Nature AOP, published online 15 September 2004; doi:10.1038/nature02980
A single population of olfactory sensory neurons mediates an innate avoidance behaviour in Drosophila
GREG S. B. SUH1,2, ALLAN M. WONG1,3, ANNE C. HERGARDEN1,2, JING W. WANG1,3, ANNE F. SIMON2,*, SEYMOUR BENZER2, RICHARD AXEL1,3 & DAVID J. ANDERSON1,2
1 Howard Hughes Medical Institute,
2 Division of Biology 216-76 and 156-29, California Institute of Technology, Pasadena, California 91125, USA
3 Columbia University College of Physicians and Surgeons 701 West 168th Street, New York 10032, USA
* Present address: Brain Research Institute University of California, Los Angeles, California, USA
Correspondence and requests for materials should be addressed to D.J.A. (wuwei@caltech.edu).
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 (Shits)1, 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.
We observed that Drosophila tend to avoid chambers in which other flies have previously been subjected to stress by mechanical shaking or electric shock. To investigate the basis of this behaviour, about 70 'emitter' flies were subjected to mechanical stress by vortexing them in a test tube (see Methods). We removed the stressed flies, and allowed naive 'responder' flies to choose between this 'conditioned tube' and a fresh tube, in a T-maze apparatus2. The majority (80–95%) of responder flies avoided the conditioned tube in a one-minute trial (Fig. 1a). A similar avoidance response was observed when emitter flies were stressed using electric shock (Fig. 1a). To determine whether the mere presence of flies in a tube causes emission of the avoidance-promoting substance, flies were gently introduced into a tube using positive phototaxis, and removed after several hours of occupancy. Despite the evident presence in the tube of fly waste, responder flies showed no avoidance response to the tube (Fig. 1a, 'no stress'). This suggests that avoidance is elicited by a substance emitted in response to mechanical or electrical stress. The emission of the substance is not observed when anaesthetized flies are vortexed, indicating that such emission requires neural activity.
Figure 1 Drosophila exhibits innate avoidance of odorants released by stressed flies. Full legend
High resolution image and legend (99k)
Surgical removal of the third antennal segment, which houses the olfactory receptor neurons (ORNs), eliminated the avoidance response (Fig. 1b). By contrast, removal of the aristae or maxillary palps had no effect. These data suggest that the olfactory system mediates the avoidance response. We therefore operationally refer to the substance evoking the avoidance response as Drosophila stress odorant (dSO).
Olfactory sensory neurons in the antennae project axons to glomeruli in the antennal lobe. Projection neurons then connect the antennal lobe to the mushroom body and lateral protocerebrum3. Conditioned olfactory avoidance responses produced by associative learning require the mushroom body4, whereas unconditioned avoidance responses to chemical repellents do not5, 6. We asked whether the mushroom body is required for the avoidance response to dSO, by ablating this structure through hydroxyurea (HU) treatment at a critical stage of development7. Alternatively, we have used a mushroom-body-specific Gal4 enhancer trap line to drive the expression of UAS-Shits, a dominant-negative mutant of dynamin that inhibits neurotransmitter release at a non-permissive temperature1. Neither treatment impaired the avoidance response to dSO (Fig. 1c), although a deficit in olfactory learning confirmed that the lesion was successful (Fig. 1c, lower panel). These data suggest that the avoidance response to dSO does not require brain structures necessary for learned olfactory avoidance.
