptic organisation of olfactory information processing, the transmitter receptors of the antennal lobe need to be characterized. Using whole-cell patch-clamp recordings, we analysed the ligand-gated ionic currents of antennal lobe neurons in primary cell culture. Pressure applications of acetylcholine (ACh), gamma-amino butyric acid (GABA) or glutamate induced rapidly activating ionic currents. The ACh-induced current flows through a cation-selective ionotropic receptor with a nicotinic profile. The ACh-induced current is partially blocked by alpha-bungarotoxin. Epibatidine and imidacloprid are partial agonists. Our data indicate the existence of an ionotropic GABA receptor which is permeable to chloride ions and sensitive to picrotoxin (PTX) and the insecticide fipronil. We also identified the existence of a chloride current activated by pressure applications of glutamate. The glutamate-induced current is sensitive to PTX. Thus, within the honeybee antennal lobe, an excitatory cholinergic transmitter system and two inhibitory networks that use GABA or glutamate as their neurotransmitter were identified.