InteractiveFly: GeneBrief

Ionotropic receptor 21a: Biological Overview | References

Gene name - Ionotropic receptor 21a

Synonyms -

Cytological map position - 21A5-21B1

Function - Ionotropic receptor

Keywords - ionotropic receptor - mediator of heat seeking in the malaria vector Anopheles gambiae - Ir21a mediates heat avoidance in Drosophila - antenna

Symbol - Ir21a

FlyBase ID: FBgn0031209

Genetic map position - chr2L:21,823-25,155

NCBI classification - Ligand-gated ion channel, Type 2 periplasmic binding fold superfamily

Cellular location - surface transmembrane

NCBI link: EntrezGene, Nucleotide, Protein
Ir21a orthologs: Biolitmine

Mosquitoes transmit pathogens that kill >700,000 people annually. These insects use body heat to locate and feed on warm-blooded hosts, but the molecular basis of such behavior is unknown. This study identified Ionotropic receptor IR21a, a receptor conserved throughout insects, as a key mediator of heat seeking in the malaria vector Anopheles gambiae. Although Ir21a mediates heat avoidance in Drosophila, it drives heat seeking and heat-stimulated blood feeding in Anopheles. At a cellular level, Ir21a is essential for the detection of cooling, suggesting that during evolution mosquito heat seeking relied on cooling-mediated repulsion. These data indicate that the evolution of blood feeding in Anopheles involves repurposing an ancestral thermoreceptor from non-blood-feeding Diptera (Greppi, 2020).

Insect-borne diseases kill over 700,000 people annually, with >400,000 deaths resulting from malaria, a disease caused by protozoan Plasmodium spp. parasites that are transmitted by blood-feeding anopheline mosquitoes. Host seeking by mosquitoes and other pathogen-spreading insects relies on the detection of host-associated cues, including carbon dioxide (CO2), odors, and body heat. Receptors for CO2 and host odors have been characterized in mosquitoes, but receptors that promote heat seeking and heat-induced blood feeding have remained elusive. As vector mosquitoes are descendants of non-blood-feeding ancestors, it remains unknown whether the emergence of heat seeking and warming-induced blood feeding in mosquitoes involved the generation of novel thermoreceptors or the repurposing of existing thermoreceptors (Greppi, 2020).

To date, mosquito orthologs of two Drosophila warmth receptors, TRPA1 and GR28b, have been tested as candidate heat-seeking receptors in the yellow fever mosquito Aedes aegypti. However, neither is required for heat seeking in Aedes. Rather, TRPA1 promotes heat avoidance in both Aedes and Drosophila. Although efforts have focused on warmth receptors, insects also possess cooling-activated receptors, which should be equally capable of supporting heat seeking through cooling-mediated repulsion. In Drosophila, cooling detection is mediated by IR21a, IR25a, and IR93a (Ni, 2016; Knecht; 2017; Budelli, 2019), three members of the ionotropic receptor (IR) family, a group of invertebrate-specific sensory receptors related to ionotropic glutamate receptors. IR21a is specifically required for cooling detection in the fly and can confer cooling sensitivity when ectopically expressed, while IR25a and IR93a are more broadly acting co-receptors that support cooling detection and other IR-dependent sensory modalities. At the behavioral level in Drosophila, IR21a, IR25a, and IR93a help the fly achieve optimal body temperatures by supporting avoidance of excessively cool and warm temperatures. Beyond Drosophila, IR21a, Ir25a, and IR93a are each widely conserved from Diptera (flies and mosquitoes) to Isoptera (termites), raising the possibility that their thermosensory functions may also be conserved. Using Anopheles gambiae, a major vector of malaria in sub-Saharan Africa, tests were performed to see whether IR21a is required for detecting cooling in mosquitoes and subsequently whether it can drive heat attraction and heat-stimulated blood feeding (Greppi, 2020).

Two mutant alleles of A. gambiae Ir21a were generated using CRISPR-Cas9. Ir21a+7bp contains a 7-base pair (bp) insertion, introducing a frameshift positioned to disrupt IR21a's translation within the second of IR21a's three transmembrane domains; this lesion is predicted to generate a nonfunctional receptor. In Ir21aEYFP, a disruption cassette containing an enhanced yellow fluorescent protein (EYFP) marker, was inserted into IR21a's fourth exon, a lesion also predicted to create a nonfunctional receptor. Both mutants lacked detectable IR21a protein expression, consistent with their acting as Ir21a null mutations (Greppi, 2020).

Genome-wide analyses of A. gambiae sensory tissues suggest that Ir21a RNA is specifically expressed in the antenna. To visualize IR21a protein expression and localization with cellular resolution, anti-IR21a antisera were generated. The antenna's most distal segment (flagellomere 13) contains three coeloconic sensilla that house sensitive thermoreceptors. In females, IR21a expression was detected in three sensory neurons in flagellomere 13, one innervating each of the coeloconic sensilla. Consistent with a role in thermosensory transduction, IR21a strongly localized to the sensory ending of each of these neurons. IR21a immunostaining was absent in Ir21a mutants, confirming antisera specificity. The male antennal tip also contains thermoreceptors, and IR21a expression was detected in sensory endings there as well (Greppi, 2020).