Analysis of the chemical components of dSO by gas chromatography and mass spectrometry (GC/MS) revealed a small peak of 44 daltons that corresponds to CO2, which was present in samples of air from conditioned tubes (Fig. 2a, left panel, arrow). Further analysis using a CO2 respirometer indicated that flies emit about three- to fourfold more CO2 during shaking than do undisturbed flies (Fig. 2b). By comparison to signals obtained by injecting known amounts of pure CO2 into the respirometer, the concentration of CO2 in conditioned tubes was estimated at 0.2% (data not shown). We next asked whether CO2 alone evoked avoidance behaviour in a T-maze assay. Flies avoid CO2 in a dose-dependent manner, at concentrations far below anaesthetic levels (30%) (Fig. 2c; see also Supplementary Fig. S1). A concentration as low as 0.1% above the ambient CO2 level (0.0376%) evoked a statistically significant avoidance response (performance index, PI = 29.6 10.9, P < 0.001 by ANOVA, n = 3, see Supplementary Fig. S1). A concentration comparable to that estimated in dSO ( 0.2%), although it did elicit significant avoidance, evoked a weaker response than did dSO itself (Fig. 2c, ' + CO2 0.02 ml' versus 'CS shaken', where 'CS' indicates the wild-type Canton S Drosophila strain), suggesting that stressed flies release additional repellent compound(s) together with CO2.
Figure 2 CO2 is a component of dSO. Full legend
High resolution image and legend (69k)
We analysed the pattern of glomerular activity in the antennal lobe elicited by CO2, using a calcium-sensitive fluorescent indicator protein (GCaMP) and two-photon microscopy8. Electrophysiological recordings have previously identified CO2-responsive ORNs in the ab1 basiconic sensilla of the antenna9-11, but the receptor expressed in these neurons, and their projections, have not been established. Experiments in Lepidoptera12, 13 and Diptera14, 15 have traced the glomerular targets innervated by axons from sensilla containing CO2-responsive ORNs, but these sensilla contain other ORNs as well. Moreover, the studies in Dipteran species have differed with respect to the number and identity of the glomeruli innervated14, 15. Ca2+ imaging permits an analysis of the glomerular activation pattern of CO2-responsive sensory neurons in the antennal lobe. We first examined flies in which the GCaMP indicator (UAS-GCaMP) is driven in all neurons by the Elav-GAL4 activator. At concentrations of CO2 up to 10%, only the most ventral pair of glomeruli, the V glomeruli2, were activated (Fig. 3a). Activation was detected by as little as 0.05% CO2 (Supplementary Fig. S1). These glomeruli were not activated by any of 26 other odorants tested (data not shown).
Figure 3 CO2 avoidance is mediated by ORNs that project to the V glomerulus. Full legend
High resolution image and legend (88k)
We have previously shown16 that axonal projections to V originate from antennal sensory neurons expressing the candidate gustatory receptor GR21A (Fig. 3d, left). GR21A+ neurons are located in the dorso-medial portion of the antenna (Fig. 3d, right), the region where CO2-responsive ab1 neurons are positioned in basiconic sensilla10, 11. Calcium imaging was thus performed with flies in which the UAS-GCaMP reporter was driven by a GR21A promoter-Gal4 activator (Fig. 3b). CO2 (Fig. 3b), as well as air from a tube conditioned by traumatized flies (Fig. 3c, left), activated GR21A sensory termini in the V glomeruli. Air from a tube that had contained undisturbed flies produced significantly lower levels of activation (Fig. 3c, right). These results indicate that both CO2 and dSO activate neurons that express the GR21A receptor and project to the V glomerulus.
We next asked whether the GR21A+ sensory neurons are necessary for the avoidance responses to CO2 and dSO. For this, we employed Shibirets to reversibly inactivate these neurons at increased temperature1. Flies bearing either of two independent GR21A-Gal4 insertions and UAS-Shits no longer exhibited the avoidance response to 1% CO2 at a non-permissive temperature (that is, a temperature at which neurotransmitter release cannot occur), but revealed a normal response at a permissive temperature (Fig. 3e, red versus blue bars labelled '2', respectively). Control flies expressing either of the GR21A-Gal4 drivers, but not UAS-Shits, showed normal CO2 avoidance at the non-permissive temperature (Fig. 3e). Furthermore, flies expressing UAS-Shits and either of two Gal4 drivers broadly expressed in other ORNs, but not in GR21A+ neurons (OR83b-Gal4, expressed by 80% of ORNs, or Or47b-Gal4; L. Vosshall, personal communication), exhibited normal CO2 avoidance at the non-permissive temperature (Fig. 3e). Similarly, flies expressing UAS-Shits and another Gal4 driver, GH146-Gal4, which is expressed in about two-thirds of antennal lobe projection neurons5, 17 (but not in those innervating V), also showed robust CO2 avoidance at the non-permissive temperature (Fig. 3e). These data indicate that GR21A+ sensory neurons that project to the V glomerulus are probably the sole population of ORNs responsive to CO2, and are required for the avoidance response. Flies expressing UAS-Shits in GR21A+ neurons still avoided dSO (Fig. 3e, red bars labelled '1'). Although a reduced response was observed using one of the two driver lines (Fig. 3e, GR21aG4(2);UAS-Shits), this reduction did not reach statistical significance. These data support the notion that dSO contains other repellent(s), in addition to CO2.
