InteractiveFly: GeneBrief

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

Gene name - Gustatory receptor 21a and Gustatory receptor 63a

Synonyms -

Cytological map position- 21E2-21E2 and 63F5-63F5

Function - G-protein coupled receptor

Keywords - C02, gustatory receptor, antenna, olfactory sensory neurons

Symbol - Gr21a and Gr63a

FlyBase ID: FBgn0041250 and FBgn0035468

Genetic map position - 2L and 3L

Classification - gustatory receptors, GPCR

Cellular location - surface transmembrane

NCBI links for Gr21a: Precomputed BLAST | EntrezGene

NCBI links for Gr63a: Precomputed BLAST | EntrezGene
Recent literature
Pan, J. W., McLaughlin, J., Yang, H., Leo, C., Rambarat, P., Okuwa, S., Monroy-Eklund, A., Clark, S., Jones, C. D. and Volkan, P. C. (2017). Comparative analysis of behavioral and transcriptional variation underlying CO2 sensory neuron function and development in Drosophila. Fly (Austin): [Epub ahead of print]. PubMed ID: 28644712
Carbon dioxide is an important environmental cue for many insects, regulating many behaviors including some that have direct human impacts. To further improve understanding of how this system varies among closely related insect species, both the behavioral response to CO2 as well as the transcriptional profile were examined of key developmental regulators of CO2 sensory neurons in the olfactory system across the Drosophila genus. CO2 was found to generally evoke repulsive behavior across most of the Drosophilids examined, but this behavior has been lost or reduced in several lineages. Comparisons of transcriptional profiles from the developing and adult antennae for subset these species suggest that behavioral differences in some species may be due to differences in the expression of the CO2 co-receptor Gr63a. Furthermore, these differences in Gr63a expression are correlated with changes in the expression of a few genes known to be involved in the development of the CO2 circuit, namely dachshund, an important regulator of sensilla fate for sensilla that house CO2 ORNs, and mip120, a member of the MMB/dREAM epigenetic regulatory complex that regulates CO2 receptor expression. In contrast, most of the other known structural, molecular, and developmental components of the peripheral Drosophila CO2 olfactory system seem to be well-conserved across all examined lineages. These findings suggest that certain components of CO2 sensory ORN development may be more evolutionarily labile, and may contribute to differences in CO2-evoked behavioral responses across species.

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).

Molecular and cellular organization of the taste system in the Drosophila larva

This study examined the molecular and cellular basis of taste perception in the Drosophila larva through a comprehensive analysis of the expression patterns of all 68 Gustatory receptors (Grs). Gr-GAL4 lines representing each Gr are examined, and 39 show expression in taste organs of the larval head, including the terminal organ (TO), the dorsal organ (DO), and the pharyngeal organs. A receptor-to-neuron map is constructed. The map defines 10 neurons of the TO and DO, and it identifies 28 receptors that map to them. Each of these neurons expresses a unique subset of Gr-GAL4 drivers, except for two neurons that express the same complement. All of these neurons express at least two drivers, and one neuron expresses 17. Many of the receptors map to only one of these cells, but some map to as many as six. Conspicuously absent from the roster of Gr-GAL4 drivers expressed in larvae are those of the sugar receptor subfamily. Coexpression analysis suggests that most larval Grs act in bitter response and that there are distinct bitter-sensing neurons. A comprehensive analysis of central projections confirms that sensory information collected from different regions (e.g., the tip of the head vs the pharynx) is processed in different regions of the subesophageal ganglion, the primary taste center of the CNS. Together, the results provide an extensive view of the molecular and cellular organization of the larval taste system (Kwon, 2011).

Of the 67 Gr-GAL4 transgenes, 43 showed expression in the larva, of which 39 were expressed in the major taste organs of the head. The 39 Gr-GAL4 drivers are expressed in combinatorial fashion. Individual Gr-GAL4 drivers are expressed in up to 12 cells, in the case of Gr33a- and Gr66a-GAL4; approximately one-half, however, are expressed in only one cell (Kwon, 2011).

