RESUMO
In flies and humans, bitter chemicals are known to inhibit sugar detection, but the adaptive role of this inhibition is often overlooked. At best, this inhibition is described as contributing to the rejection of potentially toxic food, but no studies have addressed the relative importance of the direct pathway that involves activating bitter-sensitive cells versus the indirect pathway represented by the inhibition of sugar detection. Using toxins to selectively ablate or inactivate populations of bitter-sensitive cells, we assessed the behavioral responses of flies to sucrose mixed with strychnine (which activates bitter-sensitive cells and inhibits sugar detection) or with L-canavanine (which only activates bitter-sensitive cells). As expected, flies with ablated bitter-sensitive cells failed to detect L-canavanine mixed with sucrose in three different feeding assays (proboscis extension responses, capillary feeding, and two-choice assays). However, such flies were still able to avoid strychnine mixed with sucrose. By means of electrophysiological recordings, we established that bitter molecules differ in their potency to inhibit sucrose detection and that sugar-sensing inhibition affects taste cells on the proboscis and the legs. The optogenetic response of sugar-sensitive cells was not reduced by strychnine, thus suggesting that this inhibition is linked directly to sugar transduction. We postulate that sugar-sensing inhibition represents a mechanism in insects to prevent ingesting harmful substances occurring within mixtures.
Assuntos
Aprendizagem da Esquiva/fisiologia , Drosophila melanogaster/fisiologia , Paladar/fisiologia , Animais , Comportamento Animal/fisiologia , Extremidades/inervação , Extremidades/fisiologia , Feminino , Optogenética , Rodopsina/fisiologia , Sensilas/fisiologia , Células Receptoras Sensoriais/fisiologia , Estimulação QuímicaRESUMO
For all animals, the taste sense is crucial to detect and avoid ingesting toxic molecules. Many toxins are synthesized by plants as a defense mechanism against insect predation. One example of such a natural toxic molecule is L-canavanine, a nonprotein amino acid found in the seeds of many legumes. Whether and how insects are informed that some plants contain L-canavanine remains to be elucidated. In insects, the taste sense relies on gustatory receptors forming the gustatory receptor (Gr) family. Gr proteins display highly divergent sequences, suggesting that they could cover the entire range of tastants. However, one cannot exclude the possibility of evolutionarily independent taste receptors. Here, we show that L-canavanine is not only toxic, but is also a repellent for Drosophila. Using a pharmacogenetic approach, we find that flies sense food containing this poison by the DmX receptor. DmXR is an insect orphan G-protein-coupled receptor that has partially diverged in its ligand binding pocket from the metabotropic glutamate receptor family. Blockade of DmXR function with an antagonist lowers the repulsive effect of L-canavanine. In addition, disruption of the DmXR encoding gene, called mangetout (mtt), suppresses the L-canavanine repellent effect. To avoid the ingestion of L-canavanine, DmXR expression is required in bitter-sensitive gustatory receptor neurons, where it triggers the premature retraction of the proboscis, thus leading to the end of food searching. These findings show that the DmX receptor, which does not belong to the Gr family, fulfills a gustatory function necessary to avoid eating a natural toxin.
Assuntos
Canavanina/farmacologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/fisiologia , Inseticidas/farmacologia , Plantas/metabolismo , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Canavanina/metabolismo , Linhagem Celular , Células Quimiorreceptoras/citologia , Células Quimiorreceptoras/efeitos dos fármacos , Células Quimiorreceptoras/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Comportamento Alimentar/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Hibridização In Situ , Inseticidas/metabolismo , Mutação , Interferência de RNA , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/fisiologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Glutamate is the predominant excitatory neurotransmitter in the vertebrate brain, whereas acetylcholine has been considered to play the same role in insects. Recent studies have, however, questioned the latter view by showing a rather general distribution of glutamate transporters. Here, we describe the expression pattern of the receptor DmGlu-A (DmGluRA), the unique homolog of vertebrate metabotropic glutamate receptors. Metabotropic glutamate receptors play important roles in the regulation of glutamatergic neurotransmission. Using a specific antibody, we report DmGluRA expression in most neuropile areas in both larvae and adults, but not in the lobes of the mushroom bodies. These observations suggest a key role for glutamate in the insect brain.
