Characterization of the short neuropeptide F receptor from Drosophila melanogaster

A seven transmembrane G-protein coupled receptor has been cloned from Drosophila melanogaster. This receptor shows structural similarities to vertebrate Neuropeptide Y(2) receptors and is activated by endogenous Drosophila peptides, recently designated as short neuropeptide Fs (sNPFs). sNPFs have so far been found in neuroendocrine tissues of four other insect species and of the horseshoe crab. In locusts, they accelerate ovarian maturation, and in mosquitoes, they inhibit host-seeking behavior. Expression analysis by RT-PCR shows that the sNPF receptor (Drm-sNPF-R) is present in several tissues (brain, gut, Malpighian tubules and fat body) from Drosophila larvae as well as in ovaries of adult females. All 4 Drosophila sNPFs clearly elicited a calcium response in receptor expressing mammalian Chinese hamster ovary cells. The response is dose-dependent and appeared to be very specific. The short NPF receptor was not activated by any of the other tested arthropod peptides, not even by FMRFamide-related peptides (also ending in RFamide), indicating that the Arg residue at position 4 from the amidated C-terminus appears to be crucial for the response elicited by the sNPFs.     

The olfactory circuit of the fruit fly Drosophila melanogaster

The olfactory circuit of the fruit fly Drosophila melanogaster has emerged in recent years as an excellent paradigm for studying the principles and mechanisms of information processing in neuronal circuits. We discuss here the organizational principles of the olfactory circuit that make it an attractive model for experimental manipulations, the lessons that have been learned, and future challenges.     

Processed short neuropeptide F peptides regulate growth through the ERK-insulin pathway in Drosophila melanogaster

The DrosophilasNPF gene regulates growth through the ERK-insulin pathway. sNPF encodes a precursor protein that is processed and produces biologically active sNPF peptides. However, the functions of these peptides are not known. In Drosophila neuronal cells in culture and in flies in vivo, sNPF1 and sNPF2 activated the ERK-insulin pathway and regulated body growth. In addition, the sNPF precursor and the processed sNPF peptide were co-localized in the neurons of the central nervous system. These results indicate that sNPF1 and sNPF2 peptides processed from the sNPF precursor are sufficient for regulating body growth through the ERK-insulin pathway in Drosophila.     

Ambient temperature affects free-flight performance in the fruit fly Drosophila melanogaster

To gain insight into how temperature affects locomotor performance in insects, the limits of flight performance have been estimated in freely flying fruit flies Drosophila melanogaster by determining the maximum load that a fly could carry following take-off. At a low ambient temperature of 15 °C, muscle mechanical power output matches the minimum power requirements for hovering flight. Aerodynamic force production rises with increasing temperature and eventually saturates at a flight force that is roughly equal to 2.1 times the body mass. Within the two-fold range of different body sizes, maximum flight force production during free flight does not decrease with decreasing body size as suggested by standard aerodynamic theories. Estimations of flight muscle mechanical power output yields a peak performance of 110 W kg⁻¹ muscle tissue for short-burst flight that was measured at an ambient temperature of 30 °C. With respect to the uncertainties in estimating muscle mechanical power during free flight, the estimated values are similar to those that were published for flight under tethered flight conditions. Accepted: 5 January 1999     

