RESUMO
Flies, like all animals, need to find suitable and safe food. Because the principal food source for Drosophila melanogaster is yeast growing on fermenting fruit, flies need to distinguish fruit with safe yeast from yeast covered with toxic microbes. We identify a functionally segregated olfactory circuit in flies that is activated exclusively by geosmin. This microbial odorant constitutes an ecologically relevant stimulus that alerts flies to the presence of harmful microbes. Geosmin activates only a single class of sensory neurons expressing the olfactory receptor Or56a. These neurons target the DA2 glomerulus and connect to projection neurons that respond exclusively to geosmin. Activation of DA2 is sufficient and necessary for aversion, overrides input from other olfactory pathways, and inhibits positive chemotaxis, oviposition, and feeding. The geosmin detection system is a conserved feature in the genus Drosophila that provides flies with a sensitive, specific means of identifying unsuitable feeding and breeding sites.
Assuntos
Bactérias/química , Drosophila melanogaster/fisiologia , Fungos/química , Naftóis , Células Receptoras Sensoriais/fisiologia , Animais , Células Quimiorreceptoras/metabolismo , Drosophila/fisiologia , Comportamento Alimentar , Feminino , Masculino , Naftóis/química , Condutos Olfatórios , Oviposição , Receptores Odorantes/metabolismoRESUMO
Odor detection in insects is largely mediated by structures on antennae called sensilla, which feature a strongly conserved architecture and repertoire of olfactory sensory neurons (OSNs) and various support cell types. In Drosophila, OSNs are tightly apposed to supporting cells, whose connection with neurons and functional roles in odor detection remain unclear. Coupling mechanisms between these neuronal and non-neuronal cell types have been suggested based on morphological observations, concomitant physiological activity during odor stimulation, and known interactions that occur in other chemosensory systems. For instance, it is not known whether cell-cell coupling via gap junctions between OSNs and neighboring cells exists, or whether hemichannels interconnect cellular and extracellular sensillum compartments. Here, we show that innexins, which form hemichannels and gap junctions in invertebrates, are abundantly expressed in adult drosophilid antennae. By surveying antennal transcriptomes and performing various immunohistochemical stainings in antennal tissues, we discover innexin-specific patterns of expression and localization, with a majority of innexins strongly localizing to glial and non-neuronal cells, likely support and epithelial cells. Finally, by injecting gap junction-permeable dye into a pre-identified sensillum, we observe no dye coupling between neuronal and non-neuronal cells. Together with evidence of non-neuronal innexin localization, we conclude that innexins likely do not conjoin neurons to support cells, but that junctions and hemichannels may instead couple support cells among each other or to their shared sensillum lymph to achieve synchronous activity. We discuss how coupling of sensillum microenvironments or compartments may potentially contribute to facilitate chemosensory functions of odor sensing and sensillum homeostasis.
RESUMO
In insect olfaction, sensitization refers to the amplification of a weak olfactory signal when the stimulus is repeated within a specific time window. In the vinegar fly, Drosophila melanogaster, this occurs already at the periphery, at the level of olfactory sensory neurons (OSNs) located in the antenna. In our study, we investigate whether sensitization is a widespread property in a set of seven types of OSNs, as well as the mechanisms involved. First, we characterize and compare the differences in spontaneous activity, response velocity and response dynamics, among the selected OSN types. These express different receptors with distinct tuning properties and behavioral relevance. Second, we show that sensitization is not a general property. Among our selected OSN types, it occurs in those responding to more general food odors, while OSNs involved in very specific detection of highly specific ecological cues like pheromones and warning signals show no sensitization. Moreover, we show that mitochondria play an active role in sensitization by contributing to the increase in intracellular Ca2+ upon weak receptor activation. Thus, by using a combination of single sensillum recordings (SSRs), calcium imaging and pharmacology, we widen the understanding of how the olfactory signal is processed at the periphery.
