InteractiveFly: GeneBrief

Gustatory receptor 21a and Gustatory receptor 63a: Biological Overview | References

Blood-feeding insects, including the malaria mosquito Anopheles gambiae, use highly specialized and sensitive olfactory systems to locate their hosts. This is accomplished by detecting and following plumes of volatile host emissions, which include carbon dioxide (CO₂). CO₂ is sensed by a population of olfactory sensory neurons in the maxillary palps of mosquitoes and in the antennae of Drosophila. The molecular identity of the chemosensory CO₂ receptor, however, remains unknown. This study reports that CO₂-responsive neurons in Drosophila co-express a pair of chemosensory receptors, Gr21a and Gr63a, at both larval and adult life stages. Mosquito homologues of Gr21a and Gr63a, GPRGR22 and GPRGR24 have been identified; these are co-expressed in A. gambiae maxillary palps. Gr21a and Gr63a together are sufficient for olfactory CO₂-chemosensation in Drosophila. Ectopic expression of Gr21a and Gr63a together confers CO₂ sensitivity on CO₂- insensitive olfactory neurons, but neither gustatory receptor alone has this function. Mutant flies lacking Gr63a lose both electrophysiological and behavioural responses to CO₂. Knowledge of the molecular identity of the insect olfactory CO₂ receptors may spur the development of novel mosquito control strategies designed to take advantage of this unique and critical olfactory pathway. This in turn could bolster the worldwide fight against malaria and other insect-borne diseases (Jones, 2007).

Carbon dioxide is a pervasive chemical stimulus that is important in the ecology of many insect species. Interestingly, the ethological message conveyed by this gas is highly species- and context-specific. The hawkmoth, Manduca sexta, evaluates the quality of Datura wrightii flowers by measuring the amount of CO₂ that a given flower produces (Thom, 2004); newly opened flowers emit more CO₂ and are preferred because they offer more nectar. In response to elevated CO₂ in their hives, honeybees show a stereotyped fanning response that ventilates the hive and reduces ambient CO₂ levels (Southwick, 1987). For blood-feeding female mosquitoes, CO₂ emitted in the breath of animal hosts (~4%-5%) is an arousing stimulus that synergizes with host body odour to produce host-seeking behaviours (Gillies, 1980; Takken, 1999). The ecological relevance of CO₂ to fruitflies is less clear, but CO₂ is one component of dSO, aversive Drosophila stress odorant, (Suh, 2004) and may also signal food source suitability (Faucher, 2006; Jones, 2007 and references therein).

The chemosensory neurons that are thought to underlie these CO₂-evoked behaviours have been functionally characterized on antennal, maxillary or labial appendages in a number of different insects (Stange, 1999). Drosophila antennae have a small, CO₂-sensitive subpopulation of olfactory sensory neurons that have been designated ab1C (de Bruyne, 2001). The antennal lobe of the fly brain has a single ventrally-situated glomerulus (V) that responds selectively to CO₂ (Suh, 2004). This V glomerulus is innervated by a population of olfactory neurons expressing the chemosensory receptor Gr21a (Scott, 2001); these neurons correspond to the ab1C neurons. Gr21a is a member of the gustatory receptor gene family, which includes bitter and sweet taste receptors necessary for taste recognition in the fly, along with a number of gustatory receptor genes expressed in the antenna that may function as odorant receptors. Although clearly separable into two gene families, Drosophila gustatory receptors and odorant receptors are thought to have a common phylogenetic origin and were originally assigned to taste and smell modalities by gene homology and not function. Genetic silencing or ablation of Gr21a-expressing neurons (Suh, 2004; Faucher, 2006) eliminates both adult and larval chemosensory responses to CO₂, confirming that these are the only CO₂-sensitive neurons in Drosophila (Jones, 2007 and references therein).

Is Gr21a merely a marker for the CO₂-sensitive neurons in Drosophila or is it directly involved in CO₂ detection? Since taste neurons can express multiple gustatory receptor genes, a screen was performed for additional gustatory receptor genes expressed in ab1C neurons. Two other gustatory receptor genes are known to be expressed in the antenna, and fluorescent RNA in situ hybridization reveals that Gr63a is co-expressed with Gr21a, but that Gr10a is expressed in the adjacent ab1D neuron. Confirming these in situ hybridization results, neurons labelled with genetic markers under the control of Gr21a and Gr63a promoters co-converge upon the CO₂-sensitive V glomerulus. These chemosensory receptors are therefore co-expressed in the adult ab1C sensillum. Next, the expression of Gr21a and Gr63a in the larval olfactory system was investigaed. Larvae show robust avoidance of CO₂, which is mediated by Gr21a-expressing neurons (Faucher, 2006). Both Gr21a-GAL4 and Gr63a-GAL4 transgenes drive expression of a membrane-tethered green fluorescent protein (GFP) in the same neuron that innervates the larval terminal organ, which is thought to be primarily gustatory in function. This indicates that Gr21a and Gr63a are also coexpressed in the larval chemosensory system (Jones, 2007).

To generalize these results to other insects, Gr21a and Gr63a homologues were investigated in A. gambiae. Mosquitoes, in whom CO₂ plays an important role in human-host-seeking, have closely related homologues of both Gr21a and Gr63a, called GPRGR22 and GPRGR24, respectively. RNA in situ hybridization reveals co-expression of GPRGR22 and GPRGR24 in a subset of neurons in the maxillary palp, the CO₂-sensitive organ of the mosquito. No expression is detected in the antenna or proboscis. As in the fly, these putative mosquito CO₂-responsive neurons do not express GPROR7, the A. gambiae Or83b orthologue. Thus these mosquito homologues share three key properties in common with fly Gr21a/Gr63a: they are co-expressed in the same sensory neurons; they are selectively expressed only in the olfactory appendage that responds to CO₂; and they do not express the olfactory co-receptor Or83b (Jones, 2007).