For some Gr-GAL4 drivers the observed pattern of expression may not be identical with that of the endogenous Gr gene. It was precisely with this concern in mind that a mean of 7.6 independent lines were analyzed for each of the 67 Gr drivers, and a rigorous, quantitative protocol was establised for identifying a representative line for each gene. In the absence of an effective in situ hybridization protocol, the approach used here seemed likely to be the most informative in providing a comprehensive systems-level view of larval taste reception (Kwon, 2011).

The Gr receptor-to-neuron map of the dorsal and terminal organs identified 10 neurons. Two neurons have cell bodies in the DOG and innervate the DO, two have cell bodies in the DOG and innervate the TO, and six have cell bodies in the TOG and innervate the TO (Kwon, 2011).

28 receptors were mapped to these 10 neurons. All of these neurons express at least two Gr-GAL4 drivers. Two receptors, Gr21a and Gr63a, are coreceptors for CO2; neither is sufficient to confer chemosensory function alone. It is conceivable that many other Grs may also require a coreceptor, which may explain the lack of neurons expressing a single Gr-GAL4. The number of receptors per neuron ranges up to 17, in the case of C1. This number is comparable with the maximum number of Gr-GAL4s observed in a labellar neuron (29), and much greater than the number of Ors observed in individual neurons of either the larval or adult olfactory system (Kwon, 2011).

Among the 10 identified cells, individual Gr-GAL4 drivers are expressed in as few as one cell and as many as six cells. Most of the drivers are expressed in only one of these 10 cells. The drivers expressed in six cells, Gr33a-GAL4 and Gr66a-GAL4, are expressed in all bitter neurons of the adult labellum. It is noted that Gr33a-GAL4 and Gr66a-GAL4 are the only drivers expressed in B1, arguing against the possibility that both of these receptors function exclusively as chaperones or as coreceptors that require another Gr for ligand specificity (Kwon, 2011).

There is little cellular redundancy. Only two neurons, A1 and A2, express the same complement of receptors. All other neurons contain a unique subset of the Gr repertoire. In this respect, the larval taste system differs from the adult taste system but is similar to the larval olfactory system, which also contains little if any cellular redundancy (Kwon, 2011).

Analysis of the central projections of all 39 Gr-GAL4 drivers provided evidence for a systematic difference among projection patterns between TO/DO neurons and pharyngeal neurons. These results support the conclusion that sensory information collected from the tip of the head is processed in different regions of the SOG than information collected in the pharynx, i.e., that evaluation of a potential food source before ingestion and the testing of food quality during ingestion are functionally partitioned in the brain. Similar inferences were drawn in an elegant study of a limited number of Gr-GAL4 transgenes (Colomb, 2007; Kwon, 2011 and references therein).

Conspicuously absent from the list of Gr-GAL4 drivers expressed in the larval taste system are those representing the eight members of the sugar receptor subfamily (Gr5a, Gr61a, Gr64a-f). The founding member of this family, Gr5a, mediates response to the sugar trehalose, and two other members of the subfamily have been shown to encode sugar receptors as well. No GFP expression for these genes was observed in cells of the taste organs or in neural fibers in the brain or ventral ganglion. Most of these Gr-GAL4 transgenes drive expression in the adult, but it is acknowledged that these transgenes may not faithfully reflect expression in the larva (Kwon, 2011).

Given that Drosophila larvae respond to sugars, as do larvae of other insect species, how do they detect them without members of the sugar receptor subfamily? Other Grs, including the recently identified fructose receptor Gr43a, may underlie sugar detection in the larva. It is noted that Gr59e-GAL4 and Gr59f-GAL4 are coexpressed in a cell that does not express the bitter cell markers Gr33a-GAL4 or Gr66a-GAL4. Sugar reception may also be mediated by other kinds of receptors, such as those of the TRPA family (Kwon, 2011).