Assuntos
Drosophila melanogaster , Neurônios Receptores Olfatórios , Olfato , Animais , Neurônios Receptores Olfatórios/fisiologia , Neurônios Receptores Olfatórios/metabolismo , Drosophila melanogaster/fisiologia , Olfato/fisiologia , Odorantes , Cálcio/metabolismo , Plasticidade Neuronal/fisiologia , Receptores Odorantes/metabolismo , Mitocôndrias/metabolismoRESUMO
The majority of insect olfactory receptors belong to two distinct protein families, the ionotropic receptors (IRs), which are related to the ionotropic glutamate receptor family, and the odorant receptors (ORs), which evolved from the gustatory receptor family. Both receptor types assemble to heteromeric ligand-gated cation channels composed of odor-specific receptor proteins and co-receptor proteins. We here present in short the current view on evolution, function, and regulation of IRs and ORs. Special attention is given on how their functional properties can meet the environmental and ecological challenges an insect has to face.
Assuntos
Neurônios Receptores Olfatórios/fisiologia , Receptores Ionotrópicos de Glutamato/fisiologia , Receptores Odorantes/fisiologia , Animais , InsetosRESUMO
Insect odorant receptors (ORs) show a limited functional expression in various heterologous expression systems including insect and mammalian cells. This may be in part due to the absence of key components driving the release of these proteins from the endoplasmic reticulum and directing them to the plasma membrane. In order to mitigate this problem, we took advantage of small export signals within the human HCN1 and Rhodopsin that have been shown to promote protein release from the endoplasmic reticulum and the trafficking of post-Golgi vesicles, respectively. Moreover, we designed a new vector based on a bidirectional expression cassette to drive the functional expression of the insect odorant receptor coreceptor (Orco) and an odor-binding OR, simultaneously. We show that this new method can be used to reliably express insect ORs in HEK293 cells via transient transfection and that is highly suitable for downstream applications using automated and high-throughput imaging platforms.
Assuntos
Proteínas de Drosophila/metabolismo , Receptores Odorantes/metabolismo , Animais , Membrana Celular/metabolismo , Proteínas de Drosophila/agonistas , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Células HEK293 , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Pentanóis/farmacologia , Plasmídeos/genética , Plasmídeos/metabolismo , Canais de Potássio/genética , Transporte Proteico/efeitos dos fármacos , Receptores Odorantes/agonistas , Receptores Odorantes/genética , Rodopsina/genética , TransfecçãoRESUMO
BACKGROUND: Odor information is processed through multiple receptor-glomerular channels in the first order olfactory center, the antennal lobe (AL), then reformatted into higher brain centers and eventually perceived by the fly. To reveal the logic of olfaction, it is fundamental to map odor representations from the glomerular channels into higher brain centers. RESULTS: We characterize odor response profiles of AL projection neurons (PNs) originating from 31 glomeruli using whole cell patch-clamp recordings in Drosophila melanogaster. We reveal that odor representation from olfactory sensory neurons to PNs is generally conserved, while transformation of odor tuning curves is glomerulus-dependent. Reconstructions of PNs reveal that attractive and aversive odors are represented in different clusters of glomeruli in the AL. These separate representations are preserved into higher brain centers, where attractive and aversive odors are segregated into two regions in the lateral horn and partly separated in the mushroom body calyx. CONCLUSIONS: Our study reveals spatial representation of odor valence coding from the AL to higher brain centers. These results provide a global picture of the olfactory circuit design underlying innate odor-guided behavior.