Extracellular recordings were performed from the IR21a-positive coeloconic sensilla at the antennal tip. In wild-type mosquitoes, the activity of the Cooling Cell, a thermosensory neuron stimulated by cooling and inhibited by warming, was readily detected. On rare occasions of exceptional signal to noise, a smaller-amplitude spike was also detected, corresponding to a Heating Cell activated by warming and inhibited by cooling. Cooling Cell responses were highly thermosensitive: an ~0.5°C drop from ~30°C increased spiking by ~40%, and an ~0.5°C drop from ~37°C increased spiking by ~80%. Response adaptation initiated rapidly, followed by a slower decline to baseline. Heating inhibited spiking, in a similarly transient manner. Importantly, Cooling Cells remained highly active at warm temperatures (e.g., 37°C) and were neither more active nor more thermosensitive at colder temperatures. Thus, while often referred to as Cold Cells in the classical literature, cooling and not cold is their activating stimulus. In addition, while often referred to as 'phasic-tonic' receptors, their responses to temperature shifts adapted fully, albeit slowly, requiring sustained observation (>20 s) to fully appreciate. Therefore, although they fire robustly at constant temperature, Cooling Cells are phasic thermoreceptors. Their rate of baseline firing was relatively temperature insensitive [with a fold change upon 10°C increase (Q10) of ~1.6, reflecting a slight increase with warmth], enabling the cell to respond to small temperature fluctuations over a wide range of absolute temperatures. Taken together, these data indicate that Cooling Cells are phasic thermoreceptors that respond to temperature change rather than absolute temperature and that they are capable of responding to abrupt changes in temperature over the wide range of absolute temperatures relevant for host seeking (Greppi, 2020).

Cooling Cell thermosensitivity was eliminated in A. gambiae Ir21a mutants. The large-amplitude spike detected in Ir21a mutants was neither activated by cooling nor inhibited by warming. Rather, its activity increased slightly upon warming, with a Q10 under 2, which is average for a biological process. Thus, Ir21a is essential for thermosensing by Cooling Cells in the mosquito, demonstrating that A. gambiae IR21a's molecular function is conserved with its Drosophila ortholog (Greppi, 2020).

In female mosquitoes, heat seeking is part of a multimodal host-seeking program activated upon exposure to CO2, with body heat serving as an important cue close to the host (within ~10 to 15 cm). To assess heat seeking, female mosquitoes were provided a 20-s puff of 4% CO2 and exposed to two targets, a control target at ambient temperature (~26°C) and a heated target at ~37°C. Wild-type mosquitoes exhibited robust heat seeking, with 43 ± 3% of CO2-activated mosquitoes landing on the 37°C target (average ± SEM). The loss of Ir21a greatly reduced this behavior, with only 15 ± 4% of Ir21aEYFP mutants and 14 ± 4% of Ir21a+7bp mutants landing on the 37°C target. In all cases, the control target was largely ignored, confirming temperature's importance in the assay. While heat seeking was greatly reduced in Ir21a mutants, it was not entirely eliminated. This residual activity likely reflects signaling from other as-yet-uncharacterized thermosensors. However, the strong reduction of heat seeking in Ir21a mutants identifies this receptor as a major driver of mosquito attraction to warmth (Greppi, 2020).

To test the specificity of the Ir21a mutant behavioral deficit for heat seeking, their ability to perform an activation-dependent behavior less reliant on thermosensation was tested. While body heat is a powerful short-range cue, a multimodal combination of longer-range chemosensory and visual cues mediates initial approach, suggesting that such behavior should be largely unaffected by a specific thermosensory deficit. To assess approach behavior, mosquitoes were activated by five human breaths and presented a human hand, positioned on a platform to prevent physical contact with the mosquitoes but otherwise providing host-associated cues. Hand approach was strong in the wild type, with 57 ± 2% of mosquitoes landing on the surface beneath the hand. Hand-associated cues were critical, as hand withdrawal prompted rapid dispersal. Ir21aEYFP mutants remained robustly responsive, exhibiting maximum levels of approach (55 ± 2%) similar to wild-type levels. Careful examination of response kinetics revealed that, compared to wild type, their initial accumulation rate decreased by ~25%, and their dispersal rate upon hand removal increased by ~40%, potentially reflecting subtle contributions of the warmth gradient created by the presence of the hand to the avidity of host approach. Similar results were obtained for Ir21a+7bp. Overall, these data demonstrate that the loss of Ir21a does not broadly disrupt orientation toward sensory cues and argue against the presence of global behavioral deficits in the mutants. These results are consistent with prior work indicating that the disruption of single sensory modalities is insufficient to completely eliminate host approach (Greppi, 2020).