In adult Drosophila, Gr33a-GAL4 and Gr66a-GAL4 are coexpressed with other Gr-GAL4s in bitter neurons; the simplest interpretation of expression and functional analysis is that multiple bitter receptors are coexpressed (Kwon, 2011).

In the larva, it ws found that most larval Gr-GAL4s are coexpressed with Gr33a- and Gr66a-GAL4, suggesting the possibility that most larval Grs act in bitter response. It is noted that, of the 17 Gr-GAL4s coexpressed in the C1 neuron, 15 are coexpressed in a bitter neuron of the labellum. It was also establish that there are distinct molecular classes of Gr33a-GAL4, Gr66a-GAL4-expressing neurons. The simplest interpretation of these results is that there are distinct bitter-sensing neurons in larvae (Kwon, 2011).

Larvae must determine whether to accept or reject a food source, and in principle this determination could be made by a simple binary decision-making circuit. However, the existence of six Gr33a-GAL4, Gr66a-GAL4-expressing neurons expressing distinct subsets of Gr-GAL4s suggests a greater level of complexity in the processing of gustatory information. One possibility is that C1, which expresses the largest subset of drivers among the TO/DO neurons, may activate an aversive behavior in response to many of the bitter compounds that the larva encounters, while C2, C3, C4, or B2 either potentiates the response or activates a different motor program in response to chemical cues of particular biological significance or exceptional toxicity. The existence of heterogenous bitter-sensing cells, some more specialized than others, is a common theme in insect larvae. In particular, many species contain a taste cell that responds physiologically to many aversive compounds and whose activity deters feeding. C1 could be such a cell, and its coexpression of many receptors may provide the molecular basis of a broad response spectrum (Kwon, 2011).

It is striking that the number of TO/DO neurons that express Gr-GAL4s is small compared with the total number of TO/DO neurons. Gr-GAL4 expression was mapped to only 10 cells in the TO/DO (although Gr2a-GAL4 and Gr28a-GAL4 were each expressed in two TO neurons that were not mapped). The DOG and TOG contain 36-37 and 32 sensory neurons, respectively, among which 21 in the DOG are olfactory. Thus, of the nonolfactory cells, on the order of 20%-30% express Gr-GAL4 drivers. It will be interesting to determine how many of the other DOG/TOG cells express other chemoreceptor genes, such as Ppk, Trp, or IR genes, and how many of the other neurons have mechanosensory, thermoreceptive, hygrosensory, or other sensory functions (Kwon, 2011).

The role of Gr genes in the larval pharyngeal organs is unknown. In adult pharyngeal sensilla, the TRPA1 channel, which detects irritating compounds, regulates proboscis extension. It is possible that Grs expressed in larval pharyngeal organs may also play a role in modulating feeding behavior. Of the 24 Gr-GAL4 drivers expressed in the larval pharyngeal organs, 9 are coexpressed with Gr33a-GAL4 and Gr66a-GAL4 in the TO/DO; it seems plausible that they may monitor ingested food for the presence of aversive compounds (Kwon, 2011).

In summary, this study has analyzed essential features of the molecular and cellular organization of a numerically simple taste system in a genetic model organism. Ten gustatory receptor neurons were described and evidence was provided that they express Grs in combinatorial fashion, with most of these neurons and receptors acting in the perception of bitter compounds. The results lay a foundation for a molecular and genetic analysis of how these receptors and neurons, and the downstream circuitry, underlie a critical decision: whether to accept or reject a food source (Kwon, 2011).