Assuntos
Drosophila melanogaster/fisiologia , Odorantes , Neurônios Receptores Olfatórios/fisiologia , Olfato , Animais , Encéfalo/fisiologia , Técnicas de Patch-ClampRESUMO
Manduca sexta females attract their mates with the release of a species-specific sex-pheromone blend, with bombykal (E,Z)-10,12-hexadecadienal and (E,E,Z)-10,12,14-hexadecatrienal being the two major components. Here, we searched for the hawkmoth bombykal receptor in heterologous expression systems. The putative pheromone receptor MsexOr1 coexpressed with MsexOrco in Xenopus oocytes elicited dose-dependent inward currents upon bombykal application (10-300 µmol l-1), and coexpressed in HEK293 and CHO cells caused bombykal-dependent increases in the intracellular free Ca2+ concentration. In addition, the bombykal receptor of Bombyx mori BmOr3 coexpressed with MsexOrco responded to bombykal (30-100 µmol l-1) with inward currents. In contrast, MsexOr4 coexpressed with MsexOrco responded neither to bombykal (30-100 µmol l-1) nor to the (E,E,Z)-10,12,14-hexadecatrienal mimic. Thus, MsexOr1, but not MsexOrco and probably not MsexOr4, is the bombykal-binding pheromone receptor in the hawkmoth. Finally, we obtained evidence that phospholipase C and protein kinase C activity are involved in the hawkmoth's bombykal-receptor-mediated Ca2+ signals in HEK293 and CHO cells.
Assuntos
Manduca/fisiologia , Receptores Odorantes , Atrativos Sexuais/farmacologia , Alcadienos/farmacologia , Animais , Bombyx , Sinalização do Cálcio , Cricetulus , Células HEK293 , Humanos , Manduca/citologia , Neurônios Receptores Olfatórios , Oócitos , XenopusRESUMO
Insect odorant receptors are seven transmembrane domain proteins that form cation channels, whose functional properties such as receptor sensitivity are subject to regulation by intracellular signaling cascades. Here, we used the cAMP fluorescent indicator Epac1-camps to investigate the occurrence of odor-induced cAMP production in olfactory sensory neurons (OSNs) of Drosophila melanogaster We show that stimulation of the receptor complex with an odor mixture or with the synthetic agonist VUAA1 induces a cAMP response. Moreover, we show that while the intracellular Ca(2+) concentration influences cAMP production, the OSN-specific receptor OrX is necessary to elicit cAMP responses in Ca(2+)-free conditions. These results provide direct evidence of a relationship between odorant receptor stimulation and cAMP production in olfactory sensory neurons in the fruit fly antenna and show that this method can be used to further investigate the role that this second messenger plays in insect olfaction.
Assuntos
AMP Cíclico/metabolismo , Drosophila melanogaster/fisiologia , Odorantes/análise , Neurônios Receptores Olfatórios/fisiologia , Receptores Odorantes/metabolismo , Transdução de Sinais , Animais , Proteínas de Drosophila/metabolismo , Feminino , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Tioglicolatos/farmacologia , Triazóis/farmacologiaRESUMO
Flying insects are well known for airborne odour tracking and have evolved diverse chemoreceptors. While ionotropic receptors (IRs) are found across protostomes, insect odorant receptors (ORs) have only been identified in winged insects. We therefore hypothesized that the unique signal transduction of ORs offers an advantage for odour localization in flight. Using Drosophila, we found expression and increased activity of the intracellular signalling protein PKC in antennal sensilla following odour stimulation. Odour stimulation also enhanced phosphorylation of the OR co-receptor Orco in vitro, while site-directed mutation of Orco or mutations in PKC subtypes reduced the sensitivity and dynamic range of OR-expressing neurons in vivo, but not IR-expressing neurons. We ultimately show that these mutations reduce competence for odour localization of flies in flight. We conclude that intracellular regulation of OR sensitivity is necessary for efficient odour localization, which suggests a mechanistic advantage for the evolution of the OR complex in flying insects.