To investigate the role of these gustatory receptor genes as putative CO₂ receptors, the antennal gustatory receptor genes were ectopically expressed both alone and in pairs in neurons normally unresponsive to CO₂ using the GAL4/UAS system. Or22a-GAL4 drives expression in ~75% of the electrophysiologically accessible ab3A neurons that express Or22a/b and the co-receptor Or83b. No individual gustatory receptor gene was capable of conferring CO₂ responsiveness on the ab3A neurons, but since it has been demonstrated that fly odorant receptor genes are obligate OR/ Or83b heterodimers, it was asked whether a combination of two gustatory receptor genes could function as a CO₂ receptor. Neither Gr21a nor Gr63a confer responses to CO₂ when combined with Gr10a, but the combination of Gr21a and Gr63a produces a significant response to a stimulus of ~3% CO₂. It is therefore the specific combination of these two gustatory receptor genes that is sufficient to induce CO₂ sensitivity rather than a generic requirement for the co-expression of any two antennal gustatory receptor genes. Gr21a and Gr63a together also increase the level of spontaneous activity in the ab3A neuron. The possibility is considered that this reflects activity in response to ambient CO₂ levels (0.035%), but it was found that the activity of these neurons is not reduced in response to a CO₂- free air stream. Prior results with odorant receptor genes indicate that some have substantial odour-independent activity, and this result suggests that gustatory receptor genes share this property. Further analysis of ectopically expressed Gr21a/Gr63a reveals a dose-dependent increase to stimuli of increasing CO₂ concentration, whereas animals expressing Gr21a alone do not respond to CO₂ at any concentration tested (Jones, 2007).

Next the efficacy of the ectopic CO₂ response was compared to that obtained in the native ab1C sensillum, and it was found that although the dose-response curves have a similar shape, the efficacy of the ectopic receptor is lower than the endogenous response. A number of explanations may account for this difference in efficacy. There could be a requirement for a cell-type specific co-factor, which is missing in the ab3A cell. Alternatively, lower receptor expression levels or competition for trafficking factors with the resident odorant receptors in ab3A could lead to a reduced number of functional CO₂ receptors. Such competition, leading to lower efficacy of ectopically expressed chemosensory receptors, has been noted by other investigators. Finally, there is evidence that the ab1C neuron has a uniquely specialized dendritic architecture, with considerably more branching than other chemosensory neurons. These special structural properties may be necessary for optimal Gr21a/Gr63a receptor function and may constrain its efficacy in other neurons. Taken together, it was found that Gr21a and Gr63a together are sufficient to confer dose-dependent CO₂-responsivity on olfactory neurons normally unresponsive to CO₂ (Jones, 2007).

To investigate the role of these gustatory receptor genes in the CO₂ responses of the native ab1C neuron, a screen was performed for Gr21a and Gr63a null mutants by homologous recombination. Gr21a proved to be resistant to mutagenesis, but a single null mutant allele of Gr63a was obtained. PCR analysis of Gr63a¹ indicates the selective loss of Gr63a without affecting a neighbouring gene, CG1079. Gr63a¹ flies lack the Gr63a transcript when compared with parental controls, but have normal levels of Gr21a. Electrophysiological recordings of ab1 sensilla in Gr63a¹ flies reveal a complete indifference to stimuli of ~2.25% CO₂, in stark contrast to wild-type parental control flies, whose ab1C neurons respond strongly. The Gr63a¹ allele is genetically recessive, because the sensilla of heterozygous individuals have an essentially wild-type CO₂ response. CO₂ responses in the Gr63a¹ are restored by rescuing Gr63a expression in the ab1C neurons using the GAL4/UAS system, while control Gr63a¹ flies bearing either the Gr21a-GAL4 transgene or the UAS-Gr63a transgene alone fail to respond (Jones, 2007).

Since genetic silencing of Gr21a-expressing neurons eliminates olfactory CO₂ avoidance in a T-maze (Suh, 2004), it was asked whether Gr63a¹ flies have CO₂ avoidance defects. Whereas wild-type flies robustly avoid CO₂ in a T-maze, Gr63a¹ flies fail to distinguish room air from an ~2% CO₂ stimulus. Consistent with their electrophysiological responses, Gr63a¹ heterozygotes show a wild-type avoidance response, whereas Gr63a¹ flies bearing either Gr21a-GAL4 or UAS-Gr63a transgenes fail to differentiate room air from 2% CO₂. When combined, however, these two transgenes rescue olfactory CO₂ avoidance in the mutant. The failure of the rescue to reach wild-type levels in either the electrophysiological recordings or the behaviour is probably a consequence of the lower levels of Gr63a expression in rescued ab1C neurons when compared to wild-type ab1C neurons. These loss of function results prove that Gr63a is necessary for CO₂ chemoreception in Drosophila and strengthen the hypothesis that the Drosophila CO₂ receptor is composed of both Gr21a and Gr63a (Jones, 2007).

Taken together, these results suggest that two chemosensory receptors, Gr21a and Gr63a, are necessary and sufficient for detection of CO₂ in Drosophila. Despite the fact both Gr21a and Gr63a are required for CO₂ responses, the data at present do not allow resolution of whether one subunit acts as a chaperoning co-receptor while the other subunit confers ligand specificity (as is the case for odorant receptors and Or83b), or whether both subunits are required for both functions. This is because, unfortunately, attempts to tag these proteins while retaining function have failed. Previous work in other biological systems has implicated several cytosolic proteins as gas sensors. Atypical soluble guanylate cyclases are candidate oxygen sensors in Caenorhabditis elegans, while conventional soluble guanylate cyclases are cytosolic receptors for nitric oxide and carbon monoxide. A nuclear receptor in Drosophila has been suggested as an additional receptor for nitric oxide and carbon monoxide. Bacteria utilize haem-containing myoglobin in chemotaxis towards oxygen. Although the genetic evidence strongly suggests that Gr21a/Gr63a encodes the first example of a membrane-associated gas sensor, the possibility cannot be excluded that additional secreted or cytosolic proteins are essential co-factors for CO₂ detection. Further biochemical investigation, most effectively carried out in a cell-based heterologous expression system, will be required to address this question (Jones, 2007).