Temporal response dynamics of Drosophila olfactory sensory neurons depends on receptor type and response polarity

Insect olfactory sensory neurons (OSN) express a diverse array of receptors from different protein families, i.e. ionotropic receptors (IR), gustatory receptors (GR) and odorant receptors (OR). It is well known that insects are exposed to a plethora of odor molecules that vary widely in both space and time under turbulent natural conditions. In addition to divergent ligand specificities, these different receptors might also provide an increased range of temporal dynamics and sensitivities for the olfactory system. To test this, different Drosophila OSNs were challenged with both varying stimulus durations (10-2000 ms), and repeated stimulus pulses of key ligands at various frequencies (1-10 Hz). The results show that OR-expressing OSNs responded faster and with higher sensitivity to short stimulations as compared to IR- and Gr21a-expressing OSNs. In addition, OR-expressing OSNs could respond to repeated stimulations of excitatory ligands up to 5 Hz, while IR-expressing OSNs required ~5x longer stimulations and/or higher concentrations to respond to similar stimulus durations and frequencies. Nevertheless, IR-expressing OSNs did not exhibit adaptation to longer stimulations, unlike OR- and Gr21a-OSNs. Both OR- and IR-expressing OSNs were also unable to resolve repeated pulses of inhibitory ligands as fast as excitatory ligands. These differences were independent of the peri-receptor environment in which the receptors were expressed and suggest that the receptor expressed by a given OSN affects both its sensitivity and its response to transient, intermittent chemical stimuli. OR-expressing OSNs are better at resolving low dose, intermittent stimuli, while IR-expressing OSNs respond more accurately to long-lasting odor pulses. This diversity increases the capacity of the insect olfactory system to respond to the diverse spatiotemporal signals in the natural environment (Getahun, 2012).

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, stimulus-evoked changes were examined 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. The 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).

Modification of CO2 avoidance behaviour in Drosophila by inhibitory odorants

The fruitfly Drosophila melanogaster exhibits a robust and innate olfactory-based avoidance behaviour to CO2, a component of odour emitted from stressed flies. Specialized neurons in the antenna and a dedicated neuronal circuit in the higher olfactory system mediate CO2 detection and avoidance. However, fruitflies need to overcome this avoidance response in some environments that contain CO2 such as ripening fruits and fermenting yeast, which are essential food sources. Very little is known about the molecular and neuronal basis of this unique, context-dependent modification of innate olfactory avoidance behaviour. This study identified a new class of odorants present in food that directly inhibit CO2-sensitive neurons in the antenna. Using an in vivo expression system it was established that the odorants act on the Gr21a/Gr63a CO2 receptor. The presence of these odorants significantly and specifically reduces CO2-mediated avoidance behaviour, as well as avoidance mediated by 'Drosophila stress odour'. A model is proposed in which behavioural avoidance to CO2 is directly influenced by inhibitory interactions of the novel odours with CO2 receptors. Furthermore, differences were observed in the temporal dynamics of inhibition: the effect of one of these odorants lasts several minutes beyond the initial exposure. Notably, animals that have been briefly pre-exposed to this odorant do not respond to the CO2 avoidance cue even after the odorant is no longer present. Related odorants were shown to be effective inhibitors of the CO2 response in Culex mosquitoes that transmit West Nile fever and filariasis. These findings have broader implications in highlighting the important role of inhibitory odorants in olfactory coding, and in their potential to disrupt CO2-mediated host-seeking behaviour in disease-carrying insects like mosquitoes (Turner, 2009).

CO2 is an important sensory cue for many animals, including insects, in a variety of behavioural contexts. In Drosophila, CO2 is exclusively detected by a unique heteromeric receptor encoded by Gr21a and Gr63a that is expressed in a single population of antennal olfactory receptor neurons (ORNs), called ab1C, which innervate the ab1 class of large basiconic sensilla. These neurons send stereotypical axonal projections to the V glomerulus, and activation of this dedicated uni-glomerular circuit leads to an innate avoidance of CO2 (Turner, 2009).