Assuntos
Células Quimiorreceptoras/metabolismo , Drosophila melanogaster/fisiologia , Voo Animal/fisiologia , Espaço Intracelular/metabolismo , Odorantes/análise , Animais , Comportamento Animal , Feminino , Masculino , Mutação/genética , Neurônios Receptores Olfatórios/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Transdução de SinaisRESUMO
From worm to man, many odorant signals are perceived by the binding of volatile ligands to odorant receptors that belong to the G-protein-coupled receptor (GPCR) family. They couple to heterotrimeric G-proteins, most of which induce cAMP production. This second messenger then activates cyclic-nucleotide-gated ion channels to depolarize the olfactory receptor neuron, thus providing a signal for further neuronal processing. Recent findings, however, have challenged this concept of odorant signal transduction in insects, because their odorant receptors, which lack any sequence similarity to other GPCRs, are composed of conventional odorant receptors (for example, Or22a), dimerized with a ubiquitously expressed chaperone protein, such as Or83b in Drosophila. Or83b has a structure akin to GPCRs, but has an inverted orientation in the plasma membrane. However, G proteins are expressed in insect olfactory receptor neurons, and olfactory perception is modified by mutations affecting the cAMP transduction pathway. Here we show that application of odorants to mammalian cells co-expressing Or22a and Or83b results in non-selective cation currents activated by means of an ionotropic and a metabotropic pathway, and a subsequent increase in the intracellular Ca(2+) concentration. Expression of Or83b alone leads to functional ion channels not directly responding to odorants, but being directly activated by intracellular cAMP or cGMP. Insect odorant receptors thus form ligand-gated channels as well as complexes of odorant-sensing units and cyclic-nucleotide-activated non-selective cation channels. Thereby, they provide rapid and transient as well as sensitive and prolonged odorant signalling.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Ativação do Canal Iônico/efeitos dos fármacos , Nucleotídeos Cíclicos/farmacologia , Receptores Odorantes/metabolismo , Animais , Butiratos/farmacologia , Cálcio/metabolismo , Linhagem Celular , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Condutividade Elétrica , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Humanos , Ligantes , Nucleotídeos Cíclicos/metabolismo , Odorantes/análise , Técnicas de Patch-Clamp , Receptores Odorantes/química , Receptores Odorantes/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
In recent years, considerable progress has been made in understanding the molecular mechanisms underlying olfaction in insects. Because of the diverse nature of the gene families involved, this process has largely relied on genomic data. As a consequence, studies have focused on a small subset of species with extensive genomic information. For Lepidoptera, a large order historically crucial to olfactory research, this circumstance has mostly limited advances to the domesticated species Bombyx mori, with some progress in the noctuid Heliothis virescens based on a nonpublic partial genome database. Because of the limited behavioral repertoire and nonexistent ecological importance of Bombyx, molecular data on the tobacco hornworm Manduca sexta are of utmost importance, especially with regards to its position as a classical olfactory model and its complex natural behavior. Here we present the use of transcriptomic and microarray data to identify members of the main olfactory gene families of Manduca. To assess the quality of our data, we correlate information on expressed receptor genes with detailed morphological data on the antennal lobe. Finally, we compare the expression of the near-complete transcript sets in male and female antennae.
Assuntos
Manduca , Família Multigênica/genética , Neurópilo/citologia , Receptores Odorantes/metabolismo , Animais , Biologia Computacional , Feminino , Perfilação da Expressão Gênica , Processamento de Imagem Assistida por Computador , Masculino , Análise em Microsséries , Microscopia , Neurópilo/metabolismo , Reação em Cadeia da Polimerase , Receptores Odorantes/genética , Fatores SexuaisRESUMO
Olfaction is a crucial sensory modality in insects and is underpinned by odor-sensitive sensory neurons expressing odorant receptors that function in the dendrites as odorant-gated ion channels. Along with expression, trafficking, and receptor complexing, the regulation of odorant receptor function is paramount to ensure the extraordinary sensory abilities of insects. However, the full extent of regulation of sensory neuron activity remains to be elucidated. For instance, our understanding of the intracellular effectors that mediate signaling pathways within antennal cells is incomplete within the context of olfaction in vivo. Here, with the use of optical and electrophysiological techniques in live antennal tissue, we investigate whether nitric oxide signaling occurs in the sensory periphery of Drosophila. To answer this, we first query antennal transcriptomic datasets to demonstrate the presence of nitric oxide signaling machinery in antennal tissue. Next, by applying various modulators of the NO-cGMP pathway in open antennal preparations, we show that olfactory responses are unaffected by a wide panel of NO-cGMP pathway inhibitors and activators over short and long timescales. We further examine the action of cAMP and cGMP, cyclic nucleotides previously linked to olfactory processes as intracellular potentiators of receptor functioning, and find that both long-term and short-term applications or microinjections of cGMP have no effect on olfactory responses in vivo as measured by calcium imaging and single sensillum recording. The absence of the effect of cGMP is shown in contrast to cAMP, which elicits increased responses when perfused shortly before olfactory responses in OSNs. Taken together, the apparent absence of nitric oxide signaling in olfactory neurons indicates that this gaseous messenger may play no role as a regulator of olfactory transduction in insects, though may play other physiological roles at the sensory periphery of the antenna.
