Caractérisation anatomique et fonctionnelle de deux variants d'épissage des récepteurs GluCl. Rôle dans l'apprentissage et la mémoire chez l'abeille, Apis mellifera

Chez les insectes, la transmission glutamatergique est cruciale pour l'apprentissage olfactif. Une part de cette neurotransmission s'effectue par des canaux chlore activés par du glutamate (GluCls), spécifiques des espèces invertébrées. Chez l'abeille, il existe deux variants d'épissage provenant d'un unique gène amel_glucl. Cette étude a permis de caractériser l'expression de ces variants et leurs localisations dans le cerveau, ainsi que leurs rôles dans l'apprentissage et la mémoire olfactive. Les deux variants Amel_GluCl A & B sont exprimés dans le cerveau et ont une localisation différentielle. Le variant A est principalement neuropilaire, le variant B principalement somatique. Cette différence de localisation peut suggérer une implication fonctionnelle différente entre variant. Avec l'injection d'ARN interférents, ont été analysées le taux d'expression des deux protéines d'une part, et la mémoire olfactive d'autre part. Les effets sont transitoires et réversibles. En injectant 24h avant l'apprentissage, chaque traitement ARN interférent aboutit à une diminution des taux de rappel de l'odeur conditionnée 1h après l'apprentissage. Par contre, l'ARN interférent ciblant le variant B a des effets sur la généralisation aux nouvelles odeurs. La neurotransmission glutamatergique via les récepteurs GluCls est nécessaire pour le processus de rappel dans la mémoire olfactive et aurait un rôle dans la saillance des odeurs apprises comparée aux nouvelles odeurs. En conclusion, le rôle des récepteurs est discuté par rapport aux données obtenues chez d'autres espèces. La place des deux variants est discutée dans un schéma global des voies d'apprentissage et mémoire chez l'abeille.In insects, glutamatergic transmission is crucial for olfactory learning. Part of this transmission is mediated via glutamate-gated chloride channels (GluCls), specific to invertebrate species. In honeybee, there are two spliced-variants coming from a single gene amel_glucl. This work allowed the characterization of both GluCl variants' expression and localization them in honeybee brains, and also analyze their roles in olfactory learning and memory. Both variants Amel_GluCl A & B are expressed in honeybee brain and are differentially localized. Variant A is mainly expressed in neuropiles, variant B mainly somatic. This differential localization may suggest a different functional involvement between variants. With RNA interference injections, allowing repression of each subunit's expression, protein level and olfactory memory were analyzed. Biochemical and behavioral effects of siRNAs were both transient and reversible. Injecting 24h before olfactory conditioning, each RNAi treatment induced a decrease of the conditioned odor retrieval 1h after learning; moreover, RNAi against variant B induced a faster generalization to new odors. Overall, glutamatergic transmission mediated by GluCl channels is necessary for olfactory memory retrieval, and may have a role in salience of conditioned stimuli compared to new odors. To conclude, the role of these receptors is discussed along with data available in other species. Several hypotheses will be proposed (e.g. the link between GluCl receptors and nAChRs, and variant B putative glial localization). Finally, the two variants place will be discussed in an overall outline of the learning and memory pathway in honeybee

Studies into the insecticidal activity and mode of action of monoterpenoid constituents of essential oils against the human louse, Pediculus humanus.

The incidence of head lice, Pediculus humanus capitis, in the West is increasing, with insecticide resistance the likely cause. Previous studies have explored the utility of essential oils, and some of their constituent monoterpenoids, in the treatment of head lice. This investigation examines the relative short-term toxicity of a range of different monoterpenoid structures on adult clothing lice, Pediculus humanus cmporis, and their eggs; a structure-activity series was generated for the adults, and partially for eggs. The most effective monoterpenoid against adult lice was (+)-terpinen-4-ol, with monocyclic compounds containing a single 0-atom having the highest activities. Furthermore, there appear to be important differences between the relative potencies of monoterpenoids on lice and eggs, as nerolidol was particularly effective against eggs but completely ineffective against adult lice. To investigate the insecticidal mechanism of action of monterpenoids, various pediculicidal structures were screened for activity on an insect ionotropic GABA receptor, composed of the Drosophila melanogaster subunit RDLac, expressed in Xenopus oocytes. Thymol, eugenol and carvacrol potentiated GABA responses at this receptor, and possessed agonist activity at high concentrations. This is the first documentation of monoterpenoid bioactivity at an isolated insect receptor known to be representative of an in vivo insecticidal target. Thymol also had potentiating and agonist effects on human al(33y2s GABAA receptors expressed in Xenopus oocytes, and 50 (AM thymol induced a leftwards shift of the GABA dose-response curve. Further work on this receptor examined the interaction of thymol with previously characterised modulator binding sites. The results of functional studies suggest that thymol does not share a binding site with benzodiazepines, barbiturates, steroids, propofo1,13-carbolines or loreclezole. The direct involvement of insect GABA receptors in monoterpenoid insecticidal activity remains to be confirmed, as does the location of the thymol binding site on insect and mammalian ionotropic GABA receptors