视觉和嗅觉信号对果蝇食物搜寻行为的协同作用

为了探索视觉和嗅觉信号在昆虫食物搜寻过程中的作用, 本研究利用杨梅和橘子为引诱物, 在实验室条件下测定了嗅觉和视觉信号诱集到的黑腹果蝇Drosophila melanogaster数量, 分析了嗅觉经历对果蝇嗅觉和视觉食物搜寻的影响。发现同源性嗅觉和视觉信号存在的杨梅诱集到的果蝇数量显著大于单一的视觉信号和嗅觉信号, 但异源性嗅觉和视觉信号组合诱集到的果蝇数量和单独的嗅觉信号相似。嗅觉信号预处理不仅能够显著增加嗅觉信号诱集到的果蝇数量, 其中杨梅嗅觉信号对杨梅预处理果蝇的吸引能力与视觉和嗅觉信号存在的杨梅相似, 而且异源性嗅觉和视觉信号组合诱集到的预处理果蝇数量也不低于视觉和嗅觉信号存在的杨梅。另外杨梅嗅觉信号预处理也能够显著增强杨梅视觉信号诱集到的果蝇数量。但嗅觉预处理并不会改变同源性视觉和嗅觉信号组合诱集到的果蝇数量。本研究表明, 果蝇同时利用视觉和嗅觉信号进行食物搜寻, 因此同源性视觉和嗅觉信号在果蝇诱集过程中具有协同作用。另外果蝇具有较强的记忆和学习能力, 能够将记忆中的嗅觉信号应用于食物搜寻。本研究结果不仅有利于我们了解果蝇在自然状态下的食物搜寻机制, 而且有利于开发更有效的果蝇新型诱捕器。     

Critical role for hyperpolarization-activated cyclic nucleotide-gated channel 2 in the AIF-mediated apoptosis

Cellular calcium uptake is a controlled physiological process mediated by multiple ion channels. The exposure of cells to either one of the protein kinase C (PKC) inhibitors, staurosporine (STS) or PKC412, can trigger Ca²⁺ influx leading to cell death. The precise molecular mechanisms regulating these events remain elusive. In this study, we report that the PKC inhibitors induce a prolonged Ca²⁺ import through hyperpolarization‐activated cyclic nucleotide‐gated channel 2 (HCN2) in lung carcinoma cells and in primary culture of cortical neurons, sufficient to trigger apoptosis‐inducing factor (AIF)‐mediated apoptosis. Downregulation of HCN2 prevented the drug‐induced Ca²⁺ increase and subsequent apoptosis. Importantly, the PKC inhibitors did not cause Ca²⁺ entry into HEK293 cells, which do not express the HCN channels. However, introduction of HCN2 sensitized them to STS/PKC412‐induced apoptosis. Mutagenesis of putative PKC phosphorylation sites within the C‐terminal domain of HCN2 revealed that dephosphorylation of Thr⁵⁴⁹ was critical for the prolonged Ca²⁺ entry required for AIF‐mediated apoptosis. Our findings demonstrate a novel role for the HCN2 channel by providing evidence that it can act as an upstream regulator of cell death triggered by PKC inhibitors.     

Alleviation of thermal nociception depends on heat-sensitive neurons and a TRP channel in the brain

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Specificity of olfactory responses in the tephritid fruit fly, Rhagoletis pomonella

Electroantennograms (EAG) were recorded from, and behavior observed of female apple maggot flies, Rhagoletis pomonella (Walsh) (Diptera: Tephritidae), in response to over 60 individual esters. For acetates through decanoates, 2 methylbutyrates, and isobutyrates, we tested homologous series of systematically altered chain lengths. Most of the compounds had been isolated from behaviorally active fractions derived from extracts of volatiles produced by host fruits of R. pomonella. For the acetates through hexanoates, maximum EAG amplitudes were elicited by esters with chain lengths of 9 carbons and for the heptanoates through nonanoates, by 10 carbon esters. Recovery time, or the rate at which the EAG trace returned to the baseline following maximum depolarization, was slowest for straight chain esters that were 9–11 carbons long. Branching of the chain by addition of a methyl group to either side of the ester resulted in a decline in amplitude and a faster recovery time. Compared to EAG results, only 5 esters (butyl and pentyl hexanoate; propyl and butyl heptanoate; propyl octanoate) were highly active in wind tunnel bioassays, demonstrating (1) the hazard of assigning significance to EAG-active compounds without accompanying behavioral data, but more importantly; (2) a high degree of olfactory specificity. Maximum behavioral response was contingent upon the following rules regarding size and structure of the molecule. The ester must (1) be a straight chain; (2) be 10–11 carbons in length; (3) have an acid portion of 6–8 carbons and an alcohol portion of 3–5 carbons. One of the active esters, butyl hexanoate, appears in significant concentrations in the headspace of host fruit and, because this ester elicits such a pronounced behavioral response, our results suggest that R. pomonella is adapted to perception of a compound that is typical of its hosts.