In fact, CO2 is a major component of Drosophila stress odour (dSO), which is emitted by flies subjected to vigorous shaking or electric shock, and which elicits an immediate escape response in naive flies (Suh, 2004). However, CO2 is also present in significant quantities in several important food sources that elicit behavioural attraction of Drosophila. Fruits and plants emit CO2 as a by-product of respiration, as do fruits undergoing fermentation by microorganisms and yeasts. Flies are attracted to headspace odours containing CO2 collected from over-ripe fruits, fermenting yeast and beer when presented with a choice between two tubes in a T-maze assay, one containing air and the other containing headspace odours. However, flies avoid headspace odours collected from green fruits, which also emit CO2. A subset of specialized gustatory neurons mediate a small degree of attraction to carbonated water upon contact (Fischler, 2007); however, they do not respond to CO2 in the gas phase and are not likely to contribute to long-range or short-range behavioural attraction towards a food source. Therefore, olfactory avoidance to CO2 may be modified by context for some CO2-rich sources such as over-ripe fruit, yeast and beer (Turner, 2009).

Little is known about the molecular and neuronal mechanisms that lead to such a dramatic modification of innate avoidance behaviour. Two alternative models, although not mutually exclusive, may be evoked to explain this phenomenon. In the first model, avoidance to CO2 is overcome simply by detection of attractive odorants emitted by the same food sources. In the second model, some components of food volatiles may also directly inhibit the CO2-responsive circuit, and thereby suppress avoidance behaviour to CO2 (Turner, 2009).

To test whether odorants present in fruits and other natural environments of Drosophila can directly inhibit CO2-sensitive ab1C neurons, a simple electrophysiology screen was performed. Several individual odorants were tested for their ability to inhibit the baseline activity of the ab1C neuron (to about 0.03% CO2 present in room air) using single-sensillum electrophysiology. These experiments were performed using Or83b2 mutant flies in which the ab1C neuron remains the sole functional neuron in the ab1 sensillum. In a screen with 46 odorants, two, 1-hexanol and 2,3-butanedione, were identified that strongly inhibit the baseline activity of ab1C neurons. Both of these compounds are present in Drosophila food sources including various types of fruit. More interestingly, the abundance of both these compounds is greatly increased during the ripening process of fruits: for example, in banana, 1-hexanol increases by 777% and 2,3-butanedione by 14,900%. 1-Hexanol is formed during ripening by lipid oxidation of unsaturated fatty acids, whereas 2,3-butanedione is a natural by-product of fermentation of carbohydrates through pyruvate by yeasts and bacteria and is thus also present in fermenting fruit, wine (Turner, 2009).

Both 1-hexanol and 2,3-butanedione inhibit CO2 response in a dose-dependent manner, irrespective of whether their application is initiated before, or after, the presentation of the CO2 stimulus at relatively low, physiologically relevant concentrations (Turner, 2009).

A fly approaching an odour source from a distance likely contacts plumes of CO2, which will vary widely in concentration over baseline atmospheric levels. When several concentrations of CO2 were tested, it was found that the presence of 2,3-butanedione (10-1 dilution) completely inhibits responses up to 3.2% CO2; 1-hexanol (10-1 dilution) also causes a significant reduction of CO2 response across most tested concentrations, but complete inhibition occurs only at 0.1% CO2 (Turner, 2009).

To understand odorant structural features that might have a role in inhibition, a rationally designed panel of odorants was tested that varied in the number of carbon atoms and in the nature of the functional group. On the basis of this analysis, additional odorants were identified that also inhibit CO2 response. The inhibitory effects of each of the compounds identified so far are specific to the CO2-sensitive neuron; previous studies have shown that all of them can excite other classes of Drosophila ORNs, which suggests that they are not general inhibitors of ORN function. Surprisingly, these compounds are structurally quite different from CO2, thus raising the possibility that they may act through allosteric binding sites within the Gr21a/Gr63a receptor, or on other components of the CO2 detection pathway such as factors present in the sensillar lymph or in ab1C neurons (Turner, 2009).