RESUMO
Odorant receptors (ORs) are key specialized units for mate and host finding in moths of the Ditrysia clade, to which 98% of the lepidopteran species belong. Moth ORs have evolved to respond to long unsaturated acetates, alcohols, or aldehydes (Type I sex pheromones), falling into conserved clades of pheromone receptors (PRs). These PRs might have evolved from old lineages of non-Ditrysian moths that use plant volatile-like pheromones. However, a Ditrysian moth called the greater wax moth, Galleria mellonella (a worldwide-distributed pest of beehives), uses C9-C11 saturated aldehydes as the main sex pheromone components (i.e., nonanal and undecanal). Thus, these aldehydes represent unusual components compared with the majority of moth species that use, for instance, Type I sex pheromones. Current evidence shows a lack of consensus in the amount of ORs for G. mellonella, although consistent in that the moth does not have conserved PRs. Using genomic data, 62 OR candidates were identified, 16 being new genes. Phylogeny showed no presence of ORs in conserved PR clades. However, an OR with the highest transcript abundance, GmelOR4, appeared in a conserved plant volatile-detecting clade. Functional findings from the HEK system showed the OR as sensitive to nonanal and 2-phenylacetaldehyde, but not to undecanal. It is believed that to date GmelOR4 represents the first, but likely not unique, OR with a stable function in detecting aldehydes that help maintain the life cycle of G. mellonella around honey bee colonies.
Assuntos
Mariposas , Receptores Odorantes , Atrativos Sexuais , Animais , Abelhas/genética , Mariposas/genética , Atrativos Sexuais/genética , Aldeídos , Receptores de Feromônios/genética , Receptores Odorantes/genéticaRESUMO
Insects detect volatile chemosignals with olfactory sensory neurons (OSNs) that express olfactory receptors. Among them, the most sensitive receptors are the odorant receptors (ORs), which form cation channels passing Ca2+. OSNs expressing different groups of ORs show varying optimal odor concentration ranges according to environmental needs. Certain types of OSNs, usually attuned to high odor concentrations, allow for the detection of even low signals through the process of sensitization. By increasing the sensitivity of OSNs upon repetitive subthreshold odor stimulation, Drosophila melanogaster can detect even faint and turbulent odor traces during flight. While the influx of extracellular Ca2+ has been previously shown to be a cue for sensitization, our study investigates the importance of intracellular Ca2+ management. Using an open antenna preparation that allows observation and pharmacological manipulation of OSNs, we performed Ca2+ imaging to determine the role of Ca2+ storage in mitochondria. By disturbing the mitochondrial resting potential and induction of the mitochondrial permeability transition pore (mPTP), we show that effective storage of Ca2+ in the mitochondria is vital for sensitization to occur, and release of Ca2+ from the mitochondria to the cytoplasm promptly abolishes sensitization. Our study shows the importance of cellular Ca2+ management for sensitization in an effort to better understand the underlying mechanics of OSN modulation.
RESUMO
Insects decode volatile chemical signals from its surrounding environment with the help of its olfactory system, in a fast and reliable manner for its survival. In order to accomplish this task, odorant receptors (ORs) expressed in olfactory sensory neurons (OSNs) in the fly's antenna process such odor information. In order to study such a sophisticated process, we require access to the sensory neurons to perform functional imaging. In this article, we present different preparations to monitor odor information processing in Drosophila melanogaster OSNs using functional imaging of their Ca2+ dynamics. First, we established an in vivo preparation to image specific OSN population expressing the fluorescent Ca2+ reporter GCaMP3 during OR activation with airborne odors. Next, we developed a method to extract and to embed OSNs in a silica hydrogel with OR activation by dissolved odors. The odor response dynamics under these different conditions was qualitatively similar which indicates that the reduction of complexity did not affect the concentration dependence of odor responses at OSN level.