Effects of azadirachtin on insect and mammalian cultured cells

The aim of this project was to try to find the mode of action of azadirachtin in cultured cells, and to compare its cytotoxicity with some well-known phytochemical pesticides. The results presented here showed that azadirachtin was toxic to the cultured insect cells used (Sf9 and C6/36, derived respectively from Spodoptera frugiperda and Aedes albopictus) even in very low concentrations with an EC50 for the Spodoptera cells estimated at 5x10-9 M, but that the mammalian fibroblast cell line L929 was little affected except at concentrations greater than 10-4M. The other major neem terpenoids, nimbin and salannin, showed low toxicity towards the cultured cells. The neurotoxic pyrethrum showed little effect against the cultures, except for some slight stimulatory effect on growth at 10-8 M. Rotenone, known to inhibit the electron transport chain, effectively inhibited the growth of both insect and mammalian cells. Nicotine, another neurotoxic phytochemical, had little effect on the growth of the cultured cells. It was concluded that while cell growth assessment is not appropriate for all phytochemical pesticides, it is useful for those, such as azadirachtin and rotenone, whose effect is on the essential mechanisms of insect cells in general. Rotenone was used as a positive control to investigate if azadirachtin had its effect on respiration of the cells. Only at the highest feasible concentration of azadirachtin, was there a slight but significant (15%) reduction of respiration which was the same in both insect and mammalian cells. As expected, rotenone inhibited both insect and mammalian cells even at concentrations as low as 10-11 M. When the effects of azadirachtin on the cell cycle were examined by means of cell cytometry, it was shown that the compound arrested the cell-cycle in G2/M phase, and that the effect was related to the concentration. Microscopy confirmed that there was a three-fold increase in the mitotic index after 2 hours of exposure of 2x10-6 M azadirachtin. The similarity of the of the nuclear profiles and cell-cycle distribution to Sf9 cells treated with colchicine, a well-known antimitotic phytochemical, suggested there was a similarity of action between the two compounds

Modulation of sleep and activity in Drosophila: a systems biology approach. Genetics, pharmacology and high-throughput analysis of behaviour

Drosophila melanogaster is a widely used model organism which for the past 20 years has been employed in a variety of contexts to understand aspects of sleep, activity and more complex forms of behaviour. A challenge within the field of behaviour is how to accurately classify and quantify behaviours that arise from an organism when these behaviours are observed in different contexts. Technological advances have increased the availability of quantitative tools which can be used to examine activity and sleep behaviour. With the use of these tools, we can now answer new questions about the underlying mechanisms of behaviour in different conditions. In the thesis herein, I have examined activity and sleep behaviour in two different contexts, utilising some of these new technological tools, including a novel activity monitoring device and statistical classification techniques. In the first part of this thesis, I use this activity monitoring system to elucidate some of the mechanisms involved in homeostatic sleep behaviour. Specifically, I examine the effect of two different sleep deprivation methods on mated and virgin Drosophila females to examine their responses in terms of homeostatic sleep regulation. Using the same methodology and protocol, I then extend this work to exam- ine the role of the neuropeptide, Corazonin, and its receptor, the Corazonin receptor, in these contexts. In the second part of this thesis, I use the same activity monitor- ing system to record the behavioural responses of flies exposed to different insecticide compounds. I then use both a statistical classification technique and behavioural anal- ysis to attempt to classify these compounds based on their mode of action (MoA) and symptomology. Finally, I apply this methodology to answer other biological questions of interest, classifying both rebound sleep and flies with varying genotypes.Open Acces

Dorsal unpaired median neurons of Locusta migratoria express ivermectin- and fipronil-sensitive glutamate-gated chloride channels

Together with type A GABA and strychnine-sensitive glycine receptors, glutamate-gated chloride channels ( GluCl) are members of the Cys-loop family of ionotropic receptors, which mediate fast inhibitory neurotransmission. To date, GluCls are found in invertebrates only and therefore represent potential specific targets for insecticides, such as ivermectin and fipronil. In this study, we identified the functional expression of GluCls in dorsal unpaired median ( DUM) neurons of the metathoracic ganglion of Locusta migratoria using electrophysiological and molecular biological techniques. In whole cell patch-clamped DUM neurons, glutamate-induced changes in both their membrane potentials (current-clamp) and currents (voltage-clamp) were dependent on the chloride equilibrium potential. On continuous application of glutamate, the glutamate-elicited current response became rapidly and completely desensitized. Application of glutamate in the presence of 10 mu M fipronil or 100 mu M picrotoxin reversibly decreased GluCl-mediated currents by 87 and 39%, respectively. Furthermore, 1 mu M ivermectin induced a persistent chloride current, suggesting the expression of ivermectin-sensitive GluCl alpha subunits. A degenerate PCR/RACE strategy was used to clone the full-length L. migratoria LmGlC1 alpha subunit. Finally, RT-PCR experiments demonstrated the presence of LmGluC1 alpha transcripts in locust DUM neurons. Our results provide the first direct evidence of a functional ivermectin-sensitive GluCl channel on the cell surface of DUM neurons of L. migratoria