Expression patterns of nicotinic subunits α2, α7, α8, and ß1 affect the kinetics and pharmacology of ACh-induced currents in adult bee olfactory neuropiles.

Acetylcholine (ACh) is the main excitatory neurotransmitter of the insect brain, where nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission. In the honeybee Apis mellifera, nAChRs are expressed in diverse structures including the primary olfactory centers of the brain, the antennal lobes (ALs) and the mushroom bodies (MBs), where they participate in olfactory information processing. To understand the nature and properties of the nAChRs involved in these processes, we performed a pharmacological and molecular characterization of nAChRs on cultured Kenyon cells of the MBs, using whole cell patch-clamp recordings combined with single-cell RT-PCR. In all cells, applications of ACh as well as nicotinic agonists such as nicotine and imidacloprid induced inward currents with fast desensitization. These currents were fully blocked by saturating doses of the antagonists α-bungarotoxin (α-BGT), dihydroxy-ß-erythroidine (DHE), and methyllycaconitine (MLA) (MLA ≥ α-BGT ≥ DHE). Molecular analysis of ACh-responding cells revealed that of the 11 nicotinic receptor subunits encoded within the honeybee genome, α2, α8, and ß1 subunits were expressed in adult Kenyon cells. Comparison with the expression pattern of adult AL cells revealed the supplementary presence of subunit α7, which could be responsible for the kinetic and pharmacological differences observed when comparing ACh-induced currents from AL and Kenyon cells. Together, our data demonstrate the existence of functional nAChRs on adult MB Kenyon cells that differ from nAChRs on AL cells in both their molecular composition and pharmacological properties, suggesting that changing receptor subsets could mediate different processing functions depending on the brain structure within the olfactory pathway.

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.

Early calcium increase triggers the formation of olfactory long-term memory in honeybees.

BACKGROUND: Synaptic plasticity associated with an important wave of gene transcription and protein synthesis underlies long-term memory processes. Calcium (Ca2+) plays an important role in a variety of neuronal functions and indirect evidence suggests that it may be involved in synaptic plasticity and in the regulation of gene expression correlated to long-term memory formation. The aim of this study was to determine whether Ca2+ is necessary and sufficient for inducing long-term memory formation. A suitable model to address this question is the Pavlovian appetitive conditioning of the proboscis extension reflex in the honeybee Apis mellifera, in which animals learn to associate an odor with a sucrose reward. RESULTS: By modulating the intracellular Ca2+ concentration ([Ca2+]i) in the brain, we show that: (i) blocking [Ca2+]i increase during multiple-trial conditioning selectively impairs long-term memory performance; (ii) conversely, increasing [Ca2+]i during single-trial conditioning triggers long-term memory formation; and finally, (iii) as was the case for long-term memory produced by multiple-trial conditioning, enhancement of long-term memory performance induced by a [Ca2+]i increase depends on de novo protein synthesis. CONCLUSION: Altogether our data suggest that during olfactory conditioning Ca2+ is both a necessary and a sufficient signal for the formation of protein-dependent long-term memory. Ca2+ therefore appears to act as a switch between short- and long-term storage of learned information.

Insights into the molecular basis of social behaviour from studies on the honeybee, Apis mellifera.

The honeybee, Apis mellifera, has been the most important insect species for the study of social behaviour. With the recent release of its genome sequence, the honeybee has emerged as an excellent model for molecular studies of social behaviour. A key feature of eusocial species is a complex division of labour. Adult honeybees perform a series of tasks in the hive when they are young and then shift to foraging for nectar or pollen outside the hive when they are 2-3 weeks of age. This transition from working in the hive to foraging involves changes in the expression of thousands of genes. In this review, we focus first on recent advances in understanding of the widespread changes in gene activity that accompany the transition to foraging. Thereafter, we examine three genes in particular, foraging, malvolio and vitellogenin, all implicated in this striking behavioural change in the life of the honeybee.

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.

Exploring the pharmacological properties of insect nicotinic acetylcholine receptors.