It remains to be resolved whether the Gr21a/Gr63a receptor binds gaseous CO₂ or a metabolite, such as bicarbonate. It will also be of interest to elucidate the signal transduction cascade to which these gustatory receptors couple in order to transform the absolute concentration of environmental CO₂ into precise trains of neuronal action potentials. Since CO₂ is an important stimulus for a large number of insect pests, the identification of the CO₂ receptor provides a potential target for the design of inhibitors that might be useful as insect repellents. These would be important weapons in the fight against global infectious disease by reducing the attraction of blood-feeding insects to human hosts (Jones, 2007).

The molecular basis of CO₂ reception in Drosophila

CO₂ elicits a response from many insects, including mosquito vectors of diseases such as malaria and yellow fever, but the molecular basis of CO₂ detection is unknown in insects or other higher eukaryotes. Gr21a and Gr63a, members of a large family of Drosophila seven-transmembrane-domain chemoreceptor genes, are coexpressed in chemosensory neurons of both the larva and the adult. The two genes confer CO₂ response when coexpressed in an in vivo expression system, the 'empty neuron system.' The response is highly specific for CO₂ and dependent on CO₂ concentration. The response shows an equivalent dependence on the dose of Gr21a and Gr63a. None of 39 other chemosensory receptors confers a comparable response to CO₂. The identification of these receptors may now allow the identification of agents that block or activate them. Such agents could affect the responses of insect pests to the humans they seek (Kwon, 2007; full text of article).

The simplest interpretation of these results is that Gr21a and Gr63a form a heterodimer that responds to CO₂. There is precedence for heterodimerization of Or proteins, but not Gr proteins. Most receptors of the Or family determine the ligand-specificity of the ORN in which they are expressed but are believed to require Or83b as a coreceptor for efficient transport and/or stabilization at the membrane. Based on the current results it cannot be determined whether both Gr21a and Gr63a act directly in ligand binding and/or signaling, or whether one of them acts as a cofactor in a manner analogous to Or83b. It is noted that the two genes were functionally equivalent in terms of the effects of their dosage on both CO₂ response and spontaneous firing rate (Kwon, 2007).

There is no precedence for seven-transmembrane-domain proteins that act as receptors for CO₂ or any other gas. Previously described gas sensors include soluble guanylate cyclases that have been implicated in responses to NO and CO, atypical guanylate cyclases that have been implicated in responses to O₂, and a heme-binding nuclear receptor that has been implicated in the response to NO and CO in Drosophila (Kwon, 2007).

It is not known whether CO₂ acts on the Gr receptor via the extracellular lymph that surrounds the dendrites of ORNs. One alternative possibility is that CO₂ enters the cell by an independent mechanism and activates the receptor via the cytoplasm. There is abundant genetic and physiological evidence in unicellular organisms that Amt proteins and Rh proteins act as channels for NH₃ and CO₂, respectively. It is possible that a similar channel facilitates entry of CO₂ into the ab1C cell of Drosophila (Kwon, 2007).

It is not known whether CO₂ binds directly to the receptor. CO₂ is readily hydrated to HCO₃^- (bicarbonate), which may bind to the receptor; it is also possible that CO₂, a very small molecule, binds to a larger soluble factor that activates the receptor. It is noted that CO₂ lowers the pH of water by forming a weak solution of carbonic acid, H₂CO₃. Such pH changes contribute to responses in central respiratory chemosensory cells as well as in acid-sensing taste cells in vertebrates, and could also play a role in CO₂-sensing cells of Drosophila (Kwon, 2007).

The responses to CO₂ conferred by Gr21a and Gr63a in the empty neuron are lower than that of the ab1C neuron. The lower response may be primarily a result of lower gene dosage. However, the ab1C neuron is unique among ORNs in its dendritic morphology, which may be specialized to enhance CO₂ reception. The ab1C neuron might also contain soluble factors that optimize CO₂ sensing and that are not present in the empty neuron (Kwon, 2007).

Gr21a and Gr63a are among a small number of Gr genes that have orthologs in the malaria vector mosquito Anopheles gambiae. Orthologs have been identified in the dengue and yellow fever vector mosquito Aedes aegypti, the silk moth Bombyx mori, and the flour beetle Tribolium castaneum. Interestingly, genes closely related to either Gr21a or Gr63a have not been identified in the honey bee Apis mellifera, suggesting that bees employ a different receptor for CO₂ (Kwon, 2007).

The finding that Gr21a and Gr63a confer a response to CO₂ suggests the possibility of screening for compounds that inhibit or activate these proteins. Such compounds could affect the response of insect disease vectors, which are responsible for hundreds of millions of infections each year, to CO₂ emanations from the human hosts they seek (Kwon, 2007).

