Dedicated olfactory neurons mediating attraction behavior to ammonia and amines in Drosophila
Edited by Leslie C. Griffith, Brandeis University, Waltham, MA, and accepted by the Editorial Board February 17, 2013 (received for review September 12, 2012)
Abstract
Animals across various phyla exhibit odor-evoked innate attraction behavior that is developmentally programmed. The mechanism underlying such behavior remains unclear because the odorants that elicit robust attraction responses and the neuronal circuits that mediate this behavior have not been identified. Here, we describe a functionally segregated population of olfactory sensory neurons (OSNs) and projection neurons (PNs) in Drosophila melanogaster that are highly specific to ammonia and amines, which act as potent attractants. The OSNs express IR92a, a member of the chemosensory ionotropic receptor (IR) family and project to a pair of glomeruli in the antennal lobe, termed VM1. In vivo calcium-imaging experiments showed that the OSNs and PNs innervating VM1 were activated by ammonia and amines but not by nonamine odorants. Flies in which the IR92a+ neurons or IR92a gene was inactivated had impaired amine-evoked physiological and behavioral responses. Tracing neuronal pathways to higher brain centers showed that VM1-PN axonal projections within the lateral horn are topographically segregated from those of V-PN and DC4-PN, which mediate innate avoidance behavior to carbon dioxide and acidity, respectively, suggesting that these sensory stimuli of opposing valence are represented in spatially distinct neuroanatomic loci within the lateral horn. These experiments identified the neurons and their cognate receptor for amine detection, and mapped amine attractive olfactory inputs to higher brain centers. This labeled-line mode of amine coding appears to be hardwired to attraction behavior.
Author Summary
Fig. P1.
By characterizing a neural circuit that is dedicated to mediating innate attraction behavior in flies, our findings complement the existing knowledge of innate avoidance circuits (4, 5). Our discovery of the anatomical segregation of loci activated by olfactory cues with opposing valence in the LH provides additional insight into sensory coding in both peripheral and central nervous systems and facilitates future characterization of downstream hardwired neural circuits.
To determine whether ligand specificity is preserved in the postsynaptic projection neurons (PNs), which are equivalent to the mammalian mitral/tufted neurons, we measured the calcium response of the projection neurons that innervate the VM1 glomeruli (termed “VM1-PNs”) to a panel of more than 70 odorants. We found that VM1-PNs were activated specifically by ammonia and amines but not by nonamine odorants. This remarkable odor-tuning specificity possibly ensures the faithful relay of olfactory sensory information to motor output. To characterize this neural circuit further, we used the photoactivatable GFP technique (2, 3) to map the axonal projections of VM1-PNs at single-cell resolution. We found that these VM1-PNs form synaptic connections with two higher brain centers downstream of the antennal lobe: the mushroom body (MB) and the lateral horn (LH). The MB is important for the formation of memory, and the LH is proposed to mediate innate behaviors. We found that the MB is dispensable for amine-evoked attraction behavior, suggesting that the LH is likely the brain region required for this behavior. A direct comparison of VM1-PNs with neurons that mediate innate avoidance behaviors showed that VM1-PN projection patterns in the LH are spatially segregated from those of the projection neurons innervating the V and the DC4 glomerulus in the antennal lobe (Fig. P1B), which mediate avoidance to carbon dioxide (4) and acidity (5), respectively. The observation that the VM1 circuit that controls attraction is located in an area that is topographically segregated from those of two innate avoidance circuits suggests that sensory stimuli of opposing valence are represented in spatially distinct areas within the LH.
Using a two-choice behavioral assay in a T-maze, we found that fruit flies are attracted to the smell of ammonia and amines (Fig. P1A). Using in vivo calcium imaging, we identified a single population of olfactory sensory neurons (OSNs) that were activated exclusively by ammonia and amines but not by any nonamine odorants tested so far. These OSNs express IR92a, a member of the chemosensory ionotropic glutamate receptor (IR) family, which is expressed in approximately 25% of insect OSNs in lieu of the seven-transmembrane canonical odorant receptors (1). These neurons project to a pair of glomeruli, termed “VM1,” in the antennal lobe (AL), which is equivalent to the mouse olfactory bulb (Fig. P1B). To determine whether neurons that express IR92a are required for this attraction behavior, we functionally inactivated these neurons via the targeted expression of tetanus toxin. We found that the flies with inactivated IR92a+ neurons exhibited significantly reduced attraction to ammonia and amines. RNAi knockdown of IR92a expression in IR92a+ neurons led to significantly impaired calcium responses and attraction behavior to ammonia and amines, demonstrating that IR92a is necessary for amine-evoked physiological and behavioral responses. Furthermore, ectopic expression of IR92a in other sensory neurons was sufficient to confer amine-evoked responsiveness. Thus, IR92a functions as a receptor for specifically detecting and activating behavior responses to ammonia and amines, which serve as ethologically relevant cues for animals.
Animals exhibit innate, hardwired attraction to certain odors, which differs from starvation-induced attraction to appetitive odors. The mechanisms underlying this behavior remain unclear because the odorants that elicit innate attraction and the neural circuits that mediate this behavior have not been identified. Here we describe a robust innate attraction behavior to ammonia and amines in the fruit fly, Drosophila melanogaster. The attraction to ammonia and amines is not enhanced further by food deprivation. We characterized the olfactory sensory neurons and their downstream neurons that are required for this behavior to occur. Furthermore, we traced the axonal projections of the downstream neurons and compared their projection patterns with those that mediate avoidance behavior. Our study reveals the molecular, cellular, and circuit mechanisms that mediate this behavior and supports a model in which distinct sensory valence is represented at a spatially segregated area in the brain.
This article is a PNAS Direct Submission.
See full research article on page E1321 of www.pnas.org.
Cite this Author Summary as: PNAS 10.1073/pnas.1215680110.
References
1
R Benton, KS Vannice, C Gomez-Diaz, LB Vosshall, Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila. Cell 136, 149–162 (2009).
2
SR Datta, et al., The Drosophila pheromone cVA activates a sexually dimorphic neural circuit. Nature 452, 473–477 (2008).
3
V Ruta, et al., A dimorphic pheromone circuit in Drosophila from sensory input to descending output. Nature 468, 686–690 (2010).
4
GS Suh, et al., A single population of olfactory sensory neurons mediates an innate avoidance behaviour in Drosophila. Nature 431, 854–859 (2004).
5
M Ai, et al., Acid sensing by the Drosophila olfactory system. Nature 468, 691–695 (2010).
Sign up for PNAS alerts.
Get alerts for new articles, or get an alert when an article is cited.