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Résumé Les réactions de R. pomonella à plus de 60 esters différents ont été enregistreées par électroantennogrammes (EAG) et par ovservation du comportement. Pour les acétates, avec les décanoates, les 2-méthylbutyrates et les iso butyrates, nous avons examiné des séries homologues de chaînes aux longueurs systématiquement altérées. La plupart de ces composés ont été isolés à partir des fractions actives sur le comportement, issues des extraits des substances volatiles des fruits des plantes hôtes de cette téphritidae. Pour les acétates, avec les hexanoates, les EAG aux plus grandes amplitudes ont été obtenus avec des esters dont la longueur des chaînes est de 9 carbones, et avec les heptanoates et les nonanoates pour les esters à 10 carbones. Le temps de récupération ou temps mis par l'EAG pour revenir à l'ordonnée de départ après dépolarisation maximum, a été plus lent pour les esters à chaînes droites avec 9 à 11 carbones. La ramification de la chaîne par addition d'un groupe méthyl de chaque côté de l'ester a provoqué une réduction de l'amplitude et une accélération de la récupération. 5 esters seuls provoquent une forte réaction dans le tunnel à vent (hexanoates butilique et pentylique, heptanoates propylique et butylique, octanoate propylique). Ceci montre (1) le risque qu'il y a à attribuer une signification aux substances provoquant une réaction en EAG, lorsqu'il n'y a pas d'observations comportementales parallèles, et, surtout; (2) le degré élevé de spécificité olfactive.La résponse comportementale optimale obéit aux règles suivantes concernant la taille et la structure de la molécule. L'ester doit avoir: (1) une chaîne linéaire; (2) 10–11 carbone de longueur; (3) une portion acide de 6 à 8 C et une portion alcool de 3 à 5 C. Le butyl hexanoate, l'un des esters actifs, présent en concentration suffisante près de fruits utilisés, provoque pour cette raison une réponse comportementale tellement marquée, que nos résultats suggèrent que R. pomonella est adapté à la perception des substances caractéristiques de leurs hôtes.

     

A heat-activated calcium-permeable channel--Arabidopsis cyclic nucleotide-gated ion channel 6--is involved in heat shock responses

An increased concentration of cytosolic calcium ions (Ca²⁺) is an early response by plant cells to heat shock. However, the molecular mechanism underlying the heat‐induced initial Ca²⁺ response in plants is unclear. In this study, we identified and characterized a heat‐activated Ca²⁺‐permeable channel in the plasma membrane of Arabidopsis thaliana root protoplasts using reverse genetic analysis and the whole‐cell patch‐clamp technique. The results indicated that A. thaliana cyclic nucleotide‐gated ion channel 6 (CNGC6) mediates heat‐induced Ca²⁺ influx and facilitates expression of heat shock protein (HSP) genes and the acquisition of thermotolerance. GUS and GFP reporter assays showed that CNGC6 expression is ubiquitous in A. thaliana, and the protein is localized to the plasma membrane of cells. Furthermore, it was found that the level of cytosolic cAMP was increased by a mild heat shock, that CNGC6 was activated by cytosolic cAMP, and that exogenous cAMP promoted the expression of HSP genes. The results reveal the role of cAMP in transduction of heat shock signals in plants. The correlation of an increased level of cytosolic cAMP in a heat‐shocked plant with activation of the Ca²⁺ channels and downstream expression of HSP genes sheds some light on how plants transduce a heat stimulus into a signal cascade that leads to a heat shock response.     