Light activation of an innate olfactory avoidance response in Drosophila

How specific sensory stimuli evoke specific behaviors is a fundamental problem in neurobiology. In Drosophila, most odorants elicit attraction or avoidance depending on their concentration, as well as their identity. Such odorants, moreover, typically activate combinations of glomeruli in the antennal lobe of the brain, complicating the dissection of the circuits translating odor recognition into behavior. Carbon dioxide (CO₂), in contrast, elicits avoidance over a wide range of concentrations and activates only a single glomerulus, V (Suh, 2004). The V glomerulus receives projections from olfactory receptor neurons (ORNs) that coexpress two GPCRs, Gr21a and Gr63a, that together comprise a CO₂ receptor. These CO₂-sensitive ORNs, located in the ab1 sensilla of the antenna, are called ab1c neurons. Genetic silencing of ab1c neurons indicates that they are necessary for CO₂-avoidance behavior (Suh, 2004). Whether activation of these neurons alone is sufficient to elicit this behavior, or whether CO₂ avoidance requires additional inputs (e.g., from the respiratory system), remains unclear. This study shows that artificial stimulation of ab1c neurons with light (normally attractive to flies) elicits the avoidance behavior typical of CO₂. Thus, avoidance behavior appears hardwired into the olfactory circuitry that detects CO₂ in Drosophila (Suh, 2007).

The photo-activated cation-selective channel channel rhodopsin-2 (ChR2) (Nagel, 2003; Boyden, 2005) was expressed in ab1c neurons by using a Gr21a-Gal4 driver and the flies were raised either in food supplemented with all-trans retinal or, as a control, without the supplement. To determine whether light activation of ChR2 can mimic the effect of CO₂ on ab1c activity, action potentials were recorded in single ab1 sensilla. Indeed, 470 nm blue light elicited spike trains from ab1c neurons but not from other ORNs in this sensillum. Two types of retinal-dependent spiking responses to light were seen in CO₂-responsive sensilla: a 'nonadapting' response that persisted for the duration of the stimulus and a transient response that terminated within ~200 ms after the stimulus onset. The persistent response was similar to that evoked by ~2% CO₂. Twenty-two percent of the ab1 sensilla showed no response to blue light. This may reflect variability in ChR2 expression, retinal bioavailability, or the fact that the ChR2 response is not amplified by a second-messenger system, in contrast to the response mediated by olfactory receptors (Suh, 2007).

To determine whether light activation of CO₂-sensitive ORNs is also sufficient to elicit avoidance behavior, a T maze was constructed in which one arm was outfitted with water-cooled 470 nm light-emitting diodes (LEDs). Flies were given 30–60 s to choose, and then they were counted. Flies expressing Gr21a-Gal4 and two copies of UAS-ChR2, and raised on retinal-containing food, avoided the test arm when the LEDs were illuminated. Avoidance was not observed in flies expressing the driver, the responder alone, or in flies raised without retinal. The mean performance index (PI) obtained with light was somewhat lower than that observed with 1% CO₂ and exhibited more variability, consistent with the electrophysiological data. The extent of avoidance was dependent on diode strength. Avoidance could also be obtained in flies containing a single copy of the UAS-ChR2 responder gene, although a direct comparison of performance index as a function of UAS-ChR2 copy number was not performed (Suh, 2007).

This avoidance of blue light is in stark contrast to the normally strong attraction to blue light of wild-type flies. To test whether the avoidance of blue light by Gr21a-Gal4;UAS-ChR2 flies was due exclusively to light activation of ab1c ORNs, rather than the visual pathway, the Gr21a-Gal4 and UAS-ChR2 transgenes were crossed into genetically blind flies homozygous for a mutation in the norpA (no receptor potential A) gene. Avoidance of blue light in the T maze by such flies was observed as well. norpA⁻ flies expressing UAS-ChR2 under the control of the Or83b-Gal4 driver, which is expressed in multiple classes of ORNs [but not those responding to CO₂ (Suh, 2004)], did not avoid the blue light, consistent with the behavior being specific to activation of ab1c neurons. The lack of any net response in these Or83b-Gal4; UAS-ChR2 flies may reflect integration of opponent attraction and avoidance responses, promoted by simultaneous activation of multiple classes of ORNs. Alternatively, these neurons may be less susceptible than ab1c neurons to activation by ChR2 (Suh, 2007).

The demonstration that the activation of ab1c neurons by light is sufficient to elicit avoidance in a T maze, taken together with the requirement of these neurons for CO₂ avoidance (Suh, 2004), indicates that this behavior is exclusively mediated by ab1c ORNs and does not involve combinatorial or temporal coding of odor identity. It also indicates that CO₂ avoidance does not require other sensory input, e.g., from the respiratory system. The fact that activation of a single population of ORNs suffices to trigger avoidance further suggests that this behavior is hardwired into the olfactory circuitry that detects CO₂ in Drosophila. By contrast, the concentration-dependent, combinatorial coding for most other odorants allows for more flexible behavioral responses. These two classes of olfactory stimulus-response mechanisms may be thought of as analogous to innate versus adaptive immune responses. Mosquitoes contain similar CO₂ receptor genes as Drosophila but are attracted to this odorant; whether the CO₂ response in mosquitoes is also innate, but of opposite valence, or rather is adaptive and flexible remains to be determined (Suh, 2007).

The results extend to adult Drosophila the use of ChR2 to elicit behavior in intact animals, as has been demonstrated in C. elegans. In practice, the efficacy of this system is likely to be dependent on factors such as Gal4 driver strength, position of the neurons in the brain, and their membrane surface area and biophysical properties. The use of ChR2 to activate neurons in adult Drosophila provides a valuable complement to other genetically based neuronal photo-activation techniques (Suh, 2007).

Hybrid neurons in a microRNA mutant are putative evolutionary intermediates in insect CO2 sensory systems

Carbon dioxide (CO₂) elicits different olfactory behaviors across species. In Drosophila, neurons that detect CO₂ are located in the antenna, form connections in a ventral glomerulus in the antennal lobe, and mediate avoidance. By contrast, in the mosquito these neurons are in the maxillary palps (MPs), connect to medial sites, and promote attraction. In Drosophila loss of a microRNA, miR-279, leads to formation of CO₂ neurons in the MPs. miR-279 acts through down-regulation of the transcription factor Nerfin-1. The ectopic neurons are hybrid cells. They express CO₂ receptors and form connections characteristic of CO₂ neurons, while exhibiting wiring and receptor characteristics of MP olfactory receptor neurons (ORNs). It is proposed that this hybrid ORN reveals a cellular intermediate in the evolution of species-specific behaviors elicited by CO₂ (Cayirlioglu, 2008).