The olfactory system of adult Drosophila melanogaster serves as a genetically and anatomically simple model for studying how sensory input is translated into behavior output. The majority of olfactory sensory neurons (OSNs) express one functional receptor that is composed of an odorant receptor coreceptor (ORCO) and one of ∼45 odorant receptors (ORs) (1–3). About a quarter of the OSNs that do not express ORs recently were shown to express a second family of insect olfactory receptors termed the “ionotropic receptors” (IRs) (4). Each of these OSN types expresses a single or multiple IRs together with a coreceptor, IR8a or IR25a (or rarely both) (5). OR and IR sensory neurons expressing the same receptor send their axonal projections to one or rarely two of ∼50 spatially invariant glomeruli within the antennal lobe (AL) (4, 6–8) where they provide significant synaptic inputs to second-order projection neurons (PNs) that convey the sensory information to higher brain centers such as the mushroom body (MB) and the lateral horn (LH) (9–12).
This genetic and anatomical simplicity of Drosophila has allowed us to further our understanding of a general principle of innate behavior. The emerging characteristics of a neural circuit that is hardwired to a specific behavior include (i) the specificity of the sensory neurons to a single chemical cue and (ii) the discrete spatial representation of that cue in a higher brain center. Indeed, single populations of OSNs activated by ethologically relevant odorants such as cis-vaccenyl acetate, carbon dioxide, and protons induce female receptivity and avoidance behavior (13–16). Intriguingly, each of these sensory neurons is highly specific to each of these odorants. This specificity is unusual, however, given that most odorants stimulate multiple glomeruli and thus would be expected to stimulate multiple pathways. Identifying these distinct populations of OSNs has made it possible to trace the downstream circuits that translate odor detection into behavior (17–19).
Does a single population of OSNs that mediate attraction behavior to a single chemical cue exist? The synchronous output of the AL upon stimulation by complex odorants such as pheromone mixture and floral scent was proposed to generate attraction behavior in moth (20–22). In contrast, manipulation of individual glomeruli activated by apple cider vinegar in Drosophila demonstrated that individual glomeruli, rather than a pattern of active glomeruli, are important for mediating attraction behavior (23). Some monomolecular odorants induce a modest attraction response at low concentrations. However, either the OSNs that are activated by these odorants were not identified (24) or the attraction behavior was exhibited only after prolonged periods of starvation (25).
Ammonia and amines, which are released during the decomposition of protein-containing organic materials, appear to be important for the survival of various species. Rotting fruits emit increasing levels of amines (26, 27); scavenging flies and beetles are lured to such foul odors when they are emitted by the carrion flower to accelerate the pollination process (28); mosquitoes are attracted to ammonia, a metabolite emanating from animal hosts (29, 30); ammonia serves as a cue by which procellariiform seabirds locate their nests and prey (31); and Caenorhabditis elegans displays attraction behavior to ammonium acetate (32). Our knowledge of the ORs that mediate attraction behavior varies from one species to another. Mosquitoes are known to possess grooved-peg sensilla that mediate electrophysiological responses to ammonia and amines (33, 34); unfortunately, the molecular identity of the receptor is unknown. In contrast, the mammalian ORs that detect a small subset of volatile amines have been identified as trace amine-associated receptors (35).
We found that Drosophila display a strong attraction to ammonia and volatile amines, regardless of their internal energy state. This attraction behavior is mediated by a population of OSNs that express a member of the IR family, IR92a, and send their dendrites to coeloconic sensilla in the antenna. Silencing of IR92a+ neurons or IR92a gene impaired the physiological and behavioral responses to these odorants. In vivo calcium-imaging experiments demonstrated that both OSNs and PNs innervating the ventral medial (VM1) glomerulus in the AL were highly specific for ammonia and amines. Because of the simplicity of this initial processing, we traced the VM1–PN pathway to higher brain centers and found that these PNs provide inputs to an area within the LH that is topographically segregated from another LH area that is innervated by V-PN and DC4-PN, which mediate avoidance behaviors to carbon dioxide and acidity, respectively.
Results
Ammonia and Amines Are Strong Insect Attractants.
We observed that wild-type Canton-S flies were highly attracted to ammonia in a binary-choice assay using a T-maze. Attraction to ammonia peaked at an optimal concentration of 0.7% (vol/vol) of the odorant, with an average preference index of more than 50% (Fig. 1A). As the concentration of ammonia in a tube decreased, flies became less attracted to the tube. Conversely, as the odor concentration increased, ammonia became noxious to the responding flies, often resulting in lethality. We also observed attraction behavior in another Drosophila melanogaster strain, Oregon-R, and another species, Drosophila simulans (Fig. 1B). Other insects, such as mosquitoes, were shown to exhibit attraction to ammonia (29, 30). In accord with the previous work, we found that the mosquito species Anopheles stephensi was attracted to a lower concentration of ammonia in a T-maze (Fig. 1B). The underlying mechanism for this attraction behavior is different from the starvation-induced attraction to appetitive odorants (23, 25). Indeed, flies were highly attracted to appetitive odorants such as apple cider vinegar in a T-maze after periods of food deprivation, but attraction to ammonia was not enhanced further by starvation (Fig. 1C). The difference in attractive responses to ammonia versus appetitive odorants supports a view that attraction to ammonia does not depend on the internal energy status of animals and likely is mediated by a hardwired circuit.
Fig. 1.
The ammonia molecule has one nitrogen atom and three hydrogen atoms, which can be replaced by other functional groups to become amines. We therefore asked whether amines also act as attractant to flies. As shown in Fig. 1D, flies exhibited robust attraction responses to volatile amines.
Single Population of OSNs Is Highly Specific to Ammonia and Amines.
We next sought to identify the sensory neurons that are required for attraction to ammonia and amines. Although attraction to ammonia required an olfactory appendage, the antennae, flies mutant for ORCO (36), which is required for the function of about 75% OSNs, were completely normal in attraction to ammonia (Fig. 1E), indicating that sensory neurons other than ORCO+ neurons mediate the attraction response. Because IRs are expressed in OSNs that do not overlap with ORCO expression (4), we tested whether inactivation of IR neurons impairs attraction to ammonia and amines. We engineered flies in which several populations of IR neurons were silenced by targeted expression of tetanus toxin (TNT) (37) under the control of a broadly expressed IR8a promoter driving yeast transcription factor GAL4 (IR8a-GAL4) (Fig. S1A). In a T-maze, these flies showed a significant decrease in attraction to ammonia and trimethyl amine, whereas they exhibited strong avoidance responses to a repellent, CO2 (Fig. 2A). These results demonstrated that the sensory neurons labeled by IR8a-GAL4 are important for the attraction behavior.
Fig. 2.