Assuntos
Química Encefálica/fisiologia , Drosophila/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Alelos , Animais , Biomarcadores/metabolismo , Encéfalo/crescimento & desenvolvimento , Gânglios dos Invertebrados/citologia , Gânglios dos Invertebrados/metabolismo , Imuno-Histoquímica , Larva , Receptores de Glutamato Metabotrópico/genéticaRESUMO
Identification of the neurotransmitters in clock neurons is critical for understanding the circuitry of the neuronal network that controls the daily behavioral rhythms in Drosophila. Except for the neuropeptide pigment-dispersing factor, no neurotransmitters have been clearly identified in the Drosophila clock neurons. Here we show that glutamate and its metabotropic receptor, DmGluRA, are components of the clock circuitry and modulate the rhythmic behavior pattern of Drosophila. The dorsal clock neurons, DN1s in the larval brain and some DN1s and DN3s in the adult brain, were immunolabeled with antibodies against Drosophila vesicular glutamate transporter (DvGluT), suggesting that they are glutamatergic. Because the DN1s may communicate with the primary pacemaker neurons, s-LN(v)s, we tested glutamate responses of dissociated larval s-LN(v)s by means of calcium imaging. Application of glutamate dose dependently decreased intracellular calcium in the s-LN(v)s. Pharmacology of the response suggests the presence of DmGluRA on the s-LN(v)s. Antibodies against DmGluRA labeled dissociated s-LN(v)s and the LN(v) dendrites in the intact larval and adult brain. The role of metabotropic glutamate signaling was tested in behavior assays in transgenic larvae and flies with altered DmGluRA expression in the LN(v)s and other clock neurons. Larval photophobic behavior was enhanced in DmGluRA mutants. For adults, we could induce altered activity patterns in the dark phase under LD conditions and increase the period during constant darkness by knockdown of DmGluRA expression in LN(v)s. Our results suggest that a glutamate signal from some of the DNs modulates the rhythmic behavior pattern via DmGluRA on the LN(v)s in Drosophila.
Assuntos
Proteínas de Drosophila/metabolismo , Ácido Glutâmico/metabolismo , Rede Nervosa/metabolismo , Neurônios/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Animais Geneticamente Modificados , Comportamento Animal/fisiologia , Encéfalo/citologia , Cálcio/metabolismo , Cicloleucina/análogos & derivados , Cicloleucina/farmacologia , Relação Dose-Resposta a Droga , Drosophila , Regulação da Expressão Gênica/fisiologia , Ácido Glutâmico/farmacologia , Larva , Atividade Motora/genética , Neurônios/classificação , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
Microtubules (MTs) play crucial roles during neuronal life. They are formed by heterodimers of alpha and beta-tubulins, which are subjected to several post-translational modifications (PTMs). Amongst them, glutamylation consists in the reversible addition of a variable number of glutamate residues to the C-terminal tails of tubulins. Glutamylation is the most abundant MT PTM in the mammalian adult brain, suggesting that it plays an important role in the nervous system (NS). Here, we show that the previously uncharacterized CG31108 gene encodes an alpha-tubulin glutamylase acting in the Drosophila NS. We show that this glutamylase, which we named DmTTLL5, initiates MT glutamylation specifically on alpha-tubulin, which are the only glutamylated tubulin in the Drosophila brain. In DmTTLL5 mutants, MT glutamylation was not detected in the NS, allowing for determining its potential function. DmTTLL5 mutants are viable and we did not find any defect in vesicular axonal transport, synapse morphology and larval locomotion. Moreover, DmTTLL5 mutant flies display normal negative geotaxis behavior and their lifespan is not altered. Thus, our work identifies DmTTLL5 as the major enzyme responsible for initiating neuronal MT glutamylation specifically on alpha-tubulin and we show that the absence of MT glutamylation is not detrimental for Drosophila NS function.
Assuntos
Proteínas de Drosophila/metabolismo , Sistema Nervoso/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Proteínas de Drosophila/análise , Drosophila melanogaster , Ácido Glutâmico/metabolismo , Mutação , FenótipoRESUMO
The function of the nervous system in complex animals is reflected by the achievement of specific behaviors. For years in Drosophila, both simple and complex behaviors have been studied and their genetic bases have emerged. The neuromuscular junction is maybe one of the prototypal simplest examples. A motor neuron establishes synaptic connections on its muscle cell target and elicits behavior: the muscle contraction. Different muscles in adult fly are related to specific behaviors. For example, the thoracic muscles are associated with flight and the leg muscles are associated with locomotion. However, specific tools are still lacking for the study of cellular physiology in distinct motor neuron subpopulations. Here we decided to use the abdominal muscles and in particular the ventral abdominal muscles (VAMs) in adult Drosophila as new model to link a precise behavior to specific motor neurons. Hence, we developed a new behavioral test based on the folding movement of the adult abdomen. Further, we performed a genetic screen and identify two specific Gal4 lines with restricted expression patterns to the adult motor neurons innervating the VAMs or their precursor cells. Using these genetic tools, we showed that the lack of the VAMs or the loss of the synaptic transmission in their innervating motor neurons lead to a significant impairment of the abdomen folding behavior. Altogether, our results allow establishing a direct link between specific motor neurons and muscles for the realization of particular behavior: the folding behavior of the abdomen in Drosophila.
