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. 2011 Apr 1;5(2):119–125. doi: 10.4161/fly.5.2.14284

Behavioral responses to hypoxia and hyperoxia in Drosophila larvae

Molecular and neuronal sensors

David B Morton 1,✉
PMCID: PMC3127060  PMID: 21150317

Abstract

The ability to detect changes in oxygen concentration in the environment is critical to the survival of all animals. This requires cells to express a molecular oxygen sensor that can detect shifts in oxygen levels and transmit a signal that leads to the appropriate cellular response. Recent biochemical, genetic and behavioral studies have shown that the atypical soluble guanylyl cyclases function as oxygen detectors in Drosophila larvae triggering a behavioral escape response when exposed to hypoxia. These studies also identified the sensory neurons that innervate the terminal sensory cones as likely chemosensors that mediate this response. Here I summarize the data that led to these conclusions and also highlight evidence that suggests additional, as yet unidentified, proteins are also required for detecting increases and decreases in oxygen concentrations.

Key words: soluble guanylyl cyclase, cyclic GMP, oxygen detection

All animals need to respond to changes in environmental oxygen (O2) levels in order to survive. These responses range from relatively long-term adaptive changes in response to anoxia and hypoxia to short-term behavioral changes.1 For the purposes of this discussion I will define “hypoxia” as less than 21% O2 and “hyperoxia” as greater than 21% O2. Adaptive changes to hypoxia, for example changes in gene expression that increase survival in conditions of reduced O2, are mediated via the hypoxia-inducible transcription factors (HIF) such as HIF1.2 By contrast, short-term behavioral changes require sensory neurons to detect changes in O2 levels and activate specific behavioral or physiological responses. Examples of the latter include hyperventilation and increased heart output.1,3,4

Common to both long-term and short-term responses to hypoxia, is the need for a molecular O2 sensor that detects changes in O2 and transduces these changes into the appropriate cellular response: changes in transcription for the former and changes in electrical activity in the sensory neurons for the latter. For long-term transcriptional changes, the concentrations of O2 are sensed by a prolyl hydroxylase whose activity is dependent on O2.2 In the presence of O2 this enzyme is active, hydroxylates HIF1 and targets it for ubiquitination and degradation, thus preventing its function as a transcription factor.2 During hypoxia, HIF1 is no longer hydroxylated, is not degraded and can activate transcription of a variety of genes that are important in adaption to hypoxia.2 In mammals, short-term reduction in oxygenation of the blood is detected by the glomus cells of the carotid body where it is transduced to membrane depolarization and ultimately to changes in cardiovascular and pulmonary physiology.1 The O2 sensor in this case is hemoxygenase-2 (HO-2), which in the presence of O2 generates carbon monoxide (CO), which activates specific potassium channels in the cell membrane.5 In response to reduced oxygenation, the activity of HO-2 is reduced, generating less CO, resulting in an inhibition of the potassium channels.5

The identification of molecular O2 sensors in insects and other invertebrates had received little, if any, attention until a paper from Cori Bargmann and Michael Marletta's labs was published in 2004 showing that an atypical soluble guanylyl cyclase in C. elegans could bind O2 and was required for the animals preference for 5–10% O2.6 Soluble guanylyl cyclases (sGCs) catalyze the synthesis of the intracellular messenger cyclic guanosine monophosphate (cGMP). Conventional sGCs are the main receptor for and mediate the majority of the physiological actions of the gaseous messenger, nitric oxide (NO).7 These enzymes are heterodimeric, heme-containing proteins that unlike most heme proteins cannot bind oxygen.8 In fact, this property makes the conventional sGCs ideally suited to bind and respond to changes in the levels of NO in the presence of much higher concentrations of O2. The resulting cGMP mediates a wide variety of physiological functions including vascular smooth muscle relaxation and neural plasticity. 7 The first molecularly characterized atypical sGC was identified in the insect Manduca sexta and named MsGC-β3.9 The sequence of MsGC-β3 was closely related to the conventional sGCs found in Manduca but it had two unusual biochemical characteristics that defined it as an atypical sGC—it had enzyme activity as a homodimer and it was insensitive to NO.9,10