To identify a population of the IR neurons that respond to ammonia and amines, we performed in vivo calcium imaging of the AL of flies expressing a calcium-sensitive GFP, GCaMP3 with upstream activation sequence (UAS-GCaMP3) (38), driven by the IR8a-GAL4 driver (Fig. S1A). Using this approach, we identified a pair of ventral medial glomeruli, termed “VM1” (6, 39) that was activated by ammonia but not by other odorants (see below and Table S1; also see Fig. 4B). Axonal projection to VM1 originates from sensory neurons that reside in ammonia-responsive coeloconic sensilla (40) and express the IR92a receptor (8). In addition to VM1, we found that another pair of glomeruli, termed “VC3,” was activated by ammonia as well as by other odorants such as alcohols and acetates (Fig. 2B). These glomeruli are innervated by sensory neurons that send their dendrites to coeloconic 3B sensilla and express the OR35a receptor. The broad tuning property of OR35a+ neurons to odorants as measured by calcium imaging is consistent with the previous finding from sensillar electrophysiological recordings (40).
Fig. 3.
Fig. 4.
We next investigated the odor-tuning characteristics of IR92a+ neurons by examining the VM1 glomerular responses to a panel of more than 70 odorants. In vivo calcium imaging of flies carrying IR92a-GAL4 and UAS-GCaMP3 showed that VM1 is activated by all tested amines except cyclohexylamine but not by nonamine odorants (Fig. 2C and Table S1). Ammonia and amines are good proton acceptors or bases. Thus, we tested whether the basicity of amines is the ligand that activates VM1 by measuring the glomerular responses to nonamine bases such as NaOH and NaOCl. None of these bases activated the VM1 glomerulus (Fig. 2C and Table S1). Taken together, these results demonstrated that amine is the chemical moiety that apparently activates IR92a+ neurons.
IR92a+ Neurons Are Required for Attraction to Ammonia and Amines.
Because IR92a+ neurons and OR35a+ neurons were activated by ammonia, we asked whether attraction behavior requires both populations of OSNs or only a single population of either IR92a+ neurons or OR35a+ neurons. We functionally inactivated each population of OSNs by targeted expression of TNT using an IR92a-GAL4 and/or OR35a-GAL4 driver. In a T-maze, flies in which IR92a+ neurons were silenced had significant impairment in attraction to ammonia whereas expression of TNT in OR35a+ OSNs had no effect on attraction behavior (Fig. 3A). The expression pattern of the IR92a-GAL4 is the same as two other independently generated IR92a-GAL4 drivers, which were prominently expressed in the OSNs that innervate VM1 and were weakly labeled in fibers in the subesophageal ganglion (Fig. S1B). Because attraction to ammonia is abolished in antennaless flies (Fig. 1E), the expression in the subesophageal ganglion is unlikely to contribute to the behavior. Furthermore, inactivation of both IR92a+ and OR35a+ neuronal populations led to a defect that was similar to that seen with silencing IR92a+ neurons alone (Fig. 3A). This result suggests that IR92a+ neurons have the primary role in generating attraction behavior and that the contribution from OR35a+ neurons is negligible. Likewise, IR76a+ neurons that were activated by ammonia (and some amines) (8) were not required for attraction responses to the odorant (Fig. S2A). Although an average preference index of IR92a-GAL; UAS-TNT flies was significantly different from that of control flies expressing inactivated TNT (UAS-impTNT), they still showed residual attraction responses to ammonia that were abolished by surgical removal of the antennae (Fig. 1E). The residual responses could result from a minimal contribution from other OSNs or from incomplete silencing of IR92a+ neurons by UAS-TNT.
We further tested whether IR92a+ neurons are required for attraction to amines. Flies carrying IR92a-GAL4 and UAS-TNT showed a significant decrease in attraction to several amines, whereas control flies bearing IR92a-GAL4 and UAS-impTNT exhibited normal attraction behavior in a T-maze (Fig. 3B). Although most amines were attractants, hexylamine, which contains a large side chain and a low vapor pressure, acted as a repellent to flies (Fig. 3B). However, the modest avoidance responses to hexylamine were mediated by sensory neurons other than IR92a+ neurons, because inactivation of IR92a+ neurons still produced avoidance as robust as those in control flies (Fig. 3B). The amine with an extensive hydrocarbon chain might activate other sensory neurons that lead to avoidance behavior. Attraction to ammonia was observed in a more natural setting: an open-arena/trap assay in which flies were released in a container and given a choice between a tube containing 0.7% ammonia and a control tube containing water (Fig. 3C). Consistent with the T-maze results, flies with inactivated IR92a+ neurons had impaired attraction to ammonia in the trap assay but showed normal responses to an unrelated odorant, apple juice (Fig. 3C).
We also found that some complex amines, such as spermidine and putrescine which possess more than one amine functional group, weakly activated the VM1 glomerulus and generated a modest attraction (Fig. S2 C and D). However, inactivation of IR92a+ neurons did not reduce the attraction responses to any of these odorants, indicating that these sensory neurons are not required for attraction to the complex amines (Fig. S2 C and D). These complex amines were shown to activate another population of sensory neurons that express IR41a (8). However, inactivation of IR41a+ neurons did not block attraction to these complex amines in a T-maze either (Fig. S2 C and D).
IR92a Gene Is Necessary and Sufficient for Glomerular Activation to Ammonia and Amines.
Having shown that IR92a+ neurons are necessary for mediating attraction behavior, we next determined whether the IRs expressed in these neurons are required for attraction to ammonia and amines. IR92a+ neurons were shown to coexpress IR76b in the antenna (Fig. 4A) (4). In addition to IR76b, IR8a and IR25a proteins were localized to many of IR92a+ cells in the antennae (Fig. 4A, Upper). We therefore asked whether these IR receptors are required for physiological responses to ammonia and amines. In the IR8a mutant, the VM1 glomerulus was activated normally by ammonia, trimethyl amine, and ethyl amine measured by in vivo calcium imaging, as was the IR25a mutant (Fig. 4B). Similarly, targeted expression of IR76b RNAi by the IR92a-GAL4 driver, which would knock down the endogenous IR76b mRNA levels (Fig. S3A), did not block glomerular responses to these odorants. In contrast, expression of IR92a RNAi in IR92a+ neurons, which eliminated the IR92a expression (Fig. S3B), abrogated the VM1 glomerular activation (Fig. 4B). These results demonstrated that IR92a has a cell-autonomous function as an amine-sensing receptor that is required for VM1 activation.
To determine the role of IR92a as a putative amine receptor, we ectopically expressed IR92a in several populations of IR sensory neurons and asked whether IR92a is capable of conferring amine sensitivity in neurons that normally are insensitive to ammonia and amines. We misexpressed IR92a by using IR8a-GAL4 or IR25a-GAL4 driver, which is expressed in several IR glomeruli in the antennal lobe, and measured their glomerular responses to ammonia, trimethyl amine, and ethyl amine. Consistent with the previous experiment (4), we found that most neurons ectopically expressing IR92a by IR8a-GAL4 or IR25a-GAL4 were activated by these odorants (Fig. 4C and Fig. S4A). In contrast, misexpression of IR92a in non-IR OSNs by the ORCO-GAL4 driver did not induce ectopic response to ammonia and amines (Fig. S4B). Nor were we able to reconstitute the function of IR92a in the heterologous expression systems such as Xenopus oocytes and HEK293 cells. Together, these results demonstrated that misexpression of IR92a alone in coeloconic neurons is sufficient to induce ectopic responsiveness to ammonia and amines, suggesting that IR92a functions with a cofactor specifically expressed in coeloconic neurons to act as the direct determinant. Such a cofactor is not likely to be IR8a or IR25a, because neither IR8a nor IR25a is required for the function of IR92a in ammonia detection (Fig. 4B) (5).