Heat strongly stimulates mosquito blood feeding. To assess the effect of warmth on blood feeding, artificial membrane feeders were used to present human blood meals at different temperatures. One meal was held at room temperature (RT, ~23°C) and the other warmed to ~31°C, a temperature similar to the surface temperatures (~29°C to 33°C) of human torsos and extremities in a 23°C to 24°C room. In each trial, green food coloring was added to one meal so that the consumption of warm versus RT food could be distinguished. Each class of trial was assessed independently, and each yielded similar results. In wild type, elevated temperature robustly promoted feeding, as reflected in the greater percentages of mosquitoes consuming warm versus RT meals. For both Ir21aEYFP and Ir21+7bp mosquitoes, this preference for warm blood was significantly reduced. Thus, similar to heat seeking, warmth-promoted blood feeding was reduced in the absence of Ir21a (Greppi, 2020).

These data identify IR21a as a key mediator of heat-seeking behavior in A. gambiae mosquitoes. Although a cooling-activated receptor driving heat seeking is superficially counterintuitive, repulsion from cooling would yield a similar behavioral outcome as attraction to warming. Furthermore, Cooling Cells are bidirectional and are not only activated by cooling but also inhibited by heating; each phase of the response could modulate downstream circuits to control behavior. Ultimately, the detection of temperature change by the Cooling Cells is critical, but is just one step in heat seeking, a response that involves the processing of multiple sensory inputs to generate a coherent response. Identification of a key molecular receptor for heat seeking provides a starting point for a deeper understanding of this complex behavior and its contribution to the multimodal process that culminates in mosquito blood feeding (Greppi, 2020).

The conservation of IR21a's thermosensory function between Drosophila and Anopheles, whose last common ancestor lived ~250 million years ago, suggests thermosensing is an ancestral function of IR21a. As this ancestor predates the evolution of blood feeding, its IR21a would have regulated other behaviors, such as thermoregulation. Thus, the current findings indicate that the evolution of blood feeding in A. gambiae mosquitoes involved repurposing an ancestral thermoreceptor to facilitate host seeking. Alterations in the connectivity or function of downstream circuits would likely have been crucial in this behavioral shift. Given the conservation of IR21a as well as IR25a and IR93a (IR21a's coreceptors in Drosophila) across insects, these IRs may be used in heat seeking not only by other mosquitoes but also across a range of hematophagous insect taxa (Greppi, 2020).

In addition to Ir21a's role in heat seeking, IR21a expression in the antennae of A. gambiae males suggests it continues to serve additional thermosensory functions. It will be interesting to assess whether IR21a mediates thermal preference in male and possibly female mosquitoes and the extent to which thermal preference and heat-seeking circuits overlap. Not all thermoreceptors appear to have been repurposed, as the TRPA1 warmth receptor has a similar role in flies and mosquitoes, mediating heat avoidance in both. At a practical level, exploiting and manipulating the sensory systems of vector insects offer an avenue for disease control strategies (Greppi, 2020).

Synchronous and opponent thermosensors use flexible cross-inhibition to orchestrate thermal homeostasis

Body temperature homeostasis is essential and reliant upon the integration of outputs from multiple classes of cooling- and warming-responsive cells. The computations that integrate these outputs are not understood. This study discovered a set of warming cells (WCs) and show that the outputs of these WCs combine with previously described cooling cells (CCs) in a cross-inhibition computation to drive thermal homeostasis in larval Drosophila. WCs and CCs detect temperature changes using overlapping combinations of ionotropic receptors: Ir68a, Ir93a, and Ir25a for WCs and Ir21a, Ir93a, and Ir25a for CCs. WCs mediate avoidance to warming while cross-inhibiting avoidance to cooling, and CCs mediate avoidance to cooling while cross-inhibiting avoidance to warming. Ambient temperature-dependent regulation of the strength of WC- and CC-mediated cross-inhibition keeps larvae near their homeostatic set point. Using neurophysiology, quantitative behavioral analysis, and connectomics, this study demonstrated how flexible integration between warming and cooling pathways can orchestrate homeostatic thermoregulation (Hernandez-Nunez, 2021).

This study investigate the sensory cells and computations that control larval Drosophila body temperature. Previous work uncovered three CCs in the dorsal organ ganglion (DOG). The CCs are sensitive to temperature changes at ambient temperatures from 14° to 34°C (the innocuous temperature range). The CCs are required for cooling avoidance from as low as 14°C toward 24°C (the homeostatic set point) but are not required for innocuous warming avoidance above 24°C. Larval Drosophila do not express the adult innocuous warming receptor Gr28b(d) and use the Transient receptor potential cation channel, subfamily A, member 1 (TrpA1) channel to mediate rolling escape responses to noxious heat. The molecular and cellular sensors that larval Drosophila need for innocuous warming avoidance were not known (Hernandez-Nunez, 2021).