In insects, both the position of CO₂ neurons and the behavior elicited by CO₂ differ among species. For example, olfactory detection of CO₂ through neurons positioned in or around the mouthparts of an insect, such as maxillary palps (MPs) and labial palps, correlates with feeding-related behaviors. Indeed, in some blood-feeding insects such as mosquitoes and tsetse flies, these neurons are harbored in the MPs and are important in locating hosts via plumes of CO₂ that they emit. The hawkmoth, Manduca sexta, monitors nectar profitability of newly opened Datura wrightii flowers through CO₂ receptor neurons located in their labial palps. In these examples, CO₂ acts as an attractant. Conversely, in Drosophila CO₂ is a component of a stress-induced odor that triggers avoidance behavior. This repellent response is driven by antennal neurons expressing the CO₂ receptor complex Gr21a-Gr63a. How did these diverse behavioral responses to CO₂ arise during insect evolution? It is proposed that this diversity emerged through multiple steps, including changes in cellular position (arising from elimination of CO₂ neurons in one appendage and generation of these neurons in another) and changes in circuitry (Cayirlioglu, 2008).

In the course of a genetic screen for mutants disrupting the organization of the olfactory system, a mutant (S0962-07) was isolated that resulted in the formation of ectopic Gr21a-expressing neurons in the MPs. Some 22 ± 1.5 (mean ± SEM) green fluorescent protein (GFP)-positive cells were observed in the mutant MP, whereas the number of antennal Gr21a olfactory receptor neurons (ORNs) was unaffected. In the wild type, Gr21a cell bodies were restricted to the antenna. The ectopic MP cells expressed both CO₂ receptors (Gr21a and Gr63a). Consistent with this finding, mutant cells conferred CO₂ sensitivity to the MP. Staining the MP with an antibody to the pan-neuronal marker Elav revealed an increase of 21 ± 3.4 neurons in the mutant, which suggests that all ectopic neurons expressed Gr21a (Cayirlioglu, 2008).

In wild-type MPs, each sensillum contains two ORNs. By contrast, in the mutant MP sensilla, additional neurons expressing Elav and the general receptor Or83b were observed. This was also apparent when a MP ORN marker (MPS-GAL4) expressed in a subset of MP ORNs was used. This marker labels single cells within a subset of wild-type MP sensilla; however, in mutant MPs, two additional neurons were observed, bringing the total number of neurons within these sensilla to four. Thus, the generation of ectopic Gr21a-Gr63a neurons is due to an increase in the number of neurons within sensilla rather than transformation of MP ORNs (Cayirlioglu, 2008).

In the wild type, each class of adult ORNs sends projections from both antennae or MPs to the antennal lobe (AL). ORNs expressing same odorant receptors (ORs) typically form synapses in the same glomerulus within the AL. CO₂ neurons in the antenna target the V-glomerulus. To specifically assess the targeting of ectopic MP CO₂ neurons, flies were examined where the antennae were surgically removed. It was found that ectopic CO₂ neurons targeted the V-glomerulus and other medial sites in the AL. The wiring specificity of antennal CO₂ neurons in the mutants was identical to that in the wild type. Thus, the ectopic CO₂ neurons in the MP target, at least in part, the same glomerulus innervated by the wild-type CO₂ neurons in the antennae (Cayirlioglu, 2008).

S0962-07 was mapped to a P-element insertion some 1 kb upstream of a microRNA, miR-279. MicroRNAs (miRNAs) are small noncoding RNAs of about 22 nucleotides that bind to specific sequences of the 3'-untranslated region (3'UTR) of target genes and thereby repress gene expression posttranscriptionally. In recent years, miRNAs were implied in a variety of functions in the nervous system of different organisms. To assess whether miR-279 is responsible for the observed phenotype, three small deletions were generated that uncovered the miR-279 genomic region. These deletion mutants exhibited phenotypes indistinguishable from S0962-07. The ectopic CO₂ phenotype was rescued by a 3-kb fragment of genomic DNA encoding only miR-279. Thus, miR-279 is the gene disrupted in S0962-07 and must repress targets in the MP to inhibit ectopic CO₂ neuron development (Cayirlioglu, 2008).

To assess whether miR-279 is expressed in the developing MPs, transgenic flies were generated carrying a transcriptional reporter construct (miR-279-GAL4). Expression was monitored in flies carrying this GAL4 construct and the reporter UAS-mCD8GFP. Around 40 to 50 hours after puparium formation (APF), large cells reminiscent of sensory organ precursors in other epithelia expressed miR-279. At later stages, miR-279-expressing cells were found in clusters with smaller cells, some of which expressed neuronal markers. As ORNs matured, miR-279 expression was lost (Cayirlioglu, 2008).

Attempts were identify the target gene(s) responsible for the miR-279 mutant phenotype. About 205 potential target mRNAs of miR-279 were previously predicted. One of the strongest candidates for miR-279 regulation is Nerfin-1. The Nerfin-1 3'UTR contains multiple miR-279 binding sites and encodes a transcription factor expressed in neuronal precursors and transiently in nascent neurons in the embryonic central nervous system. Nerfin-1 protein appeared in miR-279-positive cells between 50 and 60 hours APF. Nerfin-1 and miR-279 gradually redistributed, generating complementary expression patterns. Cells with high levels of Nerfin-1 expressed low levels of miR-279 and vice versa (Cayirlioglu, 2008).