To investigate whether the inhibitors act directly on the CO2 receptor, Gr21a and Gr63a were expressed in an in vivo decoder system called the 'empty neuron'. It was found that expression of Gr21a and Gr63a in the empty ab3A neuron is sufficient to impart a robust and reproducible dose-dependent CO2 response, comparable to the levels reported previously. Upon application of each of the four inhibitory odorants along with CO2, dose-dependent inhibition was observed of CO2 response of the ab3A neuron in a Gr21a/Gr63a-dependent manner. The simplest interpretation of these results is that the odorants that were identified inhibit CO2 response by direct interaction with the CO2 receptor, Gr21a/Gr63a. However, the inhibitory effect appears shorter in duration than observed in the endogenous ab1C neurons, suggesting that additional neuron- or sensillum-specific factors may also influence the temporal aspects of the inhibition (Turner, 2009).

Next it was asked whether the inhibitory odorants identified using electrophysiology could disrupt avoidance behaviour of Drosophila to CO2. Using a T-maze choice assay, it was found that wild-type Drosophila show a robust avoidance behaviour to 0.67% CO2. Inclusion of 2,3-butanedione with CO2 completely abolishes avoidance to CO2. Importantly, 2,3-butanedione by itself does not elicit any significant attraction or avoidance behaviour. In wild-type Drosophila, however, several other ORN classes are activated by 2,3-butanedione, raising the possibility that behavioural avoidance to CO2 may be overcome by activation of these other classes of ORN, and not solely by inhibition of CO2-responsive neurons (Turner, 2009).

To distinguish between these possibilities, the behaviour of Or83b2 mutant flies was tested under conditions in which most ORNs are non-functional, but electrophysiological responses to CO2 are not affected. Consistent with the electrophysiological analysis, flies lacking Or83b have a robust avoidance response to CO2, which is absent when 2,3-butanedione is included with CO2 or is presented alone. Similar results, albeit with weaker effects, are obtained using 1-hexanol. Taken together, these results show that inhibitory odorants can effectively block CO2-mediated innate avoidance behaviour (Turner, 2009).

CO2 is one of the main components of dSO, which is emitted by stressed flies, and which triggers a robust avoidance behaviour in naive flies. Therefore, whether 2,3-butanedione can disrupt avoidance to dSO was tested. It was found that naive flies avoid odour collected from a tube of vortexed flies (dSO), but not that collected from a tube of untreated flies (mock), in a T-maze assay. Remarkably, addition of 2,3-butanedione to dSO effectively abolishes avoidance behaviour (Turner, 2009).

Interestingly, it was observed that with increasing concentrations of 2,3-butanedione, the CO2 neuron is silenced well beyond the period of application. This effect is specific to 2,3-butanedione and is not observed for 1-hexanol. To investigate this further, the fly was exposed to a 3-s stimulus of 2,3-butanedione (10-1 dilution) and subsequently tests were performed for the recovery of ab1C neuron responsiveness by applying a 0.5-s stimulus of 0.3% CO2 every 30 s, over a period of 10 min. Surprisingly, the inhibitory effect of the initial exposure to 2,3-butanedione persisted for an extended period (Turner, 2009).

It was of interest to test whether behaviour was also affected in a similar manner. Flies were exposed for 1 min to 2,3-butanedione and then transferred them to clean air for 2 min before testing for CO2-mediated avoidance behaviour. Remarkably, CO2 avoidance is almost abolished in pre-treated flies. Prior exposure to another odorant 2-methyl phenol, which does not inhibit the CO2 response, does not have any effect on behaviour. Moreover, pre-exposure to 2,3-butandione does not have a significant effect on behavioural attraction towards a different odorant, ethyl acetate. Taken together, these observations show that exposure to a long-term CO2 response inhibitor can exert a profound and specific effect on the behaviour of the animal, even after it is no longer present in the environment. Similar observations were made with Or83b mutant flies (Turner, 2009).