RESUMO
In vertebrates, several groups of metabotropic glutamate receptors (mGluRs) are known to modulate synaptic properties. In contrast, the Drosophila genome encodes a single functional mGluR (DmGluRA), an ortholog of vertebrate group II mGluRs, greatly expediting the functional characterization of mGluR-mediated signaling in the nervous system. We show here that DmGluRA is expressed at the glutamatergic neuromuscular junction (NMJ), localized in periactive zones of presynaptic boutons but excluded from active sites. Null DmGluRA mutants are completely viable, and all of the basal NMJ synaptic transmission properties are normal. In contrast, DmGluRA mutants display approximately a threefold increase in synaptic facilitation during short stimulus trains. Prolonged stimulus trains result in very strongly increased ( approximately 10-fold) augmentation, including the appearance of asynchronous, bursting excitatory currents never observed in wild type. Both defects are rescued by expression of DmGluRA only in the neurons, indicating a specific presynaptic requirement. These phenotypes are reminiscent of hyperexcitable mutants, suggesting a role of DmGluRA signaling in the regulation of presynaptic excitability properties. The mutant phenotypes could not be replicated by acute application of mGluR antagonists, suggesting that DmGluRA regulates the development of presynaptic properties rather than directly controlling short-term modulation. DmGluRA mutants also display mild defects in NMJ architecture: a decreased number of synaptic boutons accompanied by an increase in mean bouton size. These morphological changes bidirectionally correlate with DmGluRA levels in the presynaptic terminal. These data reveal the following two roles for DmGluRA in presynaptic mechanisms: (1) modulation of presynaptic excitability properties important for the control of activity-dependent neurotransmitter release and (2) modulation of synaptic architecture.
Assuntos
Proteínas de Drosophila/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de Glutamato Metabotrópico/fisiologia , Sinapses/fisiologia , Potenciais de Ação/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster , Estimulação Elétrica , Retroalimentação Fisiológica/fisiologia , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Ácido Glutâmico/metabolismo , Larva , Mutação , Junção Neuromuscular/metabolismo , Junção Neuromuscular/fisiologia , Junção Neuromuscular/ultraestrutura , Plasticidade Neuronal/genética , Plasticidade Neuronal/fisiologia , Técnicas de Patch-Clamp , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Receptores de Glutamato Metabotrópico/genética , Sinapses/metabolismo , Sinapses/ultraestrutura , Transmissão Sináptica/fisiologiaRESUMO
A protein-trap screen using the Drosophila neuromuscular junction (NMJ) as a model synapse was performed to identify genes that control synaptic structure or plasticity. We found that Shaggy (Sgg), the Drosophila homolog of the mammalian glycogen synthase kinases 3 alpha and beta, two serine-threonine kinases, was concentrated at this synapse. Using various combinations of mutant alleles of shaggy, we found that Shaggy negatively controlled the NMJ growth. Moreover, tissue-specific expression of a dominant-negative Sgg indicated that this kinase is required in the motoneuron, but not in the muscle, to control NMJ growth. Finally, we show that Sgg controlled the microtubule cytoskeleton dynamics in the motoneuron and that Futsch, a microtubule-associated protein, was required for Shaggy function on synaptic growth.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila melanogaster/enzimologia , Drosophila melanogaster/crescimento & desenvolvimento , Quinase 3 da Glicogênio Sintase/fisiologia , Junção Neuromuscular/enzimologia , Junção Neuromuscular/crescimento & desenvolvimento , Animais , Proteínas de Drosophila/análise , Proteínas de Drosophila/genética , Drosophila melanogaster/ultraestrutura , Quinase 3 da Glicogênio Sintase/análise , Quinase 3 da Glicogênio Sintase/genética , Larva/enzimologia , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/ultraestrutura , Neurônios Motores/enzimologia , Mutação , Fatores de Crescimento Neural/genética , Plasticidade Neuronal , Terminações Pré-Sinápticas/enzimologia , Terminações Pré-Sinápticas/ultraestruturaRESUMO
Taste is an essential sense for the survival of most organisms. In insects, taste is particularly important as it allows to detect and avoid ingesting many plant toxins, such as L-canavanine. We previously showed that L-canavanine is toxic for Drosophila melanogaster and that flies are able to detect this toxin in the food. L-canavanine is a ligand of DmXR, a variant G-protein coupled receptor (GPCR) belonging to the metabotropic glutamate receptor subfamily that is expressed in bitter-sensitive taste neurons of Drosophila. To transduce the signal intracellularly, GPCR activate heterotrimeric G proteins constituted of α, ß and γ subunits. The aim of this study was to identify which Gα protein was required for L-canavanine detection in Drosophila. By using a pharmacological approach, we first demonstrated that DmXR has the best coupling with Gαo protein subtype. Then, by using genetic, behavioral assays and electrophysiology, we found that Gαo47A is required in bitter-sensitive taste neurons for L-canavanine sensitivity. In conclusion, our study revealed that Gαo47A plays a crucial role in L-canavanine detection.