To characterize further the neural substrates mediating dSO-responsiveness, we conducted a screen for UAS-Shits-dependent defects in dSO-avoidance, using a collection of Gal4 enhancer trap lines (K. Kaiser). A pilot screen of 250 lines yielded 12 exhibiting reduced dSO avoidance at non-permissive temperatures (G.S.B.S., unpublished work). Several of these dSO-unresponsive lines also exhibited a strong and specific reduction in CO2 avoidance in subsequent tests, including one designated c761 (Fig. 4a, b). Analysis of the c761 expression pattern revealed that it includes a subset of ORNs in the third antennal segment (Fig. 4c, right), but not projection neurons (not shown). That this line is deficient in avoidance of CO2, as well as of dSO, suggested that these ORNs might include those projecting to V, and others projecting to additional glomeruli. The projections of c761+ neurons to the antennal lobe were consistent with this expectation; labelling was observed both in the V glomerulus, and in several other glomeruli (Fig. 4c, left; Fig. 4d, left). Calcium imaging of c761;UAS-GCaMP flies revealed activation of V by CO2 (Fig. 4d) as well as by dSO (not shown), confirming expression of this enhancer trap in GR21A+ neurons.
Figure 4 A dSO-unresponsive enhancer trap line is also defective in its CO2 response. Full legend
High resolution image and legend (169k)
Together, these results indicate that c761 is expressed in, among others, CO2-responsive sensory neurons that project to V (Fig. 4c). Because c761 was isolated in a screen for dSO-unresponsive lines, these data provide additional evidence that CO2 is a behaviourally relevant component of dSO. Moreover, the observation that c761 expresses in additional populations of ORNs besides GR21A+ neurons suggests that these ORNs may respond to other active components of dSO.
We have shown that Drosophila, when stressed, emits an odorant mixture that elicits avoidance in other flies, and have identified CO2 as one active component of this mixture. Calcium imaging data suggest that a single population of primary olfactory receptor neurons, which projects to the V glomerulus16, is activated by CO2. Specific inhibition of neurotransmission in these GR21A+ sensory neurons, but not in other sensory neurons, abrogates CO2 avoidance behaviour. Together, these data identify a single population of olfactory sensory neurons that mediates robust avoidance to a naturally occurring odorant, and provide initial insight into the neural circuitry that underlies this innate behaviour.
Many insect species, when stressed or threatened, emit semiochemicals that evoke aggressive or avoidance behaviour in conspecifics18. Whether Drosophila actually uses dSO to signal stress to conspecifics in the wild, and the conditions under which they might do so, are not yet clear. We have used experimental stimuli such as mechanical agitation and electrical current to elicit release of dSO from Drosophila. Although such conditions are artificial, they afford us the ability to maintain tight control over the stimulus and the organism's response, as well as to apply molecular genetic tools to monitor and perturb neural activity. Although these stimuli increase physical and metabolic activity, it is possible that dSO could also be emitted in response to threats.