When the C. elegans genome was sequenced it was found to contain seven atypical sGCs, but no conventional sGCs or NO synthase.11 The sequences of the C. elegans atypical sGCs suggested that they would also be insensitive to NO, but this could not be confirmed as no enzymatic activity could be measured when they were expressed in heterologous cells.12 The Drosophila genome contains genes that code for NO synthase and conventional sGCs, which are potently activated by NO.13 In addition, when the Drosophila genome was sequenced it was found to also contain genes that coded for three atypical sGCs. The three Drosophila atypical sGCs were named based on their chromosomal locations: Gyc-88E, located at 88E is the orthologue to MsGC-β3, and two additional genes located adjacent to each other at 89D, named Gyc-89Da and Gyc-89Db.14 When these were expressed in heterologous cells, they also had properties distinct from those of conventional sGCs. Gyc-88E, like MsGC-β3, showed activity without co-expression of additional subunits and was weakly stimulated by NO.14 Gyc-89Da and Gyc-89Db had no activity when expressed alone, but when co-expressed with Gyc-88E, both Gyc-89Da and Gyc-89Db exhibited basal activity and weak activation by some, but not all, NO donors.14,15 Although Gyc-88E had enzyme activity in the absence of additional subunits, in situ hybridizations showed that it was always co-expressed with either Gyc-89Da or Gyc-89Db, suggesting that in vivo, the atypical sGCs functioned as heterodimers.14,15 A comparison of the Drosophila conventional and atypical sGCs showed that the heterodimeric atypical sGCs were stimulated less potently by NO donors compared to the conventional sGCs (less than 4-fold compared to 30-fold or more) and required at least 10-fold higher concentrations of donor.15 This strongly suggested that NO was not the endogenous ligand for the atypical sGCs.

A combination of biochemical, genetic and behavioral experiments showed that the atypical sGCs in C. elegans acted as molecular O2 detectors.6 There are seven atypical sGCs in C. elegans (gcy-31 through gcy-37) and the heme-binding domain of one of these, gcy-35, was shown to bind O2 in addition to NO and CO.6 This contrasted to the equivalent region of the β1 subunit of the rat conventional sGC which bound NO and CO, but not O2.6 Furthermore, the authors showed that behavioral responses to O2 required gcy-35. When exposed to an oxygen gradient of 0–21% O2 wild-type C. elegans avoid both low (<2%) and high (>12%) O2 concentrations, congregating at 7–10% O2.6 Animals mutant for gcy-35 failed to avoid the extremes and distributed themselves evenly across the gradient.6 Further studies showed that gcy-35 and gcy-36 were required for hypoxia and avoidance of 21% O2, gcy-34 mutants showed a preference for higher O2 concentrations and gcy-32 appeared to have no effect on O2 preference.16

What was not clear from these studies was how the activity of the atypical sGCs changed with changing O2 concentration as we were unable to measure any enzyme activity from the C. elegans enzymes when expressed in heterologous cells.12 By contrast, the enzyme activity of the Drosophila atypical sGCs responded robustly to changing O2 concentrations with maximal activity measured in the absence of ambient O2. Gradually decreasing activity was measured as O2 concentrations were increased.17 This contrasts to the activation of conventional sGCs which show increasing activity with increasing concentrations of NO.7

The expression patterns of the Drosophila atypical sGCs showed that in larvae they are almost exclusively expressed in sensory and central neurons14,15,18 and this distribution together with their biochemical properties suggested that they were ideal candidates for the O2 sensors mediating behavioral responses to hypoxia.19,20 Drosophila larvae respond rapidly to hypoxia by leaving their normal feeding positions (partially buried in their food) and initiating exploratory behaviors.21 By inactivating the neurons that express Gyc-89Da with tetanus toxin (TTxN) we showed that these neurons were required for this behavioral response19 but our initial efforts at establishing a role for the atypical sGCs in responses to hypoxia were hampered by the apparent redundancy between Gyc-89Da and Gyc-89Db.

At the amino acid level, Gyc-89Da and Gyc-89Db share 85% identity (96% similarity), have a similar gene structure with a single intron and are located adjacent to each other on the genome, separated by only 2 kb. When expressed in heterologous cells their biochemical properties were indistinguishable from each other.12,15,19 All twelve species of Drosophila have two genes coding for atypical sGCs that are homologous to Gyc-89Da and Gyc-89Db whereas other insects that have had their genomes sequenced have a single gene. This includes other Diptera such as the mosquitoes Anopheles gambiae and Aedes aegypti (Morton DB, unpublished observations) and indicates that Gyc-89Da and Gyc-89Db are the result of a relatively recent gene duplication and could be functionally redundant. However, their expression patterns suggested that they might have distinct functions as they are co-expressed in few neurons.18 Both Gyc-89Da and Gyc-89Db are expressed widely in the peripheral and central nervous system (CNS).18 In the CNS we did not detect any neurons that co-expressed both Gyc-89Da and Gyc-89Db.18 In the peripheral nervous system they are expressed in gustatory and olfactory neurons, external sensilla along the lateral body wall and in neurons that innervate the terminal sensory cones. The only neurons that co-express both Gyc-89Da and Gyc-89Db are two external sensilla on each side of each thoracic segment and the neurons that innervate each sensory cone.18 The likely functional differences between neurons that express Gyc-89Da and neurons that express Gyc-89Db was demonstrated by the different lethality and phenotypes observed when TTxN was expressed in each group of neurons. Expression of TTxN in Gyc-89Da neurons resulted in late pupal lethality due to a failure to complete adult eclosion whereas expression in Gyc-89Db neurons caused larvae to die at the first larval molt.18