Insect nicotinic acetylcholine (nACh) receptors are molecular targets of insecticides such as neonicotinoids that are used to control disease-carrying insects and agricultural pests. To date, several insect nACh receptor subunits have been identified, indicating different nACh receptor subtypes and pharmacological profiles. Because of the difficulty in expressing functional insect nACh receptors in heterologous systems, new research tools are needed. Studies on insects resistant to the insecticide imidacloprid and on laboratory-generated hybrid and chimaeric nACh receptors in vitro have provided information about the molecular basis of receptor diversity, neonicotinoid resistance and selectivity. Additionally, recent results indicate that the sensitivity of insect nACh receptors to imidacloprid can be modulated by intracellular phosphorylation mechanisms, which offers a new approach to studying insect nACh receptor pharmacology.

The nicotinic acetylcholine receptor gene family of the honey bee, Apis mellifera.

Nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission and play roles in many cognitive processes. They are under intense research as potential targets of drugs used to treat neurodegenerative diseases and neurological disorders such as Alzheimer's disease and schizophrenia. Invertebrate nAChRs are targets of anthelmintics as well as a major group of insecticides, the neonicotinoids. The honey bee, Apis mellifera, is one of the most beneficial insects worldwide, playing an important role in crop pollination, and is also a valuable model system for studies on social interaction, sensory processing, learning, and memory. We have used the A. mellifera genome information to characterize the complete honey bee nAChR gene family. Comparison with the fruit fly Drosophila melanogaster and the malaria mosquito Anopheles gambiae shows that the honey bee possesses the largest family of insect nAChR subunits to date (11 members). As with Drosophila and Anopheles, alternative splicing of conserved exons increases receptor diversity. Also, we show that in one honey bee nAChR subunit, six adenosine residues are targeted for RNA A-to-I editing, two of which are evolutionarily conserved in Drosophila melanogaster and Heliothis virescens orthologs, and that the extent of editing increases as the honey bee lifecycle progresses, serving to maximize receptor diversity at the adult stage. These findings on Apis mellifera enhance our understanding of nAChR functional genomics and provide a useful basis for the development of improved insecticides that spare a major beneficial insect species.

Subtype-specific actions of beta-amyloid peptides on recombinant human neuronal nicotinic acetylcholine receptors (alpha7, alpha4beta2, alpha3beta4) expressed in Xenopus laevis oocytes.

Two-electrode voltage-clamp electrophysiology has been used to study the actions of two amyloid peptides (Abeta(1-42), Abeta(1-40)) on alpha7, alpha4beta2 and alpha3beta4 recombinant human neuronal nicotinic acetylcholine receptors (nicotinic AChRs), heterologously expressed in Xenopus laevis oocytes. The application of Abeta(1-42) or Abeta(1-40) (1 pM-100 nM) for 5 s does not directly activate expressed human alpha7, alpha4beta2 or alpha3beta4 nicotinic AChRs.Abeta(1-42) and Abeta(1-40) are antagonists of alpha7 nicotinic AChRs. For example, 10 nM Abeta(1-42) and Abeta(1-40) both reduced the peak amplitude of currents recorded (3 mM ACh) to 48+/-5 and 45+/-10% (respectively) of control currents recorded in the absence of peptide. In both the cases the effect is sustained throughout a 30 min peptide application and is poorly reversible.Abeta(1-42) and Abeta(1-40) (10 nM) enhance currents recorded in response to ACh (3 mM) from oocytes expressing alpha4beta2 nicotinic AChRs by 195+/-40 and 195+/-41% respectively. This effect is transient, reaching a peak after 3 min and returning to control values after a 24 min application of 10 nM Abeta(1-42). We observe an enhancement of 157+/-22% of control ACh-evoked current amplitude in response to 100 nM Abeta(1-42) recorded from oocytes expressing alpha4beta2 nicotinic AChRs.Abeta(1-42) and Abeta(1-40) (10 nM) were without antagonist actions on the responses of alpha3beta4 nicotinic AChRs to ACh (1 nM-3 mM).