We have identified CO2 as one active component of dSO. CO2 is known to be an important chemical messenger for many insect species19. In mosquitoes, for example, CO2 is an attractant that directs the insect towards warm-blooded animals20. At all the concentrations of CO2 that we tested, we detected only avoidance responses. The different behavioural responses to CO2 exhibited by mosquitoes and Drosophila are likely to reflect hard-wired, species-specific differences in neural circuitry; nevertheless, we cannot exclude that these behavioural differences may be context-dependent. The behavioural and physiologic responses to CO2 that we have measured in the laboratory are seen at several-fold increases above ambient ( 0.0376%) that are well within the range measured for other insects19. The role of CO2 in the ethology and ecology of Drosophila remains to be explored, but the highly specific olfactory circuitry revealed by our experiments suggests that it may be important.
Current data suggest that in Drosophila, most odorant compounds excite multiple populations of olfactory sensory neurons, each expressing a single olfactory receptor gene. A given odorant will therefore activate multiple glomeruli in the antennal lobe8, 21. By contrast, our data suggest that a single population of ORNs, and therefore a single glomerulus (V), are involved in sensing and avoiding CO2. Previous electrophysiological studies identified ORNs uniquely responsive to CO2, located exclusively in ab1 basiconic sensilla10, 11. These and the present data suggest that CO2 activates a single population of ORNs, and that these ORNs respond only (or primarily) to CO2. We cannot exclude, however, that other ORNs, (or antennal lobe projection neurons innervating glomeruli other than V), are also activated by CO2 at levels below the detection limit of our imaging technology. Nevertheless, previous studies using this method have shown that multiple glomeruli are activated by most odorants tested8, so it is striking that just a single glomerulus is activated by CO2. Furthermore, the behavioural response to CO2 is extinguished by genetic silencing of GR21A+ sensory neurons. The fact that the avoidance response to CO2 is unaffected by genetic silencing of Or83b or GH146 neurons further suggests that large populations of ORNs and projection neurons not directly innervating V are unnecessary for CO2 avoidance. Taken together, these data suggest that a dedicated circuit, which involves a single population of ORNs, mediates detection of CO2 in Drosophila. The simplicity of this early-stage olfactory processing offers a great advantage in further tracing the circuits that translate CO2 detection into an avoidance response.
In general, recognition of many odorants in insects probably requires the decoding of combinatorial patterns of glomerular activation8, 22-24, perhaps combined with complex temporal dynamics in the antennal lobe25. Nevertheless, our data suggest that there is also a set of olfactory stimuli, including CO2, that release innate behaviours by activating a single class of primary sensory neurons and their associated glomerulus. In Drosophila, these stimuli may also include mating pheromones which, in other insect species, are known to activate specialized glomeruli26, 27. In Caenorhabditis elegans, the activation of a single chemosensory neuron can elicit a repulsive behaviour28. Uni-glomerular circuits dedicated to the detection of certain odorants may have evolved to provide innate behavioural responses to these stimuli, which are essential to survival or reproduction of the species.
Methods
Flies Unless otherwise indicated, Canton-S flies (Caltech stock) were used for all experiments. Flies carrying the UAS-Shits transgenes on the X and third chromosomes were reared at 20 °C. For reversible neuronal silencing, flies were incubated in a 28 °C room for 90 min and then in a 32° or 34 °C water bath for 5 min, before performing the behavioural experiments at 28 °C. The permissive temperature used in experiments with Shits was 21 °C. To generate antennaless, palpless or aristaless flies, extirpations were performed using fine tweezers under CO2 anaesthesia, and the animals were allowed to recover for 24 h before testing.