To examine the effect of each of these genes on hypoxia escape behavior we identified transposon insertions within each of the genes that resulted in loss of expression of each gene.22 In our initial trials of the hypoxia escape assay using these mutant lines we placed larvae in yeast paste, waited until they burrowed into the food and began feeding. Then we exposed the larvae to reduced O2 and counted how many larvae had exited the food at the end of each minute of exposure as described by Wingrove and O'Farrell.21 Using this approach we did not see any differences between these mutant lines and wild-type animals suggesting that Gyc-89Da and Gyc-89Db can substitute for each other in this behavior (Langlais KK, Morton DB, unpublished observations). To overcome this we generated double mutants by recombining the two insertions and also modified the hypoxia escape assay so that we measured the time it took for each individual larva to exit the food. Using this approach we showed that the double mutants took dramatically longer to exit the food when exposed to hypoxia and each of the single mutants also took slightly longer than controls to exit the food.22 An example of this type of data is shown in Figure 1 for the response to 5% O2. We also measured the response of both single and double mutants to 0, 10 and 15% O2 and showed that wild-type animals took longer to exit the food under less severe hypoxic conditions, whereas the mutants did not show as noticeable a trend, with the curves being flatter. At 15% O2 there was no difference in the response times between the single mutants and wild-type animals, although the double mutants still took significantly longer.22 An interesting feature of this data was that although we could not detect any transcript for either Gyc-89Da or Gyc-89Db in the double mutants, the majority of the animals did eventually exit the food within 5 minutes whereas wild-type animals remained in the yeast for at least 10 minutes when exposed to 21% O2.

Figure 1.

Figure 1

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Drosophila larvae lacking the atypical sGCs, Gyc-89Da and Gyc-89Db, are defective in their response to hypoxia. Third instar larvae of the genotypes shown were placed in yeast paste, allowed to burrow and begin feeding and then exposed to 5% O2. The time taken for each larva to exit the food was then logged (detailed description of the methods are provided in ref. 22). Larvae deficient in Gyc-89Da, Gyc-89Db or both Gyc-89Da and Gyc-89Db took significantly longer to exit the food compared to control larvae (*p < 0.05; **p < 0.01 one way ANOVA). Data is redrawn from previously published data.22

To confirm that the defective escape response was due to loss of the atypical sGCs we replaced them using the GAL4-UAS system and took advantage of the fact that we had GAL4 lines for each gene so we could replace either gene with the correct gene or the opposite one and could replace them either in the cells that they are normally expressed in or in the incorrect cells. In addition, we expressed these constructs in all three mutant backgrounds. Given the similar biochemical properties of Gyc-89Da and Gyc-89Db it was not surprising that both subunits could rescue either mutation when expressed in the correct cells.22 However, we could also rescue the response if we replaced either subunit in the incorrect cells for that mutation—for example the response was rescued in Gyc-89Da mutants by expressing either Gyc-89Da or Gyc-89Db using the Gyc-89Db driver.22 The simplest way to explain these observations is that the sensory neurons that mediate the hypoxia escape response are those cells that co-express both Gyc-89Da and Gyc-89Db. This restricts the likely sensory neurons to the external sensilla neurons of the thoracic segments or the neurons that innervate the terminal sensory cones. There has not been a specific function assigned to these latter neurons and their associated sensilla. They are in an ideal location for feeding larvae to detect changes in the external environment as they are located next to the terminal spiracles which remain out of contact with the semi-liquid food. We examined the external sensilla associated with the terminal sensory cones with scanning electron microscopy and identified a single pore in the tip of the sensilla—indicating a chemosensory function.22 We suggest that it is these neurons that sense reduced levels of O2 and trigger the escape response via activation of the atypical sGCs. An image of one of these sensory cones with its associated neuron is shown in Figure 2A and B. It should be possible to directly test whether the cells that express both Gyc-89Da and Gyc-89Db are required for this behavior by restricting expression of various transgenes to these cells using the split-GAL4 expression system.23

Figure 2.

