Dopamine neurons modulate pheromone responses in Drosophila courtship learning

Learning through trial-and-error interactions allows animals to adapt innate behavioural ‘rules of thumb’ to the local environment, improving their prospects for survival and reproduction. Naive Drosophila melanogaster males, for example, court both virgin and mated females, but learn through experience to selectively suppress futile courtship towards females that have already mated. Here we show that courtship learning reflects an enhanced response to the male pheromone cis-vaccenyl acetate (cVA), which is deposited on females during mating and thus distinguishes mated females from virgins. Dissociation experiments suggest a simple learning rule in which unsuccessful courtship enhances sensitivity to cVA. The learning experience can be mimicked by artificial activation of dopaminergic neurons, and we identify a specific class of dopaminergic neuron that is critical for courtship learning. These neurons provide input to the mushroom body (MB) γ lobe, and the DopR1 dopamine receptor is required in MBγ neurons for both natural and artificial courtship learning. Our work thus reveals critical behavioural, cellular and molecular components of the learning rule by which Drosophila adjusts its innate mating strategy according to experience.     

An essential role for a CD36-related receptor in pheromone detection in Drosophila

The CD36 family of transmembrane receptors is present across metazoans and has been implicated biochemically in lipid binding and transport. Several CD36 proteins function in the immune system as scavenger receptors for bacterial pathogens and seem to act as cofactors for Toll-like receptors by facilitating recognition of bacterially derived lipids. Here we show that a Drosophila melanogaster CD36 homologue, Sensory neuron membrane protein (SNMP), is expressed in a population of olfactory sensory neurons (OSNs) implicated in pheromone detection. SNMP is essential for the electrophysiological responses of OSNs expressing the receptor OR67d to (Z)-11-octadecenyl acetate (cis-vaccenyl acetate, cVA), a volatile male-specific fatty-acid-derived pheromone that regulates sexual and social aggregation behaviours. SNMP is also required for the activation of the moth pheromone receptor HR13 by its lipid-derived pheromone ligand (Z)-11-hexadecenal, but is dispensable for the responses of the conventional odorant receptor OR22a to its short hydrocarbon fruit ester ligands. Finally, we show that SNMP is required for responses of OR67d to cVA when ectopically expressed in OSNs not normally activated by pheromones. Because mammalian CD36 binds fatty acids, we suggest that SNMP acts in concert with odorant receptors to capture pheromone molecules on the surface of olfactory dendrites. Our work identifies an unanticipated cofactor for odorant receptors that is likely to have a widespread role in insect pheromone detection. Moreover, these results define a unifying model for CD36 function, coupling recognition of lipid-based extracellular ligands to signalling receptors in both pheromonal communication and pathogen recognition through the innate immune system.     

Circadian rhythms in olfactory responses of Drosophila melanogaster.

The core mechanism of circadian timekeeping in arthropods and vertebrates consists of feedback loops involving several clock genes, including period (per) and timeless (tim). In the fruitfly Drosophila, circadian oscillations in per expression occur in chemosensory cells of the antennae, even when the antennae are excised and maintained in isolated organ culture. Here we demonstrate a robust circadian rhythm in Drosophila in electrophysiological responses to two classes of olfactory stimuli. These rhythms are observed in wild-type flies during light-dark cycles and in constant darkness, but are abolished in per or tim null-mutant flies (per01 and tim01) which lack rhythms in adult emergence and locomotor behaviour. Olfactory rhythms are also abolished in the per 7.2:2 transgenic line in which per expression is restricted to the lateral neurons of the optic lobe. Because per 7.2:2 flies do not express per in peripheral oscillators, our results provide evidence that peripheral circadian oscillators are necessary for circadian rhythms in olfactory responses. As olfaction is essential for food acquisition, social interactions and predator avoidance in many animals, circadian regulation of olfactory systems could have profound effects on the behaviour of organisms that rely on this sensory modality.     

Target neuron prespecification in the olfactory map of Drosophila.

In Drosophila and mice, olfactory receptor neurons (ORNs) expressing the same receptors have convergent axonal projections to specific glomerular targets in the antennal lobe/olfactory bulb, creating an odour map in this first olfactory structure of the central nervous system. Projection neurons of the Drosophila antennal lobe send dendrites into glomeruli and axons to higher brain centres, thereby transferring this odour map further into the brain. Here we use the MARCM method to perform a systematic clonal analysis of projection neurons, allowing us to correlate lineage and birth time of projection neurons with their glomerular choice. We demonstrate that projection neurons are prespecified by lineage and birth order to form synapses with specific incoming ORN axons, and therefore to carry specific olfactory information. This prespecification could be used to hardwire the fly's olfactory system, enabling stereotyped behavioural responses to odorants. Developmental studies lead us to hypothesize that recognition molecules ensure reciprocally specific connections of ORNs and projection neurons. These studies also imply a previously unanticipated role for precise dendritic targeting by postsynaptic neurons in determining connection specificity.     

A cyclic nucleotide-gated conductance in olfactory receptor cilia

Olfactory transduction is thought to be initiated by the binding of odorants to specific receptor proteins in the cilia of olfactory receptor cells. The mechanism by which odorant binding could initiate membrane depolarization is unknown, but the recent discovery of an odorant-stimulated adenylate cyclase in purified olfactory cilia suggests that cyclic AMP may serve as an intracellular messenger for olfactory transduction. If so, then there might be a conductance in the ciliary plasma membrane which is controlled by cAMP. Here we report that excised patches of ciliary plasma membrane, obtained from dissociated receptor cells, contain a conductance which is gated directly by cAMP. This conductance resembles the cyclic GMP-gated conductance that mediates phototransduction in rod and cone outer segments, but differs in that it is activated by both cAMP and cGMP. Our data provide a mechanistic basis by which an odorant-stimulated increase in cyclic nucleotide concentration could lead to an increase in membrane conductance and therefore, to membrane depolarization. These data suggest a remarkable similarity between the mechanisms of olfactory and visual transduction and indicate considerable conservation of sensory transduction mechanisms.     

A single population of olfactory sensory neurons mediates an innate avoidance behaviour in Drosophila

All animals exhibit innate behaviours in response to specific sensory stimuli that are likely to result from the activation of developmentally programmed neural circuits. Here we observe that Drosophila exhibit robust avoidance to odours released by stressed flies. Gas chromatography and mass spectrometry identifies one component of this 'Drosophila stress odorant (dSO)' as CO2. CO2 elicits avoidance behaviour, at levels as low as 0.1%. We used two-photon imaging with the Ca2+-sensitive fluorescent protein G-CaMP to map the primary sensory neurons governing avoidance to CO2. CO2 activates only a single glomerulus in the antennal lobe, the V glomerulus; moreover, this glomerulus is not activated by any of 26 other odorants tested. Inhibition of synaptic transmission in sensory neurons that innervate the V glomerulus, using a temperature-sensitive Shibire gene (Shi(ts)), blocks the avoidance response to CO2. Inhibition of synaptic release in the vast majority of other olfactory receptor neurons has no effect on this behaviour. These data demonstrate that the activation of a single population of sensory neurons innervating one glomerulus is responsible for an innate avoidance behaviour in Drosophila.     

雄性大鼠海马多巴胺受体的增龄性改变

采用放射配基结合分析法初步观察了大鼠海马多巴胺(Dopamine,简称DA)受体的增龄性变化及腹腔注射使DA受体不可逆失活的EEDQ(N-乙氧羰基-2乙氧基-2双氧喹啉)后,DA受体的恢复情况.结果表明老年大鼠海马DA受体结合力较青年大鼠差异不明显,注射EEDQ后两者DA受体亲和力均明显降低,但恢复时青年大鼠恢复的速度快于老年鼠者.表明老年大鼠海马DA受体比青年大鼠更容易受到伤害,恢复也更困难,这些变化可能与老年期的一些表现及老年病的学习记忆紊乱有一定关系.     

ZNRF3 promotes Wnt receptor turnover in an R-spondin-sensitive manner

R-spondin proteins strongly potentiate Wnt signalling and function as stem-cell growth factors. Despite the biological and therapeutic significance, the molecular mechanism of R-spondin action remains unclear. Here we show that the cell-surface transmembrane E3 ubiquitin ligase zinc and ring finger 3 (ZNRF3) and its homologue ring finger 43 (RNF43) are negative feedback regulators of Wnt signalling. ZNRF3 is associated with the Wnt receptor complex, and inhibits Wnt signalling by promoting the turnover of frizzled and LRP6. Inhibition of ZNRF3 enhances Wnt/β-catenin signalling and disrupts Wnt/planar cell polarity signalling in vivo. Notably, R-spondin mimics ZNRF3 inhibition by increasing the membrane level of Wnt receptors. Mechanistically, R-spondin interacts with the extracellular domain of ZNRF3 and induces the association between ZNRF3 and LGR4, which results in membrane clearance of ZNRF3. These data suggest that R-spondin enhances Wnt signalling by inhibiting ZNRF3. Our study provides new mechanistic insights into the regulation of Wnt receptor turnover, and reveals ZNRF3 as a tractable target for therapeutic exploration.     

A receptor that mediates the post-mating switch in Drosophila reproductive behaviour

Mating in many species induces a dramatic switch in female reproductive behaviour. In most insects, this switch is triggered by factors present in the male's seminal fluid. How these factors exert such profound effects in females is unknown. Here we identify a receptor for the Drosophila melanogaster sex peptide (SP, also known as Acp70A), the primary trigger of post-mating responses in this species. Females that lack the sex peptide receptor (SPR, also known as CG16752), either entirely or only in the nervous system, fail to respond to SP and continue to show virgin behaviours even after mating. SPR is expressed in the female's reproductive tract and central nervous system. The behavioural functions of SPR map to the subset of neurons that also express the fruitless gene, a key determinant of sex-specific reproductive behaviour. SPR is highly conserved across insects, opening up the prospect of new strategies to control the reproductive and host-seeking behaviours of agricultural pests and human disease vectors.     

An internal thermal sensor controlling temperature preference in Drosophila

Animals from flies to humans are able to distinguish subtle gradations in temperature and show strong temperature preferences. Animals move to environments of optimal temperature and some manipulate the temperature of their surroundings, as humans do using clothing and shelter. Despite the ubiquitous influence of environmental temperature on animal behaviour, the neural circuits and strategies through which animals select a preferred temperature remain largely unknown. Here we identify a small set of warmth-activated anterior cell (AC) neurons located in the Drosophila brain, the function of which is critical for preferred temperature selection. AC neuron activation occurs just above the fly's preferred temperature and depends on dTrpA1, an ion channel that functions as a molecular sensor of warmth. Flies that selectively express dTrpA1 in the AC neurons select normal temperatures, whereas flies in which dTrpA1 function is reduced or eliminated choose warmer temperatures. This internal warmth-sensing pathway promotes avoidance of slightly elevated temperatures and acts together with a distinct pathway for cold avoidance to set the fly's preferred temperature. Thus, flies select a preferred temperature by using a thermal sensing pathway tuned to trigger avoidance of temperatures that deviate even slightly from the preferred temperature. This provides a potentially general strategy for robustly selecting a narrow temperature range optimal for survival.     

Functional genomic analysis of phagocytosis and identification of a Drosophila receptor for E. coli

The recognition and phagocytosis of microbes by macrophages is a principal aspect of innate immunity that is conserved from insects to humans. Drosophila melanogaster has circulating macrophages that phagocytose microbes similarly to mammalian macrophages, suggesting that insect macrophages can be used as a model to study cell-mediated innate immunity. We devised a double-stranded RNA interference-based screen in macrophage-like Drosophila S2 cells, and have defined 34 gene products involved in phagocytosis. These include proteins that participate in haemocyte development, vesicle transport, actin cytoskeleton regulation and a cell surface receptor. This receptor, Peptidoglycan recognition protein LC (PGRP-LC), is involved in phagocytosis of Gram-negative but not Gram-positive bacteria. Drosophila humoral immunity also distinguishes between Gram-negative and Gram-positive bacteria through the Imd and Toll pathways, respectively; however, a receptor for the Imd pathway has not been identified. Here we show that PGRP-LC is important for antibacterial peptide synthesis induced by Escherichia coli both in vitro and in vivo. Furthermore, totem mutants, which fail to express PGRP-LC, are susceptible to Gram-negative (E. coli), but not Gram-positive, bacterial infection. Our results demonstrate that PGRP-LC is an essential component for recognition and signalling of Gram-negative bacteria. Furthermore, this functional genomic approach is likely to have applications beyond phagocytosis.     

An intracellular P2X receptor required for osmoregulation in Dictyostelium discoideum

P2X receptors are membrane ion channels gated by extracellular ATP that are found widely in vertebrates, but not previously in microbes. Here we identify a weakly related gene in the genome of the social amoeba Dictyostelium discoideum, and show, with the use of heterologous expression in human embryonic kidney cells, that it encodes a membrane ion channel activated by ATP (30-100 muM). Site-directed mutagenesis revealed essential conservation of structure-function relations with P2X receptors of higher organisms. The receptor was insensitive to the usual P2X antagonists but was blocked by nanomolar concentrations of Cu2+ ions. In D. discoideum, the receptor was found on intracellular membranes, with prominent localization to an osmoregulatory organelle, the contractile vacuole. Targeted disruption of the gene in D. discoideum resulted in cells that were unable to regulate cell volume in hypotonic conditions. Cell swelling in these mutant cells was accompanied by a marked inhibition of contractile vacuole emptying. These findings demonstrate a new functional role for P2X receptors on intracellular organelles, in this case in osmoregulation.     

Dissecting a circuit for olfactory behaviour in Caenorhabditis elegans

Although many properties of the nervous system are shared among animals and systems, it is not known whether different neuronal circuits use common strategies to guide behaviour. Here we characterize information processing by Caenorhabditis elegans olfactory neurons (AWC) and interneurons (AIB and AIY) that control food- and odour-evoked behaviours. Using calcium imaging and mutations that affect specific neuronal connections, we show that AWC neurons are activated by odour removal and activate the AIB interneurons through AMPA-type glutamate receptors. The level of calcium in AIB interneurons is elevated for several minutes after odour removal, a neuronal correlate to the prolonged behavioural response to odour withdrawal. The AWC neuron inhibits AIY interneurons through glutamate-gated chloride channels; odour presentation relieves this inhibition and results in activation of AIY interneurons. The opposite regulation of AIY and AIB interneurons generates a coordinated behavioural response. Information processing by this circuit resembles information flow from vertebrate photoreceptors to 'OFF' bipolar and 'ON' bipolar neurons, indicating a conserved or convergent strategy for sensory information processing.     

An unconventional myosin in Drosophila reverses the default handedness in visceral organs

The internal organs of animals often have left-right asymmetry. Although the formation of the anterior-posterior and dorsal-ventral axes in Drosophila is well understood, left-right asymmetry has not been extensively studied. Here we find that the handedness of the embryonic gut and the adult gut and testes is reversed (not randomized) in viable and fertile homozygous Myo31DF mutants. Myo31DF encodes an unconventional myosin, Drosophila MyoIA (also referred to as MyoID in mammals; refs 3, 4), and is the first actin-based motor protein to be implicated in left-right patterning. We find that Myo31DF is required in the hindgut epithelium for normal embryonic handedness. Disruption of actin filaments in the hindgut epithelium randomizes the handedness of the embryonic gut, suggesting that Myo31DF function requires the actin cytoskeleton. Consistent with this, we find that Myo31DF colocalizes with the cytoskeleton. Overexpression of Myo61F, another myosin I (ref. 4), reverses the handedness of the embryonic gut, and its knockdown also causes a left-right patterning defect. These two unconventional myosin I proteins may have antagonistic functions in left-right patterning. We suggest that the actin cytoskeleton and myosin I proteins may be crucial for generating left-right asymmetry in invertebrates.