Ion channels: molecular targets of neuroactive insecticides.

Many of the insecticides in current use act on molecular targets in the insect nervous system. Recently, our understanding of these targets has improved as a result of the complete sequencing of an insect genome, i.e., Drosophila melanogaster. Here we examine the recent work, drawing on genetics, genomics and physiology, which has provided evidence that specific receptors and ion channels are targeted by distinct chemical classes of insect control agents. The examples discussed include, sodium channels (pyrethroids, p,p'-dichlorodiphenyl-trichloroethane (DDT), dihydropyrazoles and oxadiazines); nicotinic acetylcholine receptors (cartap, spinosad, imidacloprid and related nitromethylenes/nitroguanidines); gamma-aminobutyric acid (GABA) receptors (cyclodienes, gamma-BHC and fipronil) and L-glutamate receptors (avermectins). Finally, we have examined the molecular basis of resistance to these molecules, which in some cases involves mutations in the molecular target, and we also consider the future impact of molecular genetic technologies in our understanding of the actions of neuroactive insecticides.

Gene silencing of selected calcium-signalling molecules in a Drosophila cell line using double-stranded RNA interference.

Using the Drosophila melanogaster S2 cell line, stably expressing a cloned muscarinic acetylcholine receptor (AChR), DM1, we have applied gene silencing by double-stranded RNA interference (RNAi) to knock down gene products involved in DM1-mediated calcium signalling. We have shown that RNAi knock down of either the inositol 1,4,5-trisphosphate receptor (Ins(1,4,5)P(3)R), or the SERCA calcium pump in the S2-DM1 cells blocks the increase in intracellular calcium concentration ([Ca(2+)](i)) resulting from activation of the DM1 receptor by 100 microM carbamylcholine (CCh). When RNAi designed to knock down the ryanodine receptor (RyR) was tested, there was no change in the calcium increase detected in response to CCh, consistent with a failure to detect RyRs in S2-DM1 cells using RT-PCR. A combination of RNAi and calcium imaging has provided a direct demonstration of key roles for the Ins(1,4,5)P(3)R and the SERCA pump in the response to DM1 receptor activation.Thus, we show that silencing of individual genes by RNAi in a well characterised Drosophila S2 cell line offers experimental opportunities for cell-signalling studies. Future investigations with RNAi libraries taking full advantage of the wealth of new information available from sequencing the Drosophila genome, may help identify novel components of cell-signalling pathways and functionally linked gene products.

Action of nereistoxin on recombinant neuronal nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes.

Nereistoxin (NTX), a natural neurotoxin from the salivary glands of the marine annelid worm Lumbriconereis heteropoda, is highly toxic to insects. Its synthetic analogue, Cartap, was the first commercial insecticide based on a natural product. We have used voltage-clamp electrophysiology to compare the actions of NTX on recombinant nicotinic acetylcholine receptors (nicotinic AChRs) expressed in Xenopus laevis oocytes following nuclear injection of cDNAs. The recombinant nicotinic AChRs investigated were chicken alpha7, chicken alpha4beta2 and the Drosophila melanogaster/chicken hybrid receptors SAD/beta2 and ALS/beta2. No agonist action of NTX (0.1-100 microM) was observed on chicken alpha7, chicken alpha4beta2 and the Drosophila/chicken hybrid nicotinic AChRs. Currents elicited by ACh were reduced in amplitude by NTX in a dose-dependent manner. The toxin was slightly more potent on recombinant Drosophila/vertebrate hybrid receptors than on vertebrate homomeric (alpha7) or heteromeric (alpha4beta2) nicotinic AChRs. Block by NTX of the chicken alpha7, chicken alpha4beta2 and the SAD/beta2 and ALS/beta2 Drosophila/chicken hybrid receptors is in all cases non-competitive. Thus, the site of action on nicotinic AChRs of NTX, to which the insecticide Cartap is metabolised in insects, differs from that of the major nicotinic AChR-active insecticide, imidacloprid.