IR92a Is Required for Attraction to Ammonia and Amines.
Consistent with the physiological defects was the observation that tissue-specific knockdown of IR92a by using two different IR92a RNAi lines in IR92a+ cells impaired attraction to ammonia, trimethyl amine, and ethyl amine but did not affect attraction responses to apple cider vinegar (Fig. 4D). In contrast, IR8a and IR25a mutants as well as IR76b knock-down flies exhibited normal responses to ammonia and amines (Fig. 4 D and E) despite their expression in IR92a+ neurons. Therefore, consistent with the requirement of IR92a for amine-evoked VM1 activation measured by in vivo calcium imaging, IR92a, but not other IR receptors, is required in VM1 neurons for attraction to ammonia and amines.
Specificity of VM1-PN Responses to Ammonia and Amines.
Having shown that IR92a+ sensory neurons projecting to VM1 are highly specific to ammonia and amines, we asked whether the ligand specificity is preserved in the second-order VM1-PNs, which are postsynaptic to IR92a+ neurons. GH146-GAL4 and NP0225-GAL4 drivers are expressed in a large fraction of PNs including VM1-PNs (Fig. S5A) (17, 41). Our behavior experiments showed that flies in which the PNs were inactivated using GH146-GAL4 or NP0225-GAL4 had impaired attraction to ammonia in a T-maze (Fig. 5A and Fig. S5B). This result confirms that the PNs transmitting the odor-evoked signals to higher brain centers are contained within GH146-GAL4 and NP0225-GAL4 lines. When we measured glomerular responses of VM1-PNs to a panel of more than 70 odorants, we found that VM1-PNs were activated specifically by ammonia and amines but not by nonamine odorants (Fig. 5 B and C and Table S1), as is consistent with previous whole-cell patch-clamp recordings from a VM1-PN (11). These results illustrated that VM1-PNs have odor-tuning characteristics similar to those of VM1-OSNs and that the major inputs to VM1-PNs originate from VM1-OSNs. Because most PNs have broader odor tuning than their presynaptic OSNs (11, 42), the remarkably specific odor-tuning property of VM1-PNs implies that these neurons are dedicated for ammonia and amine attraction behavior.
Fig. 5.
VM1-PN Projection Pattern in Higher Brain Centers.
We next examined the projection patterns of VM1-PNs to higher brain centers by using a genetically encoded photoactivable GFP (PA-GFP) (18, 43). PA-GFP emits strong fluorescence after it is photoconverted by a two-photon laser and diffuses out from cell bodies to axons and dendrites. Thus, the neuronal processes become visualized. We made flies carrying a PN driver, NP0225-GAL4, and an improved version of PA-GFP (UAS-SPA/UAS-C3PA) (19) and photo-illuminated the VM1 glomerulus in these animals. Consistent with the previous work that used the mosaic analysis with a repressible cell marker labeling technique (17), we found that each VM1-PN projects to higher brain centers with a stereotypic innervation pattern. The VM1-PN innervates the MB, making arborization with approximately three discernible boutons in the calyx, and extends its axon to the lateral (and posterior) region of the LH with a stereotypic branching pattern (Fig. 6A).
Fig. 6.
To determine whether VM1-PN inputs to the MB are important for the attraction behavior, we sought to ablate the MB structure through hydroxyurea (HU) treatment at a critical period of development (44) and to test these flies in the behavior assay. In flies bearing a MB-specific driver, OK107-GAL4 (45) and UAS-mCD8GFP, the MB lobes and neuropil including the calyx were visualized by GFP fluorescence. Upon HU treatment, all these structures were thoroughly ablated, leaving a small portion of the γ lobe intact (Fig. S6). These MB-ablated flies still exhibited robust attraction to ammonia (Fig. 6B), indicating that the MB is dispensable for this attraction behavior, although VM1-PN inputs to the MB could still be important for olfactory conditioning to ammonia and amines.
Because the MB is not required for attraction behavior, VM1-PN inputs to the LH likely are important for the manifestation of this odor-evoked innate behavior, as is consistent with the notion that the LH encodes the valence of sensory information (46–48). We next asked whether the VM1-PN innervation pattern in the LH is topographically distinct from another known pathway that mediates an innate avoidance behavior. The OSNs projecting to the V glomeruli in the AL are highly dedicated to producing avoidance responses to CO2 (14, 15, 49). Photo-activating the V glomerulus of flies carrying UAS-SPA, UAS-C3PA, and a cholinergic Cha-GAL4 driver (50) uncovered at least four distinct classes of output V-PNs that extend their axonal processes via inner, medial, and outer antenno-cerebral tracts (iACT, mACT, and oACT, respectively) to different parts of the brain including the LH (Fig. S7C). To compare the projection patterns of VM1-PNs and those of V-PNs in the LH accurately, we sequentially photo-illuminated a VM1-PN and a V-PN in the brain of a fly carrying UAS-SPA/UAS-C3PA, NP0225-GAL4, and a previously reported V-PN driver, NP7273-GAL4 (51). As shown in Fig. 6 C–F and Movie S1, the medially located, crescent-shaped axonal termini of V-PN do not overlap with the laterally positioned termini of the VM1-PN axon. To test whether axonal terminals from other V-PN types also are segregated from the axonal terminals of VM1-PNs, we photo-activated the VM1 and V glomeruli in a fly carrying a pan-neuronal nSyb-GAL4 driver and UAS-SPA. We found that axonal terminals from other V-PNs do not overlap with the axonal terminals of VM1-PNs in the LH (Fig. S7 A and B).
Furthermore, the sensory neurons that project to the DC4 glomeruli in the AL also are dedicated to generating avoidance behavior to acidity (16). Using the same approach, we labeled a DC4-PN and a VM1-PN in an animal that carried UAS-SPA, UAS-C3PA, and Cha-GAL4 (Fig. 6 G–J). The projection pattern of the VM1-PN labeled by Cha-GAL4 driver is highly similar to that of the VM1-PN labeled by NP0225-GAL4 or nSyb-GAL4, which innervates the lateral (and posterior) region of the LH (Fig. 6G). In contrast, the DC4-PN innervates the medial (and anterior) region of the LH with a crescent-shaped branching pattern highly reminiscent of that of the V-PN (Fig. 6 H and I). Moreover, their axonal termini do not have any overlap with those of VM1-PNs (Fig. 6 I and J and Movie S2). These results demonstrated that the neuronal circuit mediating attraction to amines is topographically segregated from the neuronal circuits activating avoidance behaviors to CO2 and acidity within the LH.