To test whether Nerfin-1 is up-regulated in miR-279 mutants, mutant MPs were stained with antibodies to Nerfin-1. 22 ± 4.8 additional Nerfin-1-expressing cells were found in miR-279 mutant MPs relative to controls. This is similar to the number of ectopic CO₂ neurons in the MP. The vast majority of CO₂ ORNs in the MP expressed Nerfin-1. Thus, the expression pattern of Nerfin-1 protein in the wild type and in mutant MPs is consistent with nerfin-1 mRNA being a target for miR-279 in vivo (Cayirlioglu, 2008).

To determine whether miR-279 directly binds to nerfin-1 3'UTR and inhibits its expression, a luciferase reporter assay was used in cultured cells. The luciferase-coding region was fused to the full-length nerfin-1 3'UTR, which contains four conserved 8-nucleotide oligomer target sites for miR-279, as well as to a subregion containing three of these sites. Luciferase activity of both nerfin-1 sensor constructs was strongly repressed when cells were cotransfected with miR-279. By contrast, the activity of either nerfin-1 sensor was unaffected by noncognate miR-315. Antisense oligomers directed against the miR-279 core sequence specifically relieved nerfin-1 reporter repression. Thus, it is concluded that nerfin-1 is a direct target of miR-279 (Cayirlioglu, 2008).

Next whether Nerfin-1 down-regulation by miR-279 inhibits the development of CO₂ neurons in the MPs was assessed. To do this, the level of nerfin-1 was reduced by half genetically in a miR-279 mutant background. This decreased the number of CO₂ neurons in the MP relative to miR-279 mutants, providing strong in vivo evidence that miR-279 is necessary to down-regulate Nerfin-1 in MPs during normal development. Nerfin-1 up-regulation alone was not sufficient to generate a miR-279-like phenotype. Taken together, these findings suggest that miR-279 down-regulates Nerfin-1 and other targets to prevent CO₂ neuron development in the MPs (Cayirlioglu, 2008).

When analyzing the axonal projections of the CO₂ neurons in the MPs, it was observed that these neurons targeted one or more medial glomeruli in addition to the V-glomerulus, the target of antennal CO₂ neurons. These medial glomeruli are normally innervated by MP Or42a and Or59c ORNs. Double-labeling experiments revealed that mutant neurons also coexpressed Or42a and Or59c, but not other MP ORs. Analysis of subsets of MP ORNs also revealed that Or42a and Or59c classes each showed an approximate increase of 10 cells in the MPs, whereas others were unaffected. These results indicate that the ectopic CO₂ neurons are formed as additional cells within Or42a and Or59c sensilla and are hybrid in identity. They express ORs and exhibit wiring characteristics of two classes of neurons (Cayirlioglu, 2008).

It is interesting that the loss of miR-279 generates a CO₂ neuron within a sensillum harboring four neurons in the MP, given that the antennal CO₂ sensilla in Drosophila are the only sensilla in the olfactory system to harbor four ORNs. Because miR-279 acts within the precursor cells in the MP to prevent Nerfin-dependent formation of olfactory neurons, this observation raises the intriguing possibility that positioning of CO₂ neurons on different olfactory appendages might have evolved through changes at the level of precursor cell development. Thus, the evolutionary elimination of CO₂ neurons from MP sensilla might have required decreasing the number of cells with neuronal identities through down-regulation of Nerfin-1 by miR-279 (Cayirlioglu, 2008).

Although it was hypothesized that relocation of CO₂ ORNs to different appendages was important in the evolution of differences in CO₂ sensing, additional mechanisms must have evolved to modify the neural circuitry to alter species-specific behaviors in response to CO₂. The ectopic CO₂ neurons are hybrid cells, which express additional receptors (Or59c or Or42a) and also target medial glomeruli, typically innervated by wild-type ORNs expressing these ORs. This is particularly interesting given that CO₂ neurons in mosquitoes connect to medial glomeruli, driving an attractive response. It is speculated that this hybrid cell represents an evolutionary intermediate on a path leading to species-specific CO₂ behavior. Perhaps suppressing the expression of Or59c or Or42a ORs could convert this hybrid cell to one dedicated only to CO₂ reception. The nature of the behavioral output to CO₂ (i.e., attraction versus repulsion) by this cell, however, may be dictated by altering the wiring specificity to one site or the other (medial versus ventral, respectively). More generally, it is proposed that natural selection can work on such an evolutionary intermediate to generate different combinations of OR, wiring, and cellular positional specificities, depending on the insects' environmental needs. This may in turn lead to novel olfactory responses to different odorants, or to the same odorant in different species (Cayirlioglu, 2008).

Activity-dependent plasticity in an olfactory circuit

Olfactory sensory neurons (OSNs) form synapses with local interneurons and second-order projection neurons to form stereotyped olfactory glomeruli. This primary olfactory circuit is hard-wired through the action of genetic cues. It was asked whether individual glomeruli have the capacity for stimulus-evoked plasticity by focusing on the carbon dioxide (CO₂) circuit in Drosophila. Specialized OSNs detect this gas and relay the information to a dedicated circuit in the brain. Prolonged exposure to CO₂ induced a reversible volume increase in the CO₂-specific glomerulus. OSNs showed neither altered morphology nor function after chronic exposure, but one class of inhibitory local interneurons showed significantly increased responses to CO₂. Two-photon imaging of the axon terminals of a single PN innervating the CO₂ glomerulus showed significantly decreased functional output following CO₂ exposure. Behavioral responses to CO₂ were also reduced after such exposure. It is suggested that activity-dependent functional plasticity may be a general feature of the Drosophila olfactory system (Sachse, 2007).