Up-regulation of neuropeptide Y levels and modulation of glutamate release through neuropeptide Y receptors in the hippocampus of kainate-induced epileptic rats

Kainate-induced epilepsy has been shown to be associated with increased levels of neuropeptide Y (NPY) in the rat hippocampus. However, there is no information on how increased levels of this peptide might modulate excitation in kainate-induced epilepsy. In this work, we investigated the modulation of glutamate release by NPY receptors in hippocampal synaptosomes isolated from epileptic rats. In the acute phase of epilepsy, a transient decrease in the efficiency of NPY and selective NPY receptor agonists in inhibiting glutamate release was observed. Moreover, in the chronic epileptic hippocampus, a decrease in the efficiency of NPY and the Y(2) receptor agonist, NPY13-36, was also found. Simultaneously, we observed that the epileptic hippocampus expresses higher levels of NPY, which may account for an increased basal inhibition of glutamate release. Consistently, the blockade of Y(2) receptors increased KCl-evoked glutamate release, and there was an increase in Y(2) receptor mRNA levels 30 days after kainic acid injection, suggesting a basal effect of NPY through Y(2) receptors. Taken together, these results indicate that an increased function of the NPY modulatory system in the epileptic hippocampus may contribute to basal inhibition of glutamate release and control hyperexcitability.     

Natural variation at the Drosophila melanogaster Or22 odorant receptor locus is associated with changes in olfactory behaviour

In insects, many critical olfactory behaviours are mediated by the large odorant receptor (Or) gene family, which determines the response properties of different classes of olfactory receptor neurons (ORNs). While ORN responses are generally conserved within and between Drosophila species, variant alleles of the D. melanogaster Or22 locus have previously been shown to alter the response profile of an ORN class called ab3A. These alleles show potential clinal variation, suggesting that selection is acting at this locus. Here, we investigated if the changes seen in ab3A responses lead to changes in olfactory-related behaviours. We show that variation at the Or22 locus and in the ab3A neurons are not fully compensated for by other ORNs and lead to overall changes in antennal odorant detection. We further show that this correlates with differences in odorant preference behaviour and with differences in oviposition site preference, with flies that have the chimaeric short allele strongly preferring to oviposit on banana. These findings indicate that variation at the Or22 locus leads to changes in olfactory-driven behaviours, and add support to the idea that the ab3A neurons are of especial importance to the ecology of Drosophila flies.     

Resource limitation and responses to rivals in males of the fruit fly Drosophila melanogaster

Diet has a profound direct and indirect effect on reproductive success in both sexes. Variation in diet quality and quantity can significantly alter the capacity of females to lay eggs and of males to deliver courtship. Here, we tested the effect of dietary resource limitation on the ability of male Drosophila melanogaster to respond adaptively to rivals by extending their mating duration. Previous work carried out under ad libitum diet conditions showed that males exposed to rivals prior to mating significantly extend mating duration, transfer more ejaculate proteins and achieve higher reproductive success. Such adaptive responses are predicted to occur because male ejaculate production may be limited. Hence, ejaculate resources require allocation across different reproductive bouts, to balance current vs. future reproductive success. However, when males suffer dietary limitation, and potentially have fewer reproductive resources to apportion, we expect adaptive allocation of responses to rivals to be minimized. We tested this prediction and found that males held on agar‐only diets for 5–7 days lost the ability to extend mating following exposure to rivals. Interestingly, extended mating was retained in males held on low yeast/sugar: no sugar/yeast diet treatments, but was mostly lost when males were maintained on ‘imbalanced’ diets in which there was high yeast: no sugar and vice versa. Overall, the results show that males exhibit adaptive responses to rivals according to the degree of dietary resource limitation and to the ratio of individual diet components.     

Resonance assignment of the cyclic nucleotide binding domain from a cyclic nucleotide-gated K+ channel in complex with cAMP

In order to determine the structure of the 15 kDa cyclic nucleotide binding domain of a cyclic nucleotide-activated K⁺ channel from Mesorhizobium loti and its interaction with cAMP, nearly complete ¹H, ¹³C, and ¹⁵N chemical shifts were assigned.