Understanding homeostatic thermoregulation in larval Drosophila requires identifying the warming-responsive counterparts of the CCs and understanding how the outputs of CCs and WCs are combined to make behavioral decisions above, near, and below the homeostatic set point. This study uncovered a new set of WCs and warming molecular receptors with close morphological and genetic similarity to the CCs and their molecular receptors. Using optogenetics, calcium imaging, precise temperature control, sensory receptor mutants, and quantitative behavioral analysis, a sensorimotor transformation model was developed that achieves homeostatic thermoregulation. This model implements ambient temperature context-dependent cross-inhibition between the simultaneous outputs of WCs and CCs. Flexible cross-inhibition allows the net effect of WC and CC outputs to drive cooling avoidance below 24°C, suppress avoidance to temperature changes near 24°C, and drive warming avoidance above 24°C. Electron microscopy was used to reconstruct the wiring diagram of the WC and CC synaptic partners and connectome-based models to identify candidate circuits for implementing the cross-inhibitory sensorimotor transformation. This study reveals how simultaneously active opponent sensors are integrated in a context-dependent manner to achieve homeostatic regulation (Hernandez-Nunez, 2021).

In thermosensation, often one type of sensor (cooling or warming) is studied without its counterpart, making understanding of the computations' underlying thermal homeostasis incomplete. The justification for studying thermosensory cell function without their counterparts is based on the 'labeled line' hypothesis, in which cells activated by cooling exclusively regulate mechanisms that counteract cooling and cells activated by warming exclusively regulate mechanisms that counteract warming. In this view, the outputs of WCs and CCs do not necessarily integrate to shape thermoregulatory responses. This study discovered novel larval Drosophila WCs and their warming molecular receptors (Ir68a, Ir93a, and Ir25a). It was found that these WCs, together with their CC counterparts, underlie the behavioral mechanism for thermal homeostasis near the larva's set point. This study elucidated the computation that uses WC and CC outputs to determine the larva's turning responses that guide behavior toward the set point. The results rule out the labeled line hypothesis, where WCs mediate warming responses and CCs mediate cooling responses. This study found that warming and cooling are active in overlapping temperature ranges and each type of sensor contributes to both warming and cooling behavioral responses (Hernandez-Nunez, 2021).

Flexible cross-inhibition of opponent pathways explain how the larva targets its behavioral set point and achieves homeostatic thermoregulation. This study uncovered anatomical and functional evidence of an early step in the computation for homeostatic thermoregulation, calculating the time derivative of WC neural activity. Connectomic evidence was uncovered for direct integration of warming and cooling pathways in the larval brain. Both classes of thermosensors play important roles whether temperature is increasing or declining in different temperature ranges, above, near, or below the set point. These roles are quantitatively described in a C-IM, providing a framework for circuit-level dissection of the behavioral mechanisms for homeostatic thermoregulation (Hernandez-Nunez, 2021).

The new set of WCs, presented in this study, share anatomical and genetic similarities with the CCs. Each sensor requires a distinct but partly overlapping set of ionotropic receptors to detect temperature changes. The WCs require Ir68a, Ir93a, and Ir25a to detect warming. The CCs require Ir21a, Ir93a, and Ir25a to detect cooling. The opposed thermosensitive polarity of the WCs and CCs is encoded in their heteromeric expression of sets of Ir receptors. These ionotropic receptors are conserved across insects and have homologs in the disease vector mosquitoes Anopheles gambiae and Aedes aegypti, which use temperature cues to identify human hosts. Heat seeking by malaria vector mosquitoes relies on Ir21a-dependent receptors. Analogous to the role of Ir21a in the Drosophila larva, these cooling-activated mosquito receptors control behavioral responses at the elevated temperatures associated with warm-blooded prey. The combinatorial use of ionotropic receptors may be a widely used mechanism to shape the sign and sensitivity of thermosensory responses in different insects. Future structural biology studies that examine how these receptors assemble to mediate thermosensation may elucidate the transduction roles of the specific molecular receptors Ir21a and Ir68a and the co-receptors Ir93a and Ir25a (Hernandez-Nunez, 2021).

Many sensory modalities use opponent sensors to encode environmental stimuli including photosensation, hygrosensation, and thermosensation. The larval WCs and CCs respond to temperature changes with opposite polarity but with symmetric temporal dynamics. Calcium imaging revealed that WCs and CCs share the same peak times and adaptation times to a variety of sensory stimuli. When freely moving animals were exposed to optogenetic white noise stimulation of WCs or CCs and reverse-correlation analysis was conducted, identical transformations were uncovered that convert WC and CC activity into synchronous changes in behavior. This synchrony facilitates the integration of the output of WCs and CCs by downstream circuits. In particular, synchrony makes it possible to use linear combinations of WC and CC outputs to determine behavioral responses. The use of sensors with opposed polarity has been proposed to increase the optimality of information encoding. However, the dynamic properties of sensors with opposed polarity has not been analyzed from the perspective of efficient signal processing. This study underscores a potential advantage in signal processing of having synchronous sensors with opposed polarity. Synchrony may simplify downstream processing (Hernandez-Nunez, 2021).