Figure 2

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Sensory neurons and mechanism of action mediating the larval hypoxia escape response. (A) Photomicrograph of the posterior of a third instar larva showing one of the seven pairs of terminal sensory cones (arrow). Scale bar represents 250 µm. (B) Confocal micrograph showing co-expression of Gyc-89Da and Gyc-89Db (yellow where red and green overlap) in the sensory neuron innervating one of the terminal sensory cones. The larva was expressing three transgenes: Gyc-89Da-GFP, Gyc-89Db-GAL4 and UAS-dsRED.18 Note the neurite extending to the sensillum at the tip of the cone (arrow). Scale bar represents 20 µm. (C) Proposed mechanism of activation of these sensory neurons in response to hypoxia. Reduced levels of O2 activate the two heterodimeric atypical sGCs, Gyc-88E/Gyc-89Da and Gyc-88E/Gyc-89Db stimulating the production of cGMP from GTP. The increased levels of cGMP then activate the cGMP-gated ion channel cng.

We also examined possible components of the signaling cascade downstream of the atypical sGCs using RNAi lines. The physiological effects of cGMP are typically mediated by activation of a cGMP-dependent protein kinase (cGK), a cyclic nucleotide-gated ion channel (CNG) or a cGMP-regulated phosphodiesterase (PDE).24 We used a variety of RNAi lines targeted to most of these candidate targets and expressed them selectively in either the Gyc-89Da or Gyc-89Db cells. Neither of the RNAi lines targeted to two of the cGKs affected the hypoxia escape response, although each had been shown to be effective at reducing the levels of these proteins.22 By contrast one of the RNAi lines directed to one of the four predicted CNG channels in Drosophila increased the response times of larvae exposed to hypoxia.22 This channel, cng, has been shown to be selectively activated by cGMP.25 It was notable that the altered response was equivalent regardless of whether the RNAi was expressed in either the Gyc-89Da or Gyc-89Db cells. Similarly, the response could be equally ameliorated by reducing cGMP levels in either Gyc-89Da or Gyc-89Db cells by expressing a cGMP-specific PDE.22 This data again supports our model that the hypoxia escape response is mediated by neurons that express both Gyc-89Da and Gyc-89Db. A summary of our model for the activation of these neurons in shown in Figure 2C.

The results described above show that the atypical sGCs are necessary for the larval hypoxia escape response and we predict that they are necessary for the activation of the sensory neurons that innervate the terminal sensory cones. To demonstrate that activation of these neurons is sufficient to trigger the behavior we used the light-activated channel, channel- rhodopsin 2 (ChR2).26 We expressed this in either the Gyc-89Da or Gyc-89Db cells, placed the larvae in yeast paste and then activated the neurons with flashes of blue light. Control larvae remained buried within the food whereas all the larvae expressing ChR2 in the Gyc-89Da neurons rapidly exited the food (see video Files S1 in the Sup. Material in ref. 22). By contrast, animals expressing ChR2 in the Gyc-89Db neurons remained in the food. This could be due to insufficient expression of ChR2 in these cells or could reflect a different function for this population of cells. The latter explanation is more likely as observation of freely crawling ChR2-expressing larvae showed distinct differences in response to blue light. Wild-type larvae showed no obvious behavioral response, whereas larvae expressing ChR2 in the Gyc-89Da neurons actively avoided the light pulses and larvae expressing ChR2 in the Gyc-89Db neurons temporarily ceased all movement (Morton DB, unpublished observations). These results suggest that there are other behaviors, in addition to the hypoxia escape response, mediated by the cells that express the atypical sGCs. Activation of all the Gyc-89Da neurons is sufficient to trigger the hypoxia escape response, whereas activation of all the Gyc-89Db neurons activates additional behaviors that compete with the hypoxia escape response. We have preliminary evidence that the atypical sGCs are also involved in taste preference behaviors.19 In particular, Gyc-89Db is required for attraction to sucrose, whereas Gyc-89Da is required for caffeine avoidance. As hypoxia is, like caffeine, an aversive stimulus it is likely that both of these stimuli converged to trigger avoidance or escape. By, contrast sucrose is an attractive behavior, thus by activating all the neurons that express Gyc-89Db the animal received conflicting signals to escape and to remain. This might lead to a cessation of locomotion.