Discussion
Drosophila, like other insects, exhibits a strong attraction response to the monomolecular odorants ammonia and amines. These odorants often emanate from decaying flesh and organic materials and thus might serve as ethologically relevant chemical cues that animals can use to meet their nutritional needs. This attraction behavior, which was observed in both T-maze and open-arena/trap assays, is mediated by a single population of IR92a+ sensory neurons that are activated specifically by ammonia and amines. The moiety that these odorants appear to have in common is a single nitrogen atom with a lone electron pair that forms three separate single bonds with hydrogen or carbon atoms. The separate nature of these single bonds is supported by the observation that benzyl cyanide, a nitrile that possesses a nitrogen moiety with a single triple bond, failed to activate IR92a+ neurons (Table S1). Complex amines (i.e., amines containing more than one amine group) weakly excited IR92a+ neurons and induced modest attraction responses, but inactivation of these neurons did not impair the attraction behavior, indicating that attraction to complex amines is mediated through another sensory pathway.
It is important to note that the contribution of IR92a+ neurons to amine-evoked behaviors differs from that of OR35a+ or IR76a+ neurons. The difference indicates a distinction between the sensory neurons that are required for mediating a behavioral response to an odor stimulus and those that merely are activated by the stimulus. The cardinal feature of IR92a+ neurons that distinguishes them from the other neurons is their specificity to a single modality of odorants (ammonia and amines) that share a single chemical characteristic, a nitrogen atom with three chemical bonds. These odorants are released during degradation of proteins and serve as a key sensory cue to reveal an abundance of nitrogen, a primary constituent of all living organisms. To activate a hardwired behavioral response to such odorants, dedicated sensory neurons must have evolved to support a labeled-line mode of odor coding. Indeed, several lines of evidence suggest the existence of such “specialist” sensory neurons in the taste (52) and olfactory systems (13, 14, 16, 53) that are endowed with narrow tuning properties and are dedicated to mediating innate behaviors. Conversely, sensory neurons that are not specific to ammonia and amines (e.g., OR35a+ and IR76a+ neurons) do not play a role in mediating attraction behavior. Consistent with this view, we found that the DP1l glomerulus innervated by IR75a+ sensory neurons that were activated by some, but not all, acids appeared to be dispensable for avoiding acids (Fig. S2B), whereas DC4 neurons that are highly specific for all acids are indeed the determinant of acid avoidance (16).
It has been proposed that the antennal IR family emerged early in evolution as the first olfactory receptor family for detecting environmental stimuli (54). IR sensory neurons other than those expressing OR35a act as specialist neurons that are narrowly tuned to a few select stimuli such as acids, amines, or water, whereas OR neurons generally are broadly tuned to many odorants (8, 40, 55, 56). In previous studies, two other populations of IR neurons that project to the DC4 and VL2a glomeruli in the AL were found to be highly selected for the detection of single chemical cues, specifically proton and phenylacetaldehyde (or phenylacetic acid), and hardwired to specific behavioral outputs, namely avoidance and courtship (16, 53). These findings are consistent with the speculation that IR-expressing coeloconic sensilla have an ancient origin (57). Such ligand specificity and dedicated circuitry probably reflect the most basic needs of an ancestral insect (40).
In calcium-imaging experiments carried out using GCaMP3, VM1-PNs retained the extreme selectivity of their OSN inputs, a hallmark of the labeled-line mode of amine coding. These VM1-PNs were activated robustly by ammonia and amines, whereas other odorants generated little or no response. The odors to which the VM1-PNs responded were very similar to those to which the VM1-OSNs responded, with a few exceptions. For example, several complex amines, including spermidine, spermine, and putrescine, weakly excited VM1-OSNs but failed to activate VM1-PNs, possibly because the VM1-PN driver is too weak to induce the sufficient level of GCaMP expression to detect the calcium transient or because the olfactory signals evoked by complex amines are not transmitted through this pathway. In contrast, butyraldehyde and isobutyraldehyde failed to activate VM1-OSNs, but they did stimulate VM1-PNs after an approximately 1-s delay (Table S1). The delayed response was distinct from the response observed during amine-evoked activation and may have been caused by rebound effect. The narrow tuning of VM1-PNs likely is important for preventing nonamine odorants from inducing attraction behaviors and thus may ensure a tight connection between stimulus and behavior.
Our finding that the area to which VM1-PNs project within the LH is topographically segregated from the V-PN and DC4-PN target area indicates that valence—i.e., attraction and avoidance (to amines and CO2/acidity, respectively)—is represented by its own field within the LH. Chemosensory receptors of the olfactory and taste systems that are not spatially organized at the periphery converge upon a fixed cortical map where neurons with similar response profiles are clustered and probably encode the value of chemosensory signals. Taste-receptor cells, for example, provide inputs from the four basic tastes to four distinct areas in the insular cortex of the mammalian brain (58). Similarly, a gustatory map of sweet and bitter taste exists in the subesophageal ganglion of Drosophila (59, 60). The sensory pathways that mediate innate avoidance responses in mice converge onto the dorsal olfactory bulb, which projects primarily to the cortical amygdala (61, 62). Furthermore, the receptors for hot and cold sensation project onto distinct but adjacent glomeruli, thereby forming a thermotopic map in the Drosophila brain (63). Such topographic organization of sensory inputs in the higher brain center would be important for decoding sensory information and effectively activating the appropriate set of motor neurons because there are a far fewer descending neurons than sensory neurons in Drosophila. The fact that the axon termini of VM1-PNs and V-PNs (and DC4-PNs) are segregated within the LH suggests that third-order LH neurons postsynaptic to VM1-PNs may respond to similar stimuli and might be exclusive for attraction behavior. Likewise, the LH neurons postsynaptic to V-PNs and DC4-PNs would be innervated by another pathway mediating avoidance behavior. Further mapping of third-order neurons will greatly advance our understanding of how valence is encoded in the brain.
Experimental Procedures
Transgenic Flies and Fly Stocks.
IR-GAL4 constructs were generated by cloning DNA sequences immediately upstream of the ATG start codon into the pCaSpeR-AUG-Gal4 vector (1). The lengths of DNA sequence used for generating IR92a-GAL4 and IR76b-GAL4 are 9,062 bp and 663 bp, respectively. IR8a-GAL4 was generated by cloning the DNA sequence upstream of the start codon (676 bp) fused directly to the sequences from IR8a intron 1 (59 bp), intron 2 (131 bp), and intron 3 (789 bp) into pCaSpeR-AUG-Gal4. Transgenic flies were generated by Bestgene Inc. Transgenic lines expressing UAS-RNAi against IR92a (VDRC IDs: 30110 and 101920) and IR76b (VDRC ID: 8433) were obtained from the VDRC stock center. Flies carrying RNAi lines also bear UAS-Dicer2. Other flies were described previously: UAS-GCaMP 3.0 (38); ORCO−/− (36); OR35a-GAL4 (7); IR41a-GAL4, IR76a-GAL4, IR75a-GAL4, IR8a−/−, and IR25−/− (4, 5, 8); GH146-GAL4 (64); NP0225-GAL4 (41); OK107-GAL4 (45); Cha-GAL4 (50); UAS-C3PA and UAS-SPA (19); and UAS-TNT and UAS-ImpTNT (37).