Neuroanatomical, functional, and behavioral analysis suggests that the Drosophila olfactory system has the capacity for reversible activity-dependent plasticity. Evidence of this plasticity is readily seen by measuring the volume of the V glomerulus. Because the volume increase can be induced by odor activation of ORs ectopically expressed in the CO₂-activated OSNs, it is concluded that persistent stimulus-evoked activity in these neurons underlies these anatomical changes. It has been shown that stimulus-evoked plasticity is a general feature of the Drosophila olfactory system and not a peculiarity of the CO₂ circuit. For instance, the volume of DM2 is increased by chronic exposure to ethyl butyrate, a ligand for the Or22a-expressing neurons that target DM2 (Sachse, 2007).

Drosophila, CO₂ is detected by a population of approximately 25-30 OSNs in the antenna that express the chemosensory receptor Gr21a, which along with Gr63a comprises the Drosophila CO₂ receptor. These OSNs project axons that terminate in the V glomerulus in the ventral antennal lobe. The Drosophila CO₂ circuit is ideal for studying odor-evoked plasticity because Gr21a-expressing OSNs are the only neurons in the fly that respond to CO₂, and they do not respond to any other stimuli. In this work, we examined stimulus-evoked changes in the anatomy and function of the Drosophila CO₂ circuit. The results provide functional evidence that a primary olfactory center is capable of activity-dependent plasticity (Sachse, 2007).

The data are consistent with a model in which one class of inhibitory LNs and the output of the V glomerulus are the major targets of plasticity induced by sensory exposure. Under conditions of ambient CO₂, the Gr21a circuit forms normally and small amounts of CO₂ produce robust behavioral responses. When flies are exposed to elevated CO₂ early in life, it is postulated that chronic activation of Gr21a neurons promotes functional changes in the LN2 subtype of inhibitory local interneurons without affecting either the functional properties of the OSNs or the CO₂-evoked response of the LN1 neurons. It is suggested that the volume increases seen with CO₂ exposure may result from neuroanatomical changes in the LNs, although their extensive glomerular arborization made this hypothesis difficult to test experimentally. Since a majority of the LN2 population in Drosophila has been shown to be GAD1 positive and thus to release GABA, as known for antennal lobe LNs in other insects, greater CO₂-evoked activity of LN2s may lead to an increased inhibition of the PN postsynaptic to Gr21a OSNs. The finding of reduced activity in the output region of the PN innervating the V glomerulus supports this hypothesis. Thus, CO₂-evoked activity would be attenuated in the antennal lobe circuit in these animals, producing a corresponding decrease in the intensity of the behavioral response (Sachse, 2007).

It has recently been shown that LNs are not only inhibitory, as has been assumed so far. A newly described population of excitatory cholinergic LNs forms a dense network of lateral excitatory connections between different glomeruli that may boost antennal lobe output (Olsen, 2007; Shang, 2007). Future studies are necessary to investigate if excitatory LNs are also subject to activity-dependent plasticity (Sachse, 2007).

Stimulus-dependent plasticity can be induced and reversed in a critical period early in the life of a fly. Similar critical periods have been documented in selective deafferentation periods in mammalian somatosensory and visual cortex. In all these model systems, the critical period likely allows the animal to compare the genetically determined network template with external conditions and make activity-dependent adjustments that reflect the external environment. For instance, visual cortex 'expects' binocular input when it is wired in utero. If monocular input is experimentally imposed, the system is rewired to reflect this. The same rewiring occurs in the barrel cortex, in which the receptive fields of missing whiskers are invaded by neighboring whiskers, allowing the animal to maintain a continuous representation of external somatosensory space. Drosophila pupae have no sensory input during development and develop an olfactory system that relies neither on evoked activity nor the expression of ORs. The time following adult eclosion may represent a period in which the functional set point of the Drosophila olfactory system is evaluated and adapted to the local environment (Sachse, 2007).

What elements of the antennal lobe circuit are responsible for the stimulus-dependent volume increases seen here? No evidence was found that OSNs modulate their number, morphology, branching pattern, or functional properties in response to CO₂ exposure. The same neuroanatomical properties of single LNs or PNs could not be assayed due to the dense processes of these neurons in a given glomerulus. Since the observed net increase in volume cannot be ascribed to anatomical changes in OSNs, morphological plasticity is most likely occurring either at the level of LN or PN. A model is favored in which changes in the LNs underlie the observed volume increases because clear functional differences were found in LN2 responsivity in CO₂-exposed animals and because PN dendrites and axons have been shown to be extremely stable in size and morphology when deprived of OSN input. Similar stability in mitral/tufted cells has been shown in rodent olfactory bulb. The possibility that other cells, such as glia, contribute to these activity-dependent volume changes cannot be excluded (Sachse, 2007).

This work suggests that antennal lobe LNs marked with two different Gal4 enhancer traps, Gal4-LN1 and Gal4-LN2, are functionally distinct. The arborization of LN1 and LN2 processes in the V glomerulus suggests that they interact differentially with the antennal lobe circuitry. LN1 processes appear to innervate the core of a given glomerulus, while LN2 processes innervate the glomerulus more uniformly. Both LN1 and LN2 neurons show weakly concentration-dependent tuning to odor stimuli. Thus, compared to the OSNs or PNs, which transmit a precise spike-timing code that reflects absolute CO₂ concentration, these LNs appear to respond in a binary fashion, showing similar levels of activity regardless of stimulus concentration (Sachse, 2007).

There is a clear difference in how the responses of these two LN populations are modulated by CO₂ exposure. While the activity of LN1 neurons was not significantly affected by CO₂ exposure, LN2 neurons exhibited robust and significant increases in CO₂-evoked activity after CO₂ exposure. It will be of interest to examine the functional properties of these neurons in greater detail using electrophysiological approaches. It is plausible that circuit plasticity as evidenced in the LN2 neurons can be detected with electrophysiology at even lower CO₂ concentrations for shorter exposure periods (Sachse, 2007).