As described above and shown in Figure 1, although larvae that are mutant for both Gyc-89Da and Gyc-89Db were significantly impaired in their response to hypoxia, they nevertheless exited the food significantly faster than wild-type animals exit food when subjected to 21% O2. This suggests that there are additional genes coding for O2 sensors. An obvious candidate is the third atypical sGC, Gyc-88E, which we believe functions as the heterodimeric partner of both Gyc-89Da and Gyc-89Db in vivo, although it can function as a homodimer in vitro.17 This implies that Gyc-88E could function as an O2 sensor in the absence of Gyc-89Da and Gyc-89Db. To test the role of Gyc-88E in the hypoxia escape behavior we used two approaches. First we expressed RNAi targeting Gyc-88E in the Gyc-89Da and Gyc-89Db neurons. This was very effective at reducing the response to hypoxia resulting in delays longer than seen with the Gyc-89Da and Gyc-89Db double mutants at O2 concentrations between 0 and 10%22 (Fig. 3). A surprising result was that at 15% O2, larvae with reduced levels of Gyc-88E responded faster than at 10% O2,22 (Fig. 3). By contrast, wild-type larvae responded more slowly at 15% O2 compared to 10% O2, as would be expected for a weaker stimulus22 (Fig. 3). To determine whether this was an artifact of using RNAi, we also used two lines with point mutations in Gyc-88E. These lines had point mutations in the catalytic domain of Gyc-88E and one (V474M) resulted in no detectable enzyme activity and the other (S451F) greatly reduced activity.22 Unfortunately, both of these lines had second site mutations that were recessive lethal so we could not test homozygous mutant animals for their hypoxia escape response. By crossing these flies with flies containing a deficiency that covered Gyc-88E, we generated larvae that likely had non-functional Gyc-88E. These larvae also had a significantly impaired hypoxia escape response, although it was not as strongly inhibited as the RNAi line.22 Interestingly, these mutant lines also showed a faster response to 15% O2 compared to 10% O2 and at 15% O2 there was no difference in response time compared to wild-type larvae22 (Fig. 3). This is in contrast to the Gyc-89Da and Gyc-89Db double mutants, which at 15% O2 exited the food significantly more slowly that wild-type larvae22 (Fig. 3). These results strongly suggest that there are additional O2 sensors that function in particular in mild hypoxic conditions such as 15% O2.

Figure 3.

Figure 3

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Gyc-88E is required for the larval hypoxia escape response and has a larger effect at 10% than 15% O2. Third instar larvae of the genotypes shown were tested for their hypoxia escape response at 10% and 15% O2 as previously described.22 Control larvae responded more slowly to 15% than 10% O2, whereas larvae with reduced or eliminated Gyc-88E responded faster (*p < 0.05; **p < 0.01 two way ANOVA). Data is redrawn from previously published data.22

To examine additional behavioral responses to hypoxia, we monitored the response of larvae crawling on agar in the absence of food. Previous studies had shown that in the absence of food, larval locomotion is characterized by frequent stops and turns.27 When we examined this behavior under altered O2 concentrations we found that both hypoxic and hyperoxic conditions resulted in a significantly reduced number of stops and turns, marking a change from exploratory to escape behavior.22 When we examined this behavioral switch in the Gyc-89Da and Gyc-89Db mutant larvae we found that these genes were required for responding to different O2 concentrations. Larvae mutant for Gyc-89Da responded normally to mild hypoxic shifts (shifts from 21% to 17–20% O2) and all hyperoxic (21% to 22–30% O2) conditions tested, but failed to respond to stronger hypoxic shifts (from 21% to 11–16% O2). The Gyc-89Db mutant larvae showed a complementary response—they responded normally to the stronger hypoxic shifts, but failed to respond to mild hypoxia and hyperoxia.22 This suggested that there are subtle biochemical differences between these two subunits in their affinities for O2 that we were unable to observe in our in vitro experiments.