Behavioral Analysis.
Odorants were purchased from Sigma with >95% purity. Unless otherwise indicated in figure legends, the concentrations of odorants used in T-maze assay were ammonia, 0.7% (vol/vol); apple cider vinegar, 25% (vol/vol); carbon dioxide, 1% (vol/vol); acetic acid, 9% (vol/vol); other odorants, 1% in H2O (vol/vol).
Flies were maintained on conventional food containing molasses and cornmeal at 25 °C. Newly eclosed male flies were collected and aged for 10–14 d before behavioral testing. Approximately 20–25 flies were tested in each T-maze experiment. Flies were wet-starved for 23 h before conducting experiments using apple cider vinegar. All behavioral experiments were performed at 22–23 °C with 40–60% humidity.
For the T-maze assay, 10 uL of odorant solution or water as control were dispensed onto a piece of filter paper (5 × 5 mm) placed in a 14-mL tube (no. 149598; Fisher). Tubes were sealed with parafilm and allowed to stand for at least 10 min before the experiment. Flies were introduced into the elevator of a T-maze by gentle tapping. A tube containing odorant was placed onto one side of the T-maze, and a control tube was placed onto the other side. Flies in the elevator then were given a choice between the odor side and the control side for 45–55 s. The preference index was calculated as (the number of flies in odor tube − the number of flies in control tube)/the number of flies in both tubes.
The open-arena trap assay is different from those previously reported (36, 65). Our arena is smaller in size and has horizontally placed traps to facilitate fly entrance. To set the traps in the arena, 500 uL of ammonia solution (0.7%), apple juice (100%), or water as control was blotted onto a piece of KimWipe (Kimberly-Clark) that was placed at the end of a 14-mL tube (no. 149598; Fisher). The tube then was capped with three layers of parafilm and two layers of aluminum foil. A trimmed 200-uL pipette tip (∼3 cm in length) was inserted through the cap forming a one-way tunnel through which flies could enter the tube but were unlikely to exit. A pair of tubes containing odorant solution and water was placed in the middle of a glass dish with a diameter of 150 mm and a height of 20 mm (no. 89000–326; VWR). For each trial, ∼50 male flies (8–15 d old) were cold-anesthetized and transferred to the dish, which then was covered with a transparent plastic lid (no. 25384–326; VWR). Flies were allowed to choose between the odorant-containing tube and the control tube in a temperature-controlled (24–25 °C) room overnight. The preference index was calculated as (the number of flies in ammonia-containing tube − the number of flies in water-containing tube)/the number of flies in both tubes.
In Vivo Calcium Imaging.
Live fly preparation and in vivo calcium-imaging experiments were performed as previously described (16). Flies described in Fig. 4 were 13–15 d old, and their genotypes are: IR92a-RNAi (IR92a-GAL4, UAS-GCaMP3, UAS-Dicer2; UAS-IR92a-RNAi); IR76b-RNAi (IR92a-GAL4, UAS-GCaMP3, UAS-Dicer2; UAS-IR76b-RNAi); IR8a−/− (IR8a1; IR92a-GAL4, UAS-GCaMP3); and IR25a−/− (IR92a-GAL4, UAS-GCaMP3, IR25a1). Flies used in Fig. 5B were 18–20 d old carrying single copies of NP0225-GAL4 and UAS-GCaMP3.
PA-GFP Labeling.
Brains from flies that were <1 d old were dissected in a buffer similar to adult hemolymph (18) and were immobilized by pinning to silicone gel. Before photo-conversion, the low-intensity fluorescence of PA-GFP protein can be visualized by two-photon illumination at a wavelength of 925 nm. Both VM1 and V glomeruli were easily identifiable by their position within the antennal lobe.
For the labeling of a single VM1-PN, the brain from a fly carrying NP0225-GAL4, UAS-SPA; UAS-C3PA was used. The 3D structure of the VM1 glomerulus was stimulated using weak photo-converting light at a wavelength of 715 nm for 15 cycles with 30-s intervals between cycles. After the photo-conversion, PA-GFP proteins within the glomerulus enhance in fluorescent intensity, diffuse, and label the cell bodies of VM1-PNs. Then stronger photo-converting light was applied to the single-labeled PN cell body for 60 cycles with 30-s intervals, leading to robust labeling of the entire structure of the cell including axonal and dendritic terminals. For the simultaneous labeling of VM1-PN and V-PN in the same brain, a fly carrying NP0225-GAL4; NP7273-GAL4 or nSyb-GAL4 and UAS-SPA/ UAS-C3PA was used. After a single VM1-PN was labeled by photo-stimulation, a fluorescence micrograph with a 3D stack was taken to record the position of the VM1-PN axonal terminals in the MB and the LH. Then the axonal processes from the V glomerulus of the same brain were labeled by the same method. A second micrograph with a 3D stack was taken and compared with the first one. The axonal projections of the labeled PNs were traced using the Vaa3d software (66). Simultaneous labeling of VM1-PN and DC4-PN was carried out using the same approach in a fly carrying Cha-Gal4, UAS-SPA, and UAS-C3PA.
Immunohistochemistry.
Anti-IR8a and anti-IR25a antibodies were kindly provided by Richard Benton. Immunostaining of the fly brains and the cryosectioned antennae were performed as previously described (16).
HU Treatment.
Newly hatched fly larvae were subjected to treatment with HU (HU+) or mock treatment (HU−) as previously described (67). Adult flies (10–12 d old) from the HU+ and HU− groups then were tested in a T-maze. The extent of HU-mediated MB ablation was determined by the expression of UAS-mCD8GFP under the control of a MB-specific OK107-GAL4 driver.
Note
The authors declare no conflict of interest.
Acknowledgments
We thank Ruth Lehmann for sharing her two-photon microscope; Niels Ringstad and Ip Ninan for assistance with Xenopus oocyte and HEK293 cell electrophysiological recordings; Yundoo Chung for providing a work space for S.M. while waiting for his visa renewal; Richard Benton and Richard Axel for providing ionotropic receptor antibodies and fly stocks; New York University Department of Medical Parasitology for mosquitoes; and Luisa Vasconcelos and Jessica Treisman for comments on the manuscript. This work was supported by a National Research Service Award Fellowship (to M.A.), by the Whitehall Foundation and the Irma T. Hirschl/Weill Caulier Trust Award, and by R01 grants from the National Institute of General Medical Science and National Institute on Deafness and Other Communication Disorders, National Institutes of Health (to G.S.).