RESUMO
Insects detect volatile chemicals using antennae, which house a vast variety of olfactory sensory neurons (OSNs) that innervate hair-like structures called sensilla where odor detection takes place. In addition to OSNs, the antenna also hosts various support cell types. These include the triad of trichogen, tormogen, and thecogen support cells that lie adjacent to their respective OSNs. The arrangement of OSN supporting cells occurs stereotypically for all sensilla and is widely conserved in evolution. While insect chemosensory neurons have received considerable attention, little is known about the functional significance of the cells that support them. For instance, it remains unknown whether support cells play an active role in odor detection, or only passively contribute to homeostasis, e.g., by maintaining sensillum lymph composition. To investigate the functional interaction between OSNs and support cells, we used optical and electrophysiological approaches in Drosophila. First, we characterized the distribution of various supporting cells using genetic markers. By means of an ex vivo antennal preparation and genetically-encoded Ca2+ and K+ indicators, we then studied the activation of these auxiliary cells during odor presentation in adult flies. We observed acute responses and distinct differences in Ca2+ and K+ fluxes between support cell types. Finally, we observed alterations in OSN responses upon thecogen cell ablation in mature adults. Upon inducible ablation of thecogen cells, we notice a gain in mechanical responsiveness to mechanical stimulations during single-sensillum recording, but a lack of change to the neuronal resting activity. Taken together, these results demonstrate that support cells play a more active and responsive role during odor processing than previously thought. Our observations thus reveal that support cells functionally interact with OSNs and may be important for the extraordinary ability of insect olfactory systems to dynamically and sensitively discriminate between odors in the turbulent sensory landscape of insect flight.
RESUMO
Insect odorant receptors (ORs) detect volatile chemical cues with high sensitivity. These ORs operate as ligand-gated ion channels and are formed by heptahelical OrX and Orco (co-receptor) proteins. A highly conserved calmodulin (CaM) binding site (CBS) 336SAIKYWVER344 within the second intracellular loop of Drosophila melanogaster Orco constitutes a target for regulating OR performance. Here we asked how a point mutation K339N in this CBS affects the olfactory performance of Drosophila melanogaster. We first asked how this mutation would affect the odor responses of olfactory sensory neurons (OSNs). Using Ca2+ imaging in an ex-vivo antenna preparation, we activated all OR (OrX/Orco) expressing neurons using the synthetic agonist VUAA1. In a next attempt, we restricted the OR spectrum to Or22a expressing neurons (Or22a/Orco) and stimulated these OSNs with the ligand ethyl hexanoate. In both approaches, we found that flies carrying the K339N point mutation in Orco display a reduced olfactory response. We also found that the mutation abolishes the capability of OSNs to sensitize by repeated weak odor stimuli. Next, we asked whether OrcoK339N might affect the odor localization performance. Using a wind tunnel bioassay, we found that odor localization in flies carrying the OrcoK339N mutation was severely diminished.