To confirm that each subunit mediated these specific responses we tested whether either subunit could rescue the responses to each range of O2 shift. Gyc-89Da mutants were defective in the response to a 21% to 15% O2 downshift and this could be rescued by expressing either Gyc-89Da or Gyc-89Db in the Gyc-89Da neurons, but was not rescued by expressing either Gyc-89Da or Gyc-89Db in the Gyc-89Db neurons.22 Similarly, Gyc-89Db mutants were defective in their response to both a 21% to 19% O2 downshift and a 21% to 25% O2 upshift and this could be rescued by expressing either Gyc-89Da or Gyc-89Db in the Gyc-89Db neurons, but not by expressing either Gyc-89Da or Gyc-89Db in the Gyc-89Da neurons.22 This result implies that the neurons that sense these O2 shifts and mediate the change in crawling behavior are not the neurons that co-express both Gyc-89Da and Gyc-89Db but rather neurons that express Gyc-89Da, trigger changes in crawling behavior in response to 21% to 15% O2 downshifts and neurons that express Gyc-89Db, trigger changes in crawling behavior in response to milder downshifts (21% to 19% O2) and upshifts of 21% to 25% O2. A surprising result was that although the different mutations responded to different concentrations of O2, either subunit could rescue either mutation. This contrasts to recent results in C. elegans examining the role of the atypical sGCs in response to either up-shifts or down-shifts in O2 concentration.28 Up-shifts in O2 concentration were detected by specific neurons, the URX neurons, and by the atypical sGCs gcy-35 and gcy-36, whereas down-shifts were detected by the BAG neurons and gcy-31 and gcy-33.28 The ability to distinguish between up-shifts and down-shifts depends on the properties of the atypical sGCs, demonstrated by the finding that BAG neurons could be made to respond to up-shifts by replacing gcy-31 and gcy-33 with gcy-35 and gcy-36.27 This data and phylogenetic analysis of the atypical sGCs suggests that there are two subtypes of atypical sGCs—one class that includes the Drosophila atypical sGCs and the C. elegans gcy-31 and gcy-33 are activated by decreases in O2 concentration and the other class that includes the remaining C. elegans atypical sGCs are activated by increases in O2 concentration.28

Another conclusion that can be made from above experiments, is that these results provide further evidence for the requirement of additional protein(s) involved in O2 sensing. The atypical sGCs alone cannot account for the different phenotypes of the mutant larval responses to different O2 concentrations seen in the crawling assay, but must involve additional proteins/sensors expressed in the Gyc-89Da and Gyc-89Db neurons. The results from the experiments with the Gyc-88E mutants suggesting that additional proteins are required for behavioral responses to 15% O2 provides no predictions as to whether the proteins interact with the atypical sGCs or whether they are completely independent sensors. By contrast, the behavioral effects of upshifts or downshifts of O2 concentrations with the Gyc-89Da and Gyc-89Db mutants suggest that there are additional proteins that interact with these two subunits as each are required for the responses in the 15–25% O2 concentration range, whereas Gyc-88E is not required for the hypoxia escape response at 15% O2. One possibility for an interacting protein that has emerged from studies in C. elegans is globin.29 Natural variants of C. elegans strains were identified that showed different foraging behaviors in response to varying O2 levels. The locus of these variants was mapped to a globin gene, GLB-5, which is expressed in the same neurons as the atypical sGCs.29 Behavioral responses to O2 could be altered by expressing the different variants in these neurons and these changes required the presence of the atypical sGCs, suggesting that GLB-5 is able to modify the response of the sGC and the neuron to O2.29 It will be especially interesting to determine whether orthologous genes in Drosophila have similar functions.

In summary, our biochemical, genetic and behavioral experiments provide strong evidence that the atypical sGCs function as molecular O2 sensors in Drosophila larvae mediating a variety of behavioral responses to altered O2 levels. Furthermore, our experiments suggest that there are additional unidentified proteins that can also act as O2 sensors, complementing the atypical sGCs. We have identified the sensory neurons that innervate the terminal sensory cones as likely chemoreceptors mediating some of these responses. Nevertheless, there are many more cells in both the central and peripheral nervous systems that express the atypical sGCs where we do not know the effects of changes in O2 concentration. For example, we do not know the environmental conditions that lead to changes in the enzyme activity of the sGCs in neurons in the CNS. Are changes in external O2 concentrations reflected in changes in the enzyme activity of the sGCs in neurons within the CNS? If so, what effect does this have on the functions of these cells? Clearly there is much to learn about the functions of this unusual class of enzyme and how animals detect changes in environmental O2 concentration.

Acknowledgements

I'm grateful to many past and present members of my laboratory for many thoughtful discussions about this work over the years. Research from the authors laboratory was supported by NS 29740 from the National Institute for Neurological Disorders and Stroke.