Supporting Information
Supporting Information (PDF)
Supporting Information
- Download
- 1.21 MB
sm01.avi
- Download
- 2.52 MB
sm02.avi
- Download
- 2.10 MB
References
1
LB Vosshall, AM Wong, R Axel, An olfactory sensory map in the fly brain. Cell 102, 147–159 (2000).
2
PJ Clyne, et al., A novel family of divergent seven-transmembrane proteins: Candidate odorant receptors in Drosophila. Neuron 22, 327–338 (1999).
3
LB Vosshall, RF Stocker, Molecular architecture of smell and taste in Drosophila. Annu Rev Neurosci 30, 505–533 (2007).
4
R Benton, KS Vannice, C Gomez-Diaz, LB Vosshall, Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila. Cell 136, 149–162 (2009).
5
L Abuin, et al., Functional architecture of olfactory ionotropic glutamate receptors. Neuron 69, 44–60 (2011).
6
A Couto, M Alenius, BJ Dickson, Molecular, anatomical, and functional organization of the Drosophila olfactory system. Curr Biol 15, 1535–1547 (2005).
7
E Fishilevich, LB Vosshall, Genetic and functional subdivision of the Drosophila antennal lobe. Curr Biol 15, 1548–1553 (2005).
8
AF Silbering, et al., Complementary function and integrated wiring of the evolutionarily distinct Drosophila olfactory subsystems. J Neurosci 31, 13357–13375 (2011).
9
EC Marin, GS Jefferis, T Komiyama, H Zhu, L Luo, Representation of the glomerular olfactory map in the Drosophila brain. Cell 109, 243–255 (2002).
10
AM Wong, JW Wang, R Axel, Spatial representation of the glomerular map in the Drosophila protocerebrum. Cell 109, 229–241 (2002).
11
RI Wilson, GC Turner, G Laurent, Transformation of Olfactory Representations in the Drosophila Antennal Lobe. Science 303, 366–370 (2004).
12
CM Root, JL Semmelhack, AM Wong, J Flores, JW Wang, Propagation of olfactory information in Drosophila. Proc Natl Acad Sci USA 104, 11826–11831 (2007).
13
A Kurtovic, A Widmer, BJ Dickson, A single class of olfactory neurons mediates behavioural responses to a Drosophila sex pheromone. Nature 446, 542–546 (2007).
14
GS Suh, et al., A single population of olfactory sensory neurons mediates an innate avoidance behaviour in Drosophila. Nature 431, 854–859 (2004).
15
GS Suh, et al., Light activation of an innate olfactory avoidance response in Drosophila. Curr Biol 17, 905–908 (2007).
16
M Ai, et al., Acid sensing by the Drosophila olfactory system. Nature 468, 691–695 (2010).
17
GS Jefferis, et al., Comprehensive maps of Drosophila higher olfactory centers: Spatially segregated fruit and pheromone representation. Cell 128, 1187–1203 (2007).
18
SR Datta, et al., The Drosophila pheromone cVA activates a sexually dimorphic neural circuit. Nature 452, 473–477 (2008).
19
V Ruta, et al., A dimorphic pheromone circuit in Drosophila from sensory input to descending output. Nature 468, 686–690 (2010).
20
JA Riffell, H Lei, TA Christensen, JG Hildebrand, Characterization and coding of behaviorally significant odor mixtures. Curr Biol 19, 335–340 (2009).
21
JA Riffell, H Lei, JG Hildebrand, Neural correlates of behavior in the moth Manduca sexta in response to complex odors. Proc Natl Acad Sci USA 106, 19219–19226 (2009).
22
JP Martin, JG Hildebrand, Innate recognition of pheromone and food odors in moths: A common mechanism in the antennal lobe? Front Behav Neurosci 4:159. (2010).
23
JL Semmelhack, JW Wang, Select Drosophila glomeruli mediate innate olfactory attraction and aversion. Nature 459, 218–223 (2009).
24
Y Wang, et al., Blockade of neurotransmission in Drosophila mushroom bodies impairs odor attraction, but not repulsion. Curr Biol 13, 1900–1904 (2003).
25
ML Schlief, RI Wilson, Olfactory processing and behavior downstream from highly selective receptor neurons. Nat Neurosci 10, 623–630 (2007).
26
J Kiss, M Korbász, A Sass-Kiss, Study of amine composition of botrytized grape berries. J Agric Food Chem 54, 8909–8918 (2006).
27
CS Ough, CE Daudt, EA Crowell, Identification of new volatile amines in grapes and wines. J Agric Food Chem 29, 938–941 (1981).
28
CC Davis, M Latvis, DL Nickrent, KJ Wurdack, DA Baum, Floral gigantism in Rafflesiaceae. Science 315, 1812 (2007).
29
M Geier, OJ Bosch, J Boeckh, Ammonia as an attractive component of host odour for the yellow fever mosquito, Aedes aegypti. Chem Senses 24, 647–653 (1999).
30
RC Smallegange, YT Qiu, JJ van Loon, W Takken, Synergism between ammonia, lactic acid and carboxylic acids as kairomones in the host-seeking behaviour of the malaria mosquito Anopheles gambiae sensu stricto (Diptera: Culicidae). Chem Senses 30, 145–152 (2005).
31
GA Nevitt, DM Bergstrom, F Bonadonna, The potential role of ammonia as a signal molecule for procellarifform seabirds. Mar Ecol Prog Ser 315, 271–277 (2006).
32
C Frøkjaer-Jensen, M Ailion, SR Lockery, Ammonium-acetate is sensed by gustatory and olfactory neurons in Caenorhabditis elegans. PLoS ONE 3, e2467 (2008).
33
J Meijerink, MA Braks, JJ Van Loon, Olfactory receptors on the antennae of the malaria mosquito Anopheles gambiae are sensitive to ammonia and other sweat-borne components. J Insect Physiol 47, 455–464 (2001).
34
YT Qiu, JJ van Loon, W Takken, J Meijerink, HM Smid, Olfactory Coding in Antennal Neurons of the Malaria Mosquito, Anopheles gambiae. Chem Senses 31, 845–863 (2006).
35
SD Liberles, LB Buck, A second class of chemosensory receptors in the olfactory epithelium. Nature 442, 645–650 (2006).
36
MC Larsson, et al., Or83b encodes a broadly expressed odorant receptor essential for Drosophila olfaction. Neuron 43, 703–714 (2004).
37
ST Sweeney, K Broadie, J Keane, H Niemann, CJ O’Kane, Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects. Neuron 14, 341–351 (1995).
38
L Tian, et al., Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat Methods 6, 875–881 (2009).