To demonstrate unambiguously that 2,3-butanedione causes behaviour modification primarily by inhibiting CO2 responsiveness of ab1C neurons and not by other peripheral or central mechanisms, the following experiment was performed. The ab1C neuron was activated in a manner that is not inhibited by 2,3-butanedione, and it was asked whether 2,3-butanedione inhibits avoidance behaviour in this context. The odorant, butanone, which activates ab1C neurons strongly at 10-1 dilution in a Gr63a-dependent manner, was identified. It was found that Or83b mutant flies strongly avoid butanone (10-1 dilution) whereas flies lacking both Or83b and Gr63a do not, as predicted from the electrophysiology data. However, electrophysiological response to butanone is not affected by pre-exposure to, or the presence of, 2,3-butanedione, unlike what was observed for CO2. In a T-maze behaviour assay, 2,3-butanedione has no effect on behavioural avoidance of Or83b mutant flies to butanone, regardless of whether it is used to pre-treat the flies as described above or is included in a mixture with butanone. These results demonstrate that 2,3-butanedione disrupts CO2 avoidance behaviour by directly inhibiting the CO2 responsiveness of ab1C neurons, rather than by other indirect mechanisms (Turner, 2009).

CO2 emitted in human breath is a critical component of odour blends used as host-seeking cues by many vector insect species that carry deadly diseases, including Culex quinquefasciatus mosquitoes that transmit filarial parasites in tropical countries, and West Nile virus in the USA and various parts of the world. Culex mosquitoes have three conserved proteins that are closely related to the Drosophila CO2 receptors, Gr21a and Gr63a. To test whether odorants that inhibit Drosophila CO2 receptors can also inhibit CO2 response in Culex, CO2-sensitive A neurons in peg sensilla on the surface of the maxillary palps of Culex mosquitoes were tested using a panel of structurally related odours. It was found found that electrophysiological response to CO2 is not inhibited by 2,3-butanedione, but is strongly inhibited by 1-butanal and 1-hexanol. These odours are the first reported inhibitors of CO2-sensitive neurons in mosquitoes and may provide a valuable resource for the identification of economical, environmentally safe, volatile compounds that may reduce mosquito-human contact by blocking responsiveness to CO2 (Turner, 2009).


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. PubMed ID: 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 ID: 18309086

Colomb, J., Grillenzoni, N., Ramaekers, A. and Stocker, R. F. (2007). Architecture of the primary taste center of Drosophila melanogaster larvae. J. Comp. Neurol. 502: 834-847. PubMed ID: 17436288

de Bruyne, M., Foster, K. and Carlson, J. R. (2001). Odor coding in the Drosophila antenna. Neuron 30: 537-552. PubMed ID: 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. PubMed ID: 16809465

Fischler, W., Kong, P., Marella, S. and Scott, K. (2007). The detection of carbonation by the Drosophila gustatory system. Nature 448: 1054-1057. PubMed ID: 1537205

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

Getahun, M. N., Wicher, D., Hansson, B. S. and Olsson, S. B. (2012). Temporal response dynamics of Drosophila olfactory sensory neurons depends on receptor type and response polarity. Front Cell Neurosci 6: 54. PubMed ID: 23162431

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. PubMed ID: 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. PubMed ID: 17360684

Kwon, J. Y., Dahanukar, A., Weiss, L. A. and Carlson, J. R. (2011). Molecular and cellular organization of the taste system in the Drosophila larva. J. Neurosci. 31(43): 15300-9. PubMed ID: 22031876

Nagel, G., et al. (2003). Channelrhodopsin-2, a directly light-gated cation-selective membrane channel, Proc. Natl. Acad. Sci. 100: 13940-13945. PubMed ID: 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 ID: 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 ID: 18054860

Scott, K. et al. (2001). A chemosensory gene family encoding candidate gustatory and olfactory receptors in Drosophila. Cell 104: 661-673. PubMed ID: 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 ID: 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. PubMed ID: 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. PubMed ID: 15372051

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

Takken, W. and Knols, B. G. Odor-mediated behavior of Afrotropical malaria mosquitoes. Annu. Rev. Entomol. 44: 131-157. PubMed ID: 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. PubMed ID: 15303329

Turner, S. L. and Ray, A. (2009). Modification of CO2 avoidance behaviour in Drosophila by inhibitory odorants. Nature 461(7261): 277-81. PubMed ID: 19710651

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date revised: 10 February 2012

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