Assuntos
Canavanina/metabolismo , Drosophila melanogaster/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Sequência de Aminoácidos , Animais , Células Quimiorreceptoras/metabolismo , Sequência Conservada , Proteínas de Drosophila/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/antagonistas & inibidores , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Dados de Sequência Molecular , Toxina Pertussis/farmacologia , Interferência de RNA , PaladarRESUMO
BACKGROUND: The mushroom bodies (MBs) are paired brain centers located in the insect protocerebrum involved in olfactory learning and memory and other associative functions. Processes from the Kenyon cells (KCs), their intrinsic neurons, form the bulk of the MB's calyx, pedunculus and lobes. In young adult Drosophila, the last-born KCs extend their processes in the alpha/beta lobes as a thin core (alpha/beta cores) that is embedded in the surrounding matrix of other mature KC processes. A high level of L-glutamate (Glu) immunoreactivity is present in the alpha/beta cores (alpha/betac) of recently eclosed adult flies. In a Drosophila model of fragile X syndrome, the main cause of inherited mental retardation, treatment with metabotropic Glu receptor (mGluR) antagonists can rescue memory deficits and MB structural defects. RESULTS: To address the role of Glu signaling in the development and maturation of the MB, we have compared the time course of Glu immunoreactivity with the expression of various glutamatergic markers at various times, that is, 1 hour, 1 day and 10 days after adult eclosion. We observed that last-born alpha/betac KCs in young adult as well as developing KCs in late larva and at various pupal stages transiently express high level of Glu immunoreactivity in Drosophila. One day after eclosion, the Glu level was already markedly reduced in the alpha/betac neurons. Glial cell processes expressing glutamine synthetase and the Glu transporter dEAAT1 were found to surround the Glu-expressing KCs in very young adults, subsequently enwrapping the alpha/beta lobes to become distributed equally over the entire MB neuropil. The vesicular Glu transporter DVGluT was detected by immunostaining in processes that project within the MB lobes and pedunculus, but this transporter is apparently never expressed by the KCs themselves. The NMDA receptor subunit dNR1 is widely expressed in the MB neuropil just after eclosion, but was not detected in the alpha/betac neurons. In contrast, we provide evidence that DmGluRA, the only Drosophila mGluR, is specifically expressed in Glu-accumulating cells of the MB alpha/betac immediately and for a short time after eclosion. CONCLUSIONS: The distribution and dynamics of glutamatergic markers indicate that newborn KCs transiently accumulate Glu at a high level in late pupal and young eclosed Drosophila, and may locally release this amino acid by a mechanism that would not involve DVGluT. At this stage, Glu can bind to intrinsic mGluRs abundant in the alpha/betac KCs, and to NMDA receptors in the rest of the MB neuropil, before being captured and metabolized in surrounding glial cells. This suggests that Glu acts as an autocrine or paracrine agent that contributes to the structural and functional maturation of the MB during the first hours of Drosophila adult life.