39
RF Stocker, MC Lienhard, A Borst, KF Fischbach, Neuronal architecture of the antennal lobe in Drosophila melanogaster. Cell Tissue Res 262, 9–34 (1990).
40
CA Yao, R Ignell, JR Carlson, Chemosensory coding by neurons in the coeloconic sensilla of the Drosophila antenna. J Neurosci 25, 8359–8367 (2005).
41
R Okada, T Awasaki, K Ito, Gamma-aminobutyric acid (GABA)-mediated neural connections in the Drosophila antennal lobe. J Comp Neurol 514, 74–91 (2009).
42
V Bhandawat, SR Olsen, NW Gouwens, ML Schlief, RI Wilson, Sensory processing in the Drosophila antennal lobe increases reliability and separability of ensemble odor representations. Nat Neurosci 10, 1474–1482 (2007).
43
J Lippincott-Schwartz, GH Patterson, Development and use of fluorescent protein markers in living cells. Science 300, 87–91 (2003).
44
JS de Belle, M Heisenberg, Associative odor learning in Drosophila abolished by chemical ablation of mushroom bodies. Science 263, 692–695 (1994).
45
T Lee, A Lee, L Luo, Development of the Drosophila mushroom bodies: Sequential generation of three distinct types of neurons from a neuroblast. Development 126, 4065–4076 (1999).
46
SX Luo, R Axel, LF Abbott, Generating sparse and selective third-order responses in the olfactory system of the fly. Proc Natl Acad Sci USA 107, 10713–10718 (2010).
47
M Schleyer, et al., A behavior-based circuit model of how outcome expectations organize learned behavior in larval Drosophila. Learn Mem 18, 639–653 (2011).
48
M Heisenberg, Mushroom body memoir: From maps to models. Nat Rev Neurosci 4, 266–275 (2003).
49
C Faucher, M Forstreuter, M Hilker, M de Bruyne, Behavioral responses of Drosophila to biogenic levels of carbon dioxide depend on life-stage, sex and olfactory context. J Exp Biol 209, 2739–2748 (2006).
50
PM Salvaterra, T Kitamoto, Drosophila cholinergic neurons and processes visualized with Gal4/UAS-GFP. Brain Res Gene Expr Patterns 1, 73–82 (2001).
51
S Sachse, et al., Activity-dependent plasticity in an olfactory circuit. Neuron 56, 838–850 (2007).
52
DA Yarmolinsky, CS Zuker, NJ Ryba, Common sense about taste: From mammals to insects. Cell 139, 234–244 (2009).
53
Y Grosjean, et al., An olfactory receptor for food-derived odours promotes male courtship in Drosophila. Nature 478, 236–240 (2011).
54
V Croset, et al., Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction. PLoS Genet 6, e1001064 (2010).
55
EA Hallem, JR Carlson, Coding of odors by a receptor repertoire. Cell 125, 143–160 (2006).
56
EA Hallem, MG Ho, JR Carlson, The molecular basis of odor coding in the Drosophila antenna. Cell 117, 965–979 (2004).
57
RA Steinbrecht, Pore structures in insect olfactory sensilla: A review of data and concepts. Int J Insect Morphol Embryol 26, 229–245 (1997).
58
X Chen, M Gabitto, Y Peng, NJ Ryba, CS Zuker, A gustotopic map of taste qualities in the mammalian brain. Science 333, 1262–1266 (2011).
59
Z Wang, A Singhvi, P Kong, K Scott, Taste representations in the Drosophila brain. Cell 117, 981–991 (2004).
60
S Marella, et al., Imaging taste responses in the fly brain reveals a functional map of taste category and behavior. Neuron 49, 285–295 (2006).
61
K Miyamichi, et al., Cortical representations of olfactory input by trans-synaptic tracing. Nature 472, 191–196 (2011).
62
K Kobayakawa, et al., Innate versus learned odour processing in the mouse olfactory bulb. Nature 450, 503–508 (2007).
63
M Gallio, TA Ofstad, LJ Macpherson, JW Wang, CS Zuker, The coding of temperature in the Drosophila brain. Cell 144, 614–624 (2011).
64
RF Stocker, G Heimbeck, N Gendre, JS de Belle, Neuroblast ablation in Drosophila P[GAL4] lines reveals origins of olfactory interneurons. J Neurobiol 32, 443–456 (1997).
65
M Knaden, A Strutz, J Ahsan, S Sachse, BS Hansson, Spatial representation of odorant valence in an insect brain. Cell Rep 1, 392–399 (2012).
66
H Peng, Z Ruan, F Long, JH Simpson, EW Myers, V3D enables real-time 3D visualization and quantitative analysis of large-scale biological image data sets. Nat Biotechnol 28, 348–353 (2010).
67
AR Rodan, JA Kiger, U Heberlein, Functional dissection of neuroanatomical loci regulating ethanol sensitivity in Drosophila. J Neurosci 22, 9490–9501 (2002).
Information & Authors
Information
Published in
Classifications
Submission history
Published online: March 18, 2013
Published in issue: April 2, 2013
Keywords
Acknowledgments
We thank Ruth Lehmann for sharing her two-photon microscope; Niels Ringstad and Ip Ninan for assistance with Xenopus oocyte and HEK293 cell electrophysiological recordings; Yundoo Chung for providing a work space for S.M. while waiting for his visa renewal; Richard Benton and Richard Axel for providing ionotropic receptor antibodies and fly stocks; New York University Department of Medical Parasitology for mosquitoes; and Luisa Vasconcelos and Jessica Treisman for comments on the manuscript. This work was supported by a National Research Service Award Fellowship (to M.A.), by the Whitehall Foundation and the Irma T. Hirschl/Weill Caulier Trust Award, and by R01 grants from the National Institute of General Medical Science and National Institute on Deafness and Other Communication Disorders, National Institutes of Health (to G.S.).
Notes
This article is a PNAS Direct Submission.
See full research article on page E1321 of www.pnas.org.
This article is a PNAS Direct Submission. L.C.G. is a guest editor invited by the Editorial Board.
See Author Summary on page 5292 (volume 110, number 14).
Authors
Competing Interests
The authors declare no conflict of interest.
Metrics & Citations
Metrics
Altmetrics
Citations
Cite this article
Dedicated olfactory neurons mediating attraction behavior to ammonia and amines in Drosophila, Proc. Natl. Acad. Sci. U.S.A.
110 (14) E1321-E1329,
https://doi.org/10.1073/pnas.1215680110
(2013).
Copied!
Copying failed.
Export the article citation data by selecting a format from the list below and clicking Export.
Cited by
Loading...
View Options
View options
PDF format
Download this article as a PDF file
DOWNLOAD PDFLogin options
Check if you have access through your login credentials or your institution to get full access on this article.
Personal login Institutional LoginRecommend to a librarian
Recommend PNAS to a LibrarianPurchase options
Purchase this article to access the full text.