Assuntos
Calmodulina/metabolismo , Proteínas de Drosophila/genética , Receptores Odorantes/metabolismo , Animais , Sítios de Ligação , Calmodulina/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Feminino , Masculino , Odorantes , Percepção Olfatória/fisiologia , Neurônios Receptores Olfatórios/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Ligação Proteica , Receptores Odorantes/genética , Olfato/fisiologia , Tioglicolatos/farmacologia , Triazóis/farmacologiaRESUMO
The Drosophila antennal lobe (AL) has become an excellent model for studying early olfactory processing mechanisms. Local interneurons (LNs) connect a large number of glomeruli and are ideally positioned to increase computational capabilities of odor information processing in the AL. Although the neural circuit of the Drosophila AL has been intensively studied at both the input and the output level, the internal circuit is not yet well understood. An unambiguous characterization of LNs is essential to remedy this lack of knowledge. We used whole cell patch-clamp recordings and characterized four classes of LNs in detail using electrophysiological and morphological properties at the single neuron level. Each class of LN displayed unique characteristics in intrinsic electrophysiological properties, showing differences in firing patterns, degree of spike adaptation, and amplitude of spike afterhyperpolarization. Notably, one class of LNs had characteristic burst firing properties, whereas the others were tonically active. Morphologically, neurons from three classes innervated almost all glomeruli, while LNs from one class innervated a specific subpopulation of glomeruli. Three-dimensional reconstruction analyses revealed general characteristics of LN morphology and further differences in dendritic density and distribution within specific glomeruli between the different classes of LNs. Additionally, we found that LNs labeled by a specific enhancer trap line (GAL4-Krasavietz), which had previously been reported as cholinergic LNs, were mostly GABAergic. The current study provides a systematic characterization of olfactory LNs in Drosophila and demonstrates that a variety of inhibitory LNs, characterized by class-specific electrophysiological and morphological properties, construct the neural circuit of the AL.
Assuntos
Potenciais de Ação/fisiologia , Antenas de Artrópodes/citologia , Interneurônios/citologia , Interneurônios/fisiologia , Animais , Animais Geneticamente Modificados , Biofísica , Colina O-Acetiltransferase/metabolismo , Análise por Conglomerados , Drosophila , Proteínas de Drosophila/genética , Estimulação Elétrica/métodos , Feminino , Proteínas de Fluorescência Verde/genética , Técnicas In Vitro , Modelos Neurológicos , Rede Nervosa/citologia , Rede Nervosa/fisiologia , Análise Numérica Assistida por Computador , Técnicas de Patch-Clamp/métodos , Peptídeos/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Insects detect volatile chemosignals with olfactory sensory neurons (OSNs) that express olfactory receptors. Among them, the most sensitive receptors are the odorant receptors (ORs), which form cation channels passing also Ca2+. Here, we investigate if and how odor-induced Ca2+ signals in Drosophila melanogaster OSNs are controlled by intracellular Ca2+ stores, especially by mitochondria. Using an open antenna preparation that allows observation and pharmacological manipulation of OSNs we performed Ca2+ imaging to determine the role of Ca2+ influx and efflux pathways in OSN mitochondria. The results indicate that mitochondria participate in shaping the OR responses. The major players of this modulation are the mitochondrial Ca2+ uniporter and the mitochondrial permeability transition pore. Intriguingly, OR-induced Ca2+ signals were only mildly affected by modulating the Ca2+ management of the endoplasmic reticulum.
Assuntos
Drosophila melanogaster/metabolismo , Mitocôndrias/metabolismo , Odorantes , Neurônios Receptores Olfatórios/metabolismo , Animais , Canais de Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Transporte de Íons , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Receptores Odorantes/metabolismo , Trocador de Sódio e Cálcio/metabolismoRESUMO
BACKGROUND: Heterologous expression of insect odorant receptors (ORs) in mammalian or insect cells is challenging due to the insufficient intracellular trafficking of ORs and their ability to form leak ion channels. NEW METHOD: We tested whether reducing the Ca2+ levels in the cell culture medium after electroporation by means of a Dulbecco's Modified Eagle Medium (DMEM) without calcium, in a 1:1 ratio with Ham's F12 nutrient mixture, together with 10% fetal calf serum, can improve the success rate of insect OR expression in HEK293 cells. RESULTS: We show that a reduced extracellular Ca2+ level supports functional expression of insect ORs by increasing the fraction of cells responding to the co-receptor agonist VUAA1 and by reducing the intracellular Ca2+ base level of transfected cells. COMPARISON WITH EXISTING METHOD(S): A DMEM formula without calcium outperforms standard DMEM in a 1:1 ratio with Ham's F12 mix and 10% serum, when culturing HEK293 cells transiently expressing insect OR proteins. CONCLUSIONS: Reducing the extracellular Ca2+ level of HEK293 cell culture media after transfection increases the success of functional insect OR expression.