Behavioural tests For behavioural testing, 40 'responder' and 70 'emitter' adult flies (3- to 6-days post-eclosion, mixed gender) were anaesthetized using CO2 and sorted into separate vials 24–48 h before use, or were sorted using an aspirator. A 16-inch 15-W fluorescent bulb was horizontally centred on the bench-top, behind the testing apparatus, in an otherwise dark room. Responder flies were transferred into the T-maze by first placing them into a 15-ml plastic tube (Fisher no. 149598), and tapping them into the elevator of the T-maze. While the responders were in the elevator, emitter flies were vortexed in a clean 15-ml tube in 3-s bouts, at 5-s intervals, over a 1-min period. As an alternative method of stressing flies, electric shocks were applied across a copper grid at 60 V (direct current) for 1 min. The emitter flies were discarded, the conditioned tube immediately inserted into one side of the T-maze, a fresh tube being inserted on the other side. The elevator containing responder flies was lowered, and the flies given one minute to choose between the two tubes, after which the elevator was partially lifted to block any further choices, and the number of flies in each tube counted. For testing responses to other odorants, 10 µl of 0.01% octanol, 10 µl of 0.01% methylcyclohexanol (MCH) or 10 µl of 0.3 mg ml-1 freshly prepared freeze-dried instant coffee (Taster's Choice) were dripped onto pieces of Whatman filer paper (0.5 0.25 in), placed at the end of the 15-ml plastic tube inserted into the T-maze apparatus. The control tube contained filter paper and vehicle alone. The avoidance response was analysed by calculating the PI, that is, the percentage of flies avoiding minus the percentage of flies entering. PI = 0 indicates an equal distribution of flies between the two tubes. PI = 100% indicates that all flies avoided the conditioned tube. Statistical significance was calculated using analysis of variance, ANOVA.
Ca2+ imaging and odour delivery Sample preparation and calcium imaging were as described in ref. 8. CO2 was diluted to 1% and delivered at a flow rate of 81 ml min-1. dSO and air from non-traumatized flies were prepared as described above, and delivered undiluted.
Gas chromatography and mass spectrometry Air samples from tubes containing dSO and from fresh tubes were analysed using a gas chromatograph (Agilent 6890) interfaced with a quadruple mass spectrometer (Agilent 5970B). Five microlitres of air from a tube in which 250 flies had been shaken, or from a fresh tube, were injected with a Hamilton syringe into the GC column at 50 °C. The column was then heated to 270 °C at a rate of 10 °C min-1 with normal inlet temperature of 250 °C (splitless mode). The GC column was equipped with a column from J&W, DB5-MS, 30 cm 0.25 mm (i.d.) 0.25 µm film thickness. Each molecule eluted from the GC column was detected and its molecular mass and abundance measured by the MS. The operating conditions for the MS were:10 to 500 m/z; 1.64 scans s-1; ionization energy 70 eV.
Respirometer measurements Emission of carbon dioxide was measured using a Sable System TR-2 carbon dioxide gas respirometry system (Model LI-6251). Groups of 20 flies were placed in a 2.2-ml glass chamber, which was flushed with a constant flow of CO2-free air through a CO2 detector. The amount of CO2 produced by each group of flies was calculated by using DATACAN software (Sable Systems International).
Visualization of murine CD8GFP Adult fly brains were dissected, fixed in 2% paraformaldehyde, and mounted in Vectashield (Vecta Labs). Native green fluorescence protein (GFP) fluorescence of whole-mount brains was visualized by confocal microscopy (Leica). Olfactory axonal projections of flies bearing GR21A-Gal4 and UAS-mCD8GFP were visualized by fluorescent immunohistochemistry, as described in ref. 16.
Hydroxyurea treatment The HU protocol7 was used to block development of the mushroom body (MB) structure. To check the ablation of the MB, we used the 253Y enhancer trap line carrying UAS-mCD8GFP, which expresses in the MB, as well as in other regions. The survival of the other structures after treatment serves as an internal control for the specificity of HU ablation.
Learning assay The 'Long Program' training protocol described in ref. 30 was used to train flies. Flies were exposed to 60 s of odour A associated with a 90-V 1.5-s shock delivered every 5 s for 60 s (CS + ) followed by 60 s of odor B with no shock (CS - ). The trained flies were then given a choice in the T-maze between odours A and B. Both training and testing were done at room temperature (23–25 °C) and humidity (20–50%).