Larval Drosophila uses a flexible cross-inhibitory computation to achieve thermal homeostasis. Above 24.5°C warming is unfavorable because it carries the animal further from the homeostatic set point. In this temperature range, avoidance responses during warming are strongly stimulated by WCs and moderately cross-inhibited by CCs. Below 24.5°C, cooling is unfavorable. In this temperature range, avoidance responses during cooling are strongly stimulated by CCs and moderately cross-inhibited by WCs. Near the homeostatic set point, balanced cross-inhibition from WCs and CCs suppresses avoidance responses (Hernandez-Nunez, 2021).

Cross-inhibition is prevalent in perceptual choice models. In these models, cross-inhibition between competing groups of neurons often enhances accuracy in decision-making. For example, in the Usher-McClelland model of primate decision-making, different neuron groups are used to represent different choices. These neurons mutually cross-inhibit their output pathways. The most strongly activated group that represents a specific choice thus biases the decisions toward one outcome by suppressing all others (Hernandez-Nunez, 2021).

In larval Drosophila thermoregulation, the choice is whether to avoid cooling or warming: At high temperatures, warming should be avoided; at low temperatures, cooling should be avoided. At all temperatures, however, the CCs are always more active during cooling and the WCs are always more active during warming. Any cross-inhibition in the outputs of the WCs and CCs has to be flexible for these neurons to contribute differently to behavior in different contexts. Unlike the Usher-McClelland model, the current model stipulates that flexibility is encoded in the ambient temperature-dependent weights of the WC and CC contributions to behavior and not in the WC and CC neural responses. This type of flexibility requires additional information from a sensor of absolute ambient temperature, which remains to be identified in larval Drosophila (Hernandez-Nunez, 2021).

The cross-inhibition model helps understanding of how WC and CC neural dynamics are transformed into behavioral dynamics at ambient temperatures in the innocuous range (between 14° and 34°C). However, outside the innocuous temperature range, in the noxious range, the WCs and CCs also contribute to behavioral responses. This study found that at ambient temperatures below 14°C, WCs contribute to cooling avoidance, and above 34°C, CCs contribute to warming avoidance. The change in behavioral role of the CCs might be related to the reduction in responsiveness of the B-CCs at high temperatures. Examining the behavioral roles of the C-A-PNs and C-B-PNs, which preferentially receive inputs from A- and B-type CCs, respectively, may help explain this behavioral switch (Hernandez-Nunez, 2021).

The flexible integration of warming and cooling pathways is captured in the cross-inhibition model by the ambient temperature-dependent weights wWC and wCC. A neural implementation of this flexibility requires the input from an absolute ambient temperature sensor. Future studies that explore the molecular identity of this sensor may gain additional insight into the mechanisms underlying flexible warming and cooling integration (Hernandez-Nunez, 2021).

The reconstruction of the wiring diagram of the WC and CC synaptic partners revealed that the main synaptic partners of the WCs are the bLNs. This connectivity pattern suggests potential thermosensory normalization of olfactory inputs, as the bLNs are the population of GABAergic interneurons that provide panglomerular inhibition to the olfactory system. Similarly, in the mushroom body connectome, the Kenyon cells that receive inputs exclusively from thermosensory pathways synapse strongly onto the GABAergic anterior paired lateral neurons, suggesting thermosensory normalization of chemosensory pathways. This anatomical evidence, coupled with the molecular access to thermosensory and olfactory receptors, makes the Drosophila larva an ideal model organism for the study of multisensory integration between olfactory and thermosensory pathways (Hernandez-Nunez, 2021).

In mammals, prevailing models of thermoregulation propose that signals from CCs and WCs are integrated in the preoptic area of the hypothalamus. GABAergic and glutamatergic neurons are proposed to play a role in the modulation of hypothalamic WCs. However, the computations driving thermal homeostasis in mammals remain obscure and are challenging to dissect because overlapping autonomic and behavioral mechanisms contribute to thermoregulation, making the output of the computation multidimensional (Hernandez-Nunez, 2021).

Because poikilotherms strictly use behavior for thermoregulation, measurements of behavior constitute the full output of the thermoregulatory computation. Similar to mammals, Drosophila integrates the outputs of bidirectional sensors of cooling and warming to regulate body temperature. The computation underlying thermal homeostasis in the larva may represent a general means of maintaining a set point using opponent sensors (Hernandez-Nunez, 2021).