Assuntos
Drosophila melanogaster/crescimento & desenvolvimento , Ácido Glutâmico/metabolismo , Corpos Pedunculados/crescimento & desenvolvimento , Neurogênese/fisiologia , Neurônios/metabolismo , Transdução de Sinais/fisiologia , Animais , Comunicação Autócrina/fisiologia , Biomarcadores/metabolismo , Comunicação Celular/fisiologia , Diferenciação Celular/fisiologia , Transportador 1 de Aminoácido Excitatório/metabolismo , Glutamato-Amônia Ligase/metabolismo , Imuno-Histoquímica , Corpos Pedunculados/citologia , Neuroglia/metabolismo , Neurônios/citologia , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas Vesiculares de Transporte de Glutamato/metabolismoRESUMO
BACKGROUND: The Dystrophin-glycoprotein complex (DGC) comprises dystrophin, dystroglycan, sarcoglycan, dystrobrevin and syntrophin subunits. In muscle fibers, it is thought to provide an essential mechanical link between the intracellular cytoskeleton and the extracellular matrix and to protect the sarcolemma during muscle contraction. Mutations affecting the DGC cause muscular dystrophies. Most members of the DGC are also concentrated at the neuromuscular junction (NMJ), where their deficiency is often associated with NMJ structural defects. Hence, synaptic dysfunction may also intervene in the pathology of dystrophic muscles. Dystroglycan is a central component of the DGC because it establishes a link between the extracellular matrix and Dystrophin. In this study, we focused on the synaptic role of Dystroglycan (Dg) in Drosophila. METHODOLOGY/PRINCIPAL FINDINGS: We show that Dg was concentrated postsynaptically at the glutamatergic NMJ, where, like in vertebrates, it controls the concentration of synaptic Laminin and Dystrophin homologues. We also found that synaptic Dg controlled the amount of postsynaptic 4.1 protein Coracle and alpha-Spectrin, as well as the relative subunit composition of glutamate receptors. In addition, both Dystrophin and Coracle were required for normal Dg concentration at the synapse. In electrophysiological recordings, loss of postsynaptic Dg did not affect postsynaptic response, but, surprisingly, led to a decrease in glutamate release from the presynaptic site. CONCLUSION/SIGNIFICANCE: Altogether, our study illustrates a conservation of DGC composition and interactions between Drosophila and vertebrates at the synapse, highlights new proteins associated with this complex and suggests an unsuspected trans-synaptic function of Dg.
Assuntos
Drosophila melanogaster/metabolismo , Distroglicanas/metabolismo , Músculo Esquelético/metabolismo , Junção Neuromuscular/metabolismo , Neurotransmissores/metabolismo , Terminações Pré-Sinápticas/metabolismo , Animais , Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Distrofina/metabolismo , Espaço Intracelular/metabolismo , Laminina/metabolismo , Proteínas de Membrana/metabolismo , Junção Neuromuscular/citologia , Subunidades Proteicas/metabolismo , Transporte Proteico , Receptores de Glutamato/metabolismo , Espectrina/metabolismoRESUMO
The metabotropic glutamate receptors (mGluRs) are G-protein-coupled receptors involved in the regulation of glutamatergic synapses. Surprisingly, the evolution-arily distant Drosophila mGluR shares a very similar pharmacological profile with its mammalian orthologues (mGlu2R and mGlu3R). Such a conservation in ligand recognition indicates a strong selective pressure during evolution to maintain the ligand recognition selectivity of mGluRs and suggests that structural constraints within the ligand binding pocket (LBP) would hinder divergent evolution. Here we report the identification of a new receptor homologous to mGluRs found in Anopheles gambiae, Apis mellifera, and Drosophila melanogaster genomes and called AmXR, HBmXR, and DmXR, respectively (the mXRs group). Sequence comparison associated with three-dimensional modeling of the LBP revealed that the residues contacting the amino acid moiety of glutamate (the alpha-COO(-) and NH(3)(+) groups) were conserved in mXRs, whereas the residues interacting with the gamma-carboxylic group were not. This suggested that the mXRs evolved to recognize an amino acid different from glutamate. The Drosophila cDNA encoding DmXR was isolated and found to be insensitive to glutamate or any other standard amino acid. However, a chimeric receptor with the heptahelical and intracellular domains of DmXR coupled to G-protein. We found that the DmX receptor was activated by a ligand containing an amino group, which was extracted from Drosophila head and from other insects (Anopheles and Schistocerca). No orthologue of mXR could be detected in Caenorhabditis elegans or human genomes. These data indicate that the LBP of the mGluRs has diverged in insects to recognize a new ligand.