Abbreviations

cGK

cGMP-dependent protein kinase

cGMP

cyclic guanosine monophosphate

HO-2

hemoxygenase-2

ChR2

channel-rhodopsin 2

CNG

cyclic nucleotide-gated ion channel

HIF1

hypoxia-inducible transcription factor

PDE

phosphodiesterase

sGCs

soluble guanylyl cyclases

TTxN

tetanus toxin

Extra View to: Vermehren A, Ainsley JA, Johnson WA, Davies SA, Morton DB. Behavioral responses to hypoxia in Drosophila larvae are mediated by atypical soluble guanylyl cyclases. Genetics. 2010;186:183–196. doi: 10.1534/genetics.110.118166.

References

  • 1.López-Barneo J, Pardal R, Ortega-Sáenz P. Cellular mechanisms of oxygen sensing. Annu Rev Physiol. 2001;63:259–287. doi: 10.1146/annurev.physiol.63.1.259. [DOI] [PubMed] [Google Scholar]
  • 2.Kaelin WG, Ratcliffe PJ. Oxygen sensing by metazoans: The central role of the HIF hydroxylase pathway. Mol Cell. 2008;30:393–402. doi: 10.1016/j.molcel.2008.04.009. [DOI] [PubMed] [Google Scholar]
  • 3.Ramirez JM, Zuperku EJ, Alheid GF, Lieske SP, Ptak K, McCrimmon DR. Respiratory rhythm generation: converging concepts from in vitro and in vivo approaches? Respir Physiol Neurobiol. 2002;131:43–56. doi: 10.1016/s1569-9048(02)00036-8. [DOI] [PubMed] [Google Scholar]
  • 4.Pingitore A, Gemignani A, Menicucci D, Di Bella G, De Marchi D, Passera M, et al. Cardiovascular response to acute hypoxemia induced by prolonged breath holding in air. Am J Physiol Heart Circ Physiol. 2007;294:449–455. doi: 10.1152/ajpheart.00607.2007. [DOI] [PubMed] [Google Scholar]
  • 5.Williams SEJ, Wootton P, Mason HS, Bould J, Iles DE, Riccardi D, et al. Hemoxygenase-2 is an oxygen sensor for a calcium-sensitive potassium channel. Science. 2004;306:2093–2097. doi: 10.1126/science.1105010. [DOI] [PubMed] [Google Scholar]
  • 6.Gray JM, Karow DS, Lu H, Chang AJ, Chang JS, Ellis RE, et al. Oxygen sensation and social feeding mediated by a C. elegans guanylate cyclase homologue. Nature. 2004;430:317–322. doi: 10.1038/nature02714. [DOI] [PubMed] [Google Scholar]
  • 7.Garthwaite J. New insight into the functioning of nitric oxide-receptive guanylyl cyclase: physiological and pharmacological implications. Mol Cell Biochem. 2010;334:221–232. doi: 10.1007/s11010-009-0318-8. [DOI] [PubMed] [Google Scholar]
  • 8.Boon EM, Huang SH, Marletta MA. A molecular basis for NO selectivity in soluble guanylate cyclase. Nature Chem Biol. 2005;1:53–59. doi: 10.1038/nchembio704. [DOI] [PubMed] [Google Scholar]
  • 9.Nighorn A, Byrnes KA, Morton DB. Identification and characterization of a novel beta subunit of soluble guanylyl cyclase that is active in the absence of additional subunits and relatively insensitive to nitric oxide. J Biol Chem. 1999;274:2525–2531. doi: 10.1074/jbc.274.4.2525. [DOI] [PubMed] [Google Scholar]
  • 10.Morton DB, Anderson E. MsGC-β3 forms active homodimers and inactive heterodimers with NO-sensitive soluble guanylyl cyclase subunits. J exp Biol. 2003;206:937–947. doi: 10.1242/jeb.00160. [DOI] [PubMed] [Google Scholar]
  • 11.Morton DB, Hudson ML, Waters E, O'Shea M. Soluble guanylyl cyclases in C. elegans—NO is not the answer. Current Biology. 1999;9:546–547. doi: 10.1016/s0960-9822(99)80349-2. [DOI] [PubMed] [Google Scholar]
  • 12.Morton DB. Invertebrates yield a plethora of atypical guanylyl cyclases. Mol Neurobiol. 2004;29:97–115. doi: 10.1385/MN:29:2:097. [DOI] [PubMed] [Google Scholar]
  • 13.Shah S, Hyde DR. Two Drosophila genes that encode the a and β subunits of the brain soluble guanylyl cyclase. J Biol Chem. 1995;270:15368–15376. doi: 10.1074/jbc.270.25.15368. [DOI] [PubMed] [Google Scholar]
  • 14.Langlais KK, Stewart JA, Morton DB. Preliminary characterization of two atypical soluble guanylyl cyclases in the central and peripheral nervous system of Drosophila melanogaster. J exp Biol. 2004;207:2323–2338. doi: 10.1242/jeb.01025. [DOI] [PubMed] [Google Scholar]
  • 15.Morton DB, Langlais KK, Stewart JA, Vermehren A. Comparison of the properties and distribution of the five soluble guanylyl cyclase subunits in Drosophila melanogaster. J Insect Sci. 2005;5:12. doi: 10.1093/jis/5.1.12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Chang AJ, Chronis N, Karow DS, Marletta MA, Bargmann CI. A distributed chemosensory circuit for oxygen preference in C. elegans. PLoS Biology. 2006;4:1588–1602. doi: 10.1371/journal.pbio.0040274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Morton DB. Atypical soluble guanylyl cyclases in Drosophila can function as molecular oxygen sensors. J Biol Chem. 2004;279:50651–50653. doi: 10.1074/jbc.C400461200. [DOI] [PubMed] [Google Scholar]
  • 18.Morton DB, Stewart JA, Langlais KK, Clemens-Grisham R, Vermehren A. Synaptic transmission in neurons that express the Drosophila atypical soluble guanylyl cyclases, Gyc-89Da and Gyc-89Db, is necessary for the successful completion of larval and adult ecdysis. J exp Biol. 2008;211:1645–1656. doi: 10.1242/jeb.014472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Vermehren A, Langlais KK, Morton DB. Oxygen-sensitive guanylyl cyclases in insects and their potential roles in oxygen detection and in feeding behaviors. J Insect Physiol. 2006;52:340–348. doi: 10.1016/j.jinsphys.2005.12.001. [DOI] [PubMed] [Google Scholar]
  • 20.Morton DB, Vermehren A. Soluble guanylyl cyclases in invertebrates: targets for NO and O2. In: Trimmer BA, Tota B, editors. Advances in Experimental Biology on Nitric Oxide. London: Elsevier Press; 2007. pp. 65–82. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Wingrove JA, O'Farrell PH. Nitric oxide contributes to behavioral, cellular and developmental responses to low oxygen in Drosophila. Cell. 1999;98:105–114. doi: 10.1016/S0092-8674(00)80610-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Vermehren A, Ainsley JA, Johnson WA, Davies SA, Morton DB. Behavioral responses to hypoxia in Drosophila larvae are mediated by atypical soluble guanylyl cyclases. Genetics. 2010;186:183–196. doi: 10.1534/genetics.110.118166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Luann H, Peabody NC, Vinson CR, White BH. Refined spatial manipulation of neuronal function by combinatorial restriction of transgene expression. Neuron. 2006;52:425–436. doi: 10.1016/j.neuron.2006.08.028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Lucas KA, Pitari GM, Kazerounian S, Ruiz-Stewart I, Park J, Schulz S, et al. Guanylyl cyclases and signaling by cyclic GMP. Pharmacol Rev. 2000;52:375–413. [PubMed] [Google Scholar]
  • 25.Baumann A, Frings S, Godde M, Seifert R, Kaupp UB. Primary structure and functional expression of a Drosophila cyclic nucleotide-gated channel present in eyes and antennae. EMBO J. 1994;13:5040–5050. doi: 10.1002/j.1460-2075.1994.tb06833.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Nagel G, Szellas T, Huhn W, Kateriya S, Adeishvili N, Berthold P, et al. Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc Natl Acad Sci. 2003;100:13940–13945. doi: 10.1073/pnas.1936192100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Ainsley JA, Pettus JM, Bosenko D, Gerstein CE, Zinkevich N, Anderson MG, et al. Enhanced locomotion caused by loss of the Drosophila DEG/ENaC protein Pickpocket1. Curr Biol. 2003;13:1557–1563. doi: 10.1016/s0960-9822(03)00596-7. [DOI] [PubMed] [Google Scholar]
  • 28.Zimmer M, Gray JM, Pokala N, Chang AJ, Karow DS, Marletta MA, et al. Neurons detect increases and decreases in oxygen levels using distinct guanylate cyclases. Neuron. 2009;61:865–879. doi: 10.1016/j.neuron.2009.02.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Persson A, Gross E, Laurent P, Busch KE, Bretes H, de Bono M. Natural variation in a neural globin tunes oxygen sensing in wild Caenorhabditis elegans. Nature. 2009;458:1030–1033. doi: 10.1038/nature07820. [DOI] [PubMed] [Google Scholar]

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