How might chronic activation of CO₂-sensitive OSNs specifically affect the physiology of LN2 neurons? It is speculated that due to the broader innervation of LN2 processes, these neurons would receive greater presynaptic innervation from Gr21a-expressing OSNs. Thus, with chronic CO₂ exposure, the LN2 neurons would be chronically activated. This might cause long-term plasticity leading to greater GABA release from LN2 neurons. In cerebellar stellate cells, such an increase in inhibitory transmitter release has been documented and coined 'inhibitory-long term potentiation' (I-LTP). I-LTP is induced in stellate cells by glutamate released from parallel fibers acting on presynaptic NMDA receptors in these inhibitory interneurons and producing a long-lasting increase in the release of GABA from these cells. Like stellate neurons, at least one population of Drosophila LNs is pharmacologically GABAergic (Sachse, 2007).

How might alterations in LN2 pharmacology affect downstream circuit elements and ultimately CO₂-evoked behavior? Drawing on the same cerebellar analogy discussed above, it is plausible that PNs exhibit a type of 'rebound potentiation' that has been observed in Purkinje cells responding to inhibitory input. GABA released from LNs would regulate the excitability of PNs, such that greater GABA release from LN2 would tend to decrease the excitability of CO₂-specific PNs. Our finding that the output from the V glomerulus to the lateral horn is reduced following CO₂ exposure supports the idea that downstream activity in higher processing centers is modulated by the antennal lobe network. However, it still needs to be shown that LN2 neurons form direct inhibitory synapses onto PNs in the V glomerulus. Reduced PN activity in the lateral horn in turn may produce a reduced behavioral sensitivity to this stimulus. Future experiments that examine this stimulus-dependent plasticity at the cellular level using pharmacology and electrophysiology will be necessary to test this model (Sachse, 2007).

Search PubMed for articles about Drosophila Gr21a and Gr61a

Boyden, E. S., et al. (2005). Millisecond-timescale, genetically targeted optical control of neural activity, Nat. Neurosci. 8: 1263-1268. Medline abstract: 16116447

Cayirlioglu, P., et al. (2008). Hybrid neurons in a microRNA mutant are putative evolutionary intermediates in insect CO2 sensory systems. Science 319(5867): 1256-60. PubMed citation: 18309086

de Bruyne, M., Foster, K. and Carlson, J. R. (2001). Odor coding in the Drosophila antenna. Neuron 30: 537-552. Medline abstract: 11395013

Faucher, C., Forstreuter, M., Hilker, M. and de Bruyne, M. (2006). Behavioral responses of Drosophila to biogenic levels of carbon dioxide depend on life-stage, sex and olfactory context. J. Exp. Biol. 209: 2739-2748. Medline abstract: 16809465

Gillies, M. T. (1980). The role of carbon dioxide in host-finding in mosquitoes (Diptera:Culicidae): a review. Bull. Entomol. Res. 70: 525-532

Jones, W. D., Cayirlioglu, P., Kadow, I. G. and Vosshall, L. B. (2007). Two chemosensory receptors together mediate carbon dioxide detection in Drosophila. Nature 445(7123): 86-90. Medline abstract: 17167414

Kwon, J. Y., Dahanukar, A., Weiss, L. A. and Carlson, J. R. (2007). The molecular basis of CO₂ reception in Drosophila. Proc. Natl. Acad. Sci. 104(9): 3574-8. Medline abstract: 17360684

Nagel, G., et al. (2003). Channelrhodopsin-2, a directly light-gated cation-selective membrane channel, Proc. Natl. Acad. Sci. 100: 13940-13945. Medline abstract: 14615590

Olsen, S. R., Bhandawat, V. and Wilson, R. I. (2007). Excitatory interactions between olfactory processing channels in the Drosophila antennal lobe. Neuron 54: 89-103. PubMed citation: 17408580

Sachse, S., Rueckert, E., Keller, A., Okada, R., Tanaka, N. K., Ito, K. and Vosshall, L. B. (2007). Activity-dependent plasticity in an olfactory circuit. Neuron 56(5): 838-50. PubMed citation: 18054860

Scott, K. et al. (2001). A chemosensory gene family encoding candidate gustatory and olfactory receptors in Drosophila. Cell 104: 661-673. Medline abstract: 11257221

Shang, Y., Claridge-Chang, A., Sjulson, L., Pypaert, M. and Miesenbock, G. (2007). Excitatory local circuits and their implications for olfactory processing in the fly antennal lobe. Cell 128: 601-612. PubMed citation: 17289577

Southwick, E. E. and Moritz, R. F. A. (1987). Social control of air ventilation in colonies of honey bees, Apis mellifera. J. Insect Physiol. 33: 623-626

Stange, G. and Stowe, S. (1999). Carbon-dioxide sensing structures in terrestrial arthropods. Microsc. Res. Tech 47: 416-427. Medline abstract: 10607381

Suh, G. S. et al. (2004). A single population of olfactory sensory neurons mediates an innate avoidance behaviour in Drosophila. Nature 431: 854-859. Medline abstract: 15372051

Suh, G. S., et al. (2007). Light activation of an innate olfactory avoidance response in Drosophila. Curr. Biol. 17(10): 905-8. Medline abstract: 17493811

Takken, W. and Knols, B. G. Odor-mediated behavior of Afrotropical malaria mosquitoes. Annu. Rev. Entomol. 44: 131-157. Medline abstract: 9990718

Thom, C., Guerenstein, P. G., Mechaber, W. L. and Hildebrand, J. G. (2004). Floral CO₂ reveals flower profitability to moths. J. Chem. Ecol. 30: 1285-1288. Medline abstract: 15303329

date revised: 10 July 2008

Home page: The Interactive Fly © 2006 Thomas Brody, Ph.D.