Many poikilotherms, including cave beetles and python vipers, have neural and behavioral responses to temperature changes that are as robust as the ones displayed by larval Drosophila. The significance of understanding the computations underlying thermal homeostasis in D. melanogaster is that, unlike other poikilotherms, Drosophila's genetic accessibility allows one to manipulate with precision the individual receptors and neurons underlying thermal homeostasis. In addition, the recent advances in larval Drosophila connectomics and the numerical simplicity of its thermosensory system enabled tracing the second-order warming and cooling circuits. A circuit was found that integrates inputs from bilateral warming and cooling PNs and a circuit that extracts the derivative of the WCs, both of which constitute potential implementations of components of the cross-inhibition sensorimotor transformation model. However, several other undiscovered downstream circuits may implement the full sensorimotor transformation. The combination of connectome tracing and genetic identification of downstream thermosensory neurons will enable access to organism-level neural circuits underlying thermal homeostasis (Hernandez-Nunez, 2021).

Homeostatic control is pervasive in biology, including homeostatic control of synaptic plasticity in connections between neurons, homeostatic control of cardiac output, and homeostatic control of glucose. All homeostatic processes regulate a physiological variable near an optimal set point. This study has identified the control system for homeostatic thermal regulation in larval Drosophila. The computation was determined that integrates the outputs of WCs and CCs and how this computation leads to control over the homeostatic variable was established. This analysis establishes a framework for gaining quantitative insight into a homeostatic control system (Hernandez-Nunez, 2021).

Ionotropic receptors specify the morphogenesis of phasic sensors controlling rapid thermal preference in Drosophila

Thermosensation is critical for avoiding thermal extremes and regulating body temperature. While thermosensors activated by noxious temperatures respond to hot or cold, many innocuous thermosensors exhibit robust baseline activity and lack discrete temperature thresholds, suggesting they are not simply warm and cool detectors. This study investigated how the aristal Cold Cells encode innocuous temperatures in Drosophila. They are not cold sensors but cooling-activated and warming-inhibited phasic thermosensors that operate similarly at warm and cool temperatures; it is proposed renaming them 'Cooling Cells.' Unexpectedly, Cooling Cell thermosensing does not require the previously reported Brivido Transient Receptor Potential (TRP) channels. Instead, three Ionotropic Receptors (IRs), IR21a, IR25a, and IR93a, specify both the unique structure of Cooling Cell cilia endings and their thermosensitivity. Behaviorally, Cooling Cells promote both warm and cool avoidance. These findings reveal a morphogenetic role for IRs and demonstrate the central role of phasic thermosensing in innocuous thermosensation (Budelli, 2019).

Animals rely on thermosensation to maintain appropriate body temperatures; avoid thermal extremes; and, in vipers, bats, and blood-feeding insects, locate warm-blooded prey. From insects to vertebrates, thermosensing depends on multiple classes of thermosensors with distinct thermal sensitivities and behavioral roles. Thermosensors activated by noxious heat or cold commonly exhibit temperature thresholds beyond which they drive aversive responses. On the other hand, thermosensors responsive to innocuous temperatures commonly lack temperature thresholds. They instead exhibit robust baseline spiking and are more responsive to changes in temperature than its absolute value. For example, in mammalian skin, innocuous cooling sensors primarily exhibit transient increases in firing upon cooling and decreases upon warming, while innocuous warming sensors exhibit the converse behavior. While it is clear that innocuous thermosensors have key roles in thermoregulation, how they encode temperature information and control thermoregulatory responses remains a major area of inquiry (Budelli, 2019).

The relative anatomical simplicity of the Drosophila thermosensory system has made it a powerful model for studying thermosensation. At the molecular level, multiple receptors have been implicated in innocuous thermosensing and behavioral thermoregulation in Drosophila. The Transient Receptor Potential (TRP) channel TRPA1 and the Gustatory Receptor (GR) GR28b mediate warmth-sensing in distinct sets of thermosensory neurons, while adult cool-sensing has been reported to involve Brivido family TRP channels. In addition, a trio of Ionotropic Receptors (IRs), IR21a, IR25a, and IR93a, was recently shown to mediate the detection of cooling in the larva (Knecht, 2016, Ni, 2016). The IRs are a large family of invertebrate-specific sensory receptors related to variant ionotropic glutamate receptors. Many IRs have roles in chemosensing, but a subset is involved in hygrosensing and thermosensing. Among the IRs involved in thermosensing, IR25a and IR93a serve as co-receptors and act in multiple classes of sensory neurons, while IR21a appears specific for cooling detection. How the information provided by these multiple classes of molecular receptors supports thermosensory behavior remains an open question (Budelli, 2019).

At a cellular level, rapid responses to innocuous temperatures in Drosophila rely on peripheral thermosensors, including Hot Cells and Cold Cells, named based on their putative hot- and cold-sensing abilities. Hot and Cold Cells are located in the arista, an extension of the antenna, and provide thermosensory input to target neurons in the antennal lobe of the fly brain. How Drosophila Hot and Cold Cells encode thermosensory information, including whether their activities primarily reflect absolute temperature (tonic signaling), temperature change (phasic signaling), or both (phasic-tonic signaling), has not been determined (Budelli, 2019).

At an anatomical level, the sensory endings of Hot Cells and Cold Cells have very different morphologies. Hot Cell outer segments are small and finger-like, while Cold Cell outer segments are large and terminate in elaborate lamellae, layers of infolded plasma membrane thought to contain the thermotransduction machinery. The extent of lamellation varies among Cold Cells within and between insect species and correlates with the neuron's thermosensitivity. Many vertebrate thermosensory neurons also have elaborate morphologies -- from free nerve endings in mammalian skin to mitochondria-packed termini in rattlesnake pit organs. Despite the potential importance of these structures for thermotransduction, the molecules specifying them are unknown (Budelli, 2019).

This study used a combination of electrophysiology, molecular genetics, ultrastructure, and behavior to investigate how the Drosophila thermosensory system encodes thermosensory input and to identify key regulators of thermosensor development and function. These studies reveal that the cold cells are phasic detectors of temperature change, not cold sensors. Their steady-state activity does not increase at colder temperatures. Instead, they are transiently activated upon cooling and transiently inhibited upon warming, exhibiting this activity at temperatures both above and below the fly's preferred temperature. Furthermore, although activated by cooling, they mediate both warm and cool avoidance behavior. It is suggested that cold cells be renamed 'Cooling Cells' to accurately reflect their properties. At the molecular level, this study finda they rely upon a trio of IR family receptors to detect temperature, and ultrastructural analyses reveal a role for IR family sensory receptors in specifying sensory neuron morphogenesis as well as thermosensitivity (Budelli, 2019).

Animals from flies to humans show preferences for specific temperatures. While thermal preference could result from the combined effects of hot- and cold-sensing pathways, the current findings indicate this is not the case in Drosophila. Instead, thermal preference depends on a set of phasic thermosensors that respond to temperature change rather than hot or cold and that participate in both warm and cool avoidance. Although originally named 'Cold Cells', the data show that they are not cold sensors. It is therefore proposed their name be changed to 'Cooling Cells' to more accurately reflect their activity. An advantage of phasic over tonic sensors is that their consistent baseline activity helps ensure sufficient dynamic range is available to respond to small changes in temperature with large changes in firing rate across a range of baseline temperatures. Phasic thermosensors in the ant, for example, respond to milli-degree fluctuations in temperature over a wide range of innocuous temperatures. Such phasic thermosensing enables an animal retain sensitivity to small temperature changes as its overall thermal environment shifts (Budelli, 2019).

A potential limitation of the phasic nature of Cooling Cells is that their responses are similar both above and below the preferred temperature. As the relative desirability of cooling versus warming is different in these two conditions, interpreting Cooling Cell activity as signifying an improving or deteriorating situation likely requires additional thermosensory input. Such context-dependent interpretation of Cooling Cell activity is also suggested by their importance in avoiding both overly cool and warm temperatures, even though their responses to temperature are similar throughout the innocuous range. Consistent with the involvement of multiple thermosensors, the loss of Hot Cell thermosensitivity in Gr28b mutants alters warm avoidance, although the defect effect is partial, suggesting additional contributors. The context-dependent interpretation of phasic thermosensory input is a potentially general challenge. For example, innocuous thermosensors in mammalian skin, particularly cooling-sensitive neurons, are primarily phasic, raising similar concerns about how their activity is interpreted to trigger responses that either raise or lower body temperature. Interestingly, current evidence indicates that such peripheral input is combined with additional thermosensory input in the hypothalamus to control thermoregulatory responses. A potentially analogous scenario has also been observed in the nematode C. elegans. In this system, the bidirectional AFD thermosensor mediates both warm and cool avoidance, acting through circuits that receive input from additional sensory neurons (Budelli, 2019).

The discovery that Cooling Cell thermosensing depends on IRs rather than Brividos provides a unified view of cooling sensation in Drosophila and potentially other insects. Recent work has shown that larval Dorsal Organ Cool Cells (DOCCs) require IR21a, IR25a, and IR93a to detect cooling (Knecht, 2016, Ni, 2016) and that IR21a (along with its co-receptors) is not only necessary but also sufficient to confer cooling sensitivity when ectopically expressed in Hot Cells (Ni, 2016). Taken together, these data indicate that the combination of IR21a, IR25a, and IR93a constitutes a major pathway for cooling detection. In addition, like Cooling Cells, DOCCs show no requirement for Brivido TRP channels to sense temperature (Ni, 2016). The cellular origin of the behavioral defects reported in brivido mutants remains unclear (Budelli, 2019).

Electrophysiological analysis of arista thermoreceptors also demonstrates that 'Hot Cells' are not simple hot sensors. Instead, they are active at cool as well as warm temperatures, transiently responding to temperature changes in a robust and bidirectional manner (warming activated and cooling inhibited). To more accurately reflect their properties, it is proposed their name be changed to 'Heating Cells.' In addition, their steady-state firing rates rise with temperature (Q10 ~4.4). Warming accelerates most processes, but such thermosensitivity lies near the 90th percentile of biological processes. Thus, while their tonic responses are less thermosensitive than their phasic responses, both could contribute to Heating Cell function (Budelli, 2019).

The overlapping contributions of Heating and Cooling Cells to thermosensory behavior is consistent with recent physiological analyses that demonstrate interactions between cooling- and warming-responsive peripheral inputs in the fly brain. The involvement of both Heating and Cooling cells in warm avoidance is in agreement with the regulation of warming-activated projection neurons by both peripheral warming and cooling inputs, the latter mediated via inhibitory interneurons. Furthermore, warm avoidance's greater reliance on Ir21a-dependent cooling detection than Gr28b-dependent warming detection is also consistent with the larger observed contribution of cooling-responsive inhibition to the control of warming-activated projection neurons. For cool avoidance, the dependence on Ir21a-mediated cooling detection, but not Gr28b-mediated warming detection, mirrors the control of cooling-activated projection neurons by cooling-activated, but not warming-activated, inputs. Together, these findings provide a consistent picture in which cooling- and warming-activated peripheral inputs are not equal but opposite influences. Instead, the two inputs potentially communicate distinct information to the thermosensory system, with phasic Cooling Cells playing an essential role in both warm and cool avoidance and Heating Cells showing a narrower domain of influence on thermotactic behavior (Budelli, 2019).

At the molecular level, the contributions of IRs to sensory detection have been viewed previously solely through their potential to act as stimulus-responsive ion channels. This study shows that IRs are also key regulators of neuronal morphogenesis, demonstrating that IR expression is necessary and can suffice to drive the formation of the specialized membrane-membrane contacts characteristic of Cooling Cell sensory endings. As IR family members also mediate olfaction, gustation, and hygrosensation, it will be of interest to determine whether IR-dependent morphogenesis supports these other sensory modalities as well (Budelli, 2019).

From an evolutionary perspective, thermosensory neurons with similar morphologies are found throughout arthropods. This morphological conservation parallels the widespread conservation of IR21a, IR25a, and IR93a sequences, raising the possibility that their role in morphogenesis could be an ancient and conserved feature of the IR family. How the IRs contribute to the formation of lamellae and BOSS elements is not yet known, nor is the mechanistic contribution of these structures to thermotransduction. As IR proteins concentrate in the lamellar region of Cooling Cells, they potentially reside in or near these structures. However, additional Cooling Cell-specific factors likely contribute to lamellae formation, as the structural transformation of the Heating Cell dendrite upon IR misexpression is only partial. Specifically, while IR21a misexpression converts Heating Cells into cooling-activated thermoreceptors (without affecting their warmth sensitivity; Ni, 2016) and promotes membrane contacts with Cooling Cells, the Heating Cell dendrite remains finger-like and does not form internal lamellae. In addition, initial attempts to drive cell-cell association and dendritic remodeling by IR misexpression in other contexts have so far been unsuccessful (Budelli, 2019).

Establishing the appropriate morphology to support sensory receptor function is a general challenge for sensory cells. While the function of sensory receptors like rhodopsin are known to be necessary for sensory neuron morphogenesis, the current analysis demonstrates that sensory receptors can play an instructive role in establishing the structural characteristics of a sensory ending. The use of a sensory receptor to specify morphology provides a simple mechanism by which the structure and function of a sensory neuron can be coordinated (Budelli, 2019).

The ionotropic receptors IR21a and IR25a mediate cool sensing in Drosophila

Animals rely on highly sensitive thermoreceptors to seek out optimal temperatures, but the molecular mechanisms of thermosensing are not well understood. The Dorsal Organ Cool Cells (DOCCs) of the Drosophila larva are a set of exceptionally thermosensitive neurons critical for larval cool avoidance. This study shows that DOCC cool-sensing is mediated by Ionotropic Receptors (IRs), a family of sensory receptors widely studied in invertebrate chemical sensing. Two IRs, IR21a and IR25a, are required to mediate DOCC responses to cooling and are required for cool avoidance behavior. Furthermore, ectopic expression of IR21a was found to confer cool-responsiveness in an Ir25a-dependent manner, suggesting an instructive role for IR21a in thermosensing. Together, these data show that IR family receptors can function together to mediate thermosensation of exquisite sensitivity (Ni, 2016).

These data demonstrate that the ionotropic receptors IR21a and IR25a have critical roles in thermosensation in Drosophila, mediating cool detection by the larval dorsal organ cool cells (DOCCs) and the avoidance of cool temperatures. Combinations of IRs have been previously found to contribute to a wide range of chemosensory responses, including the detection of acids and amines. These findings extend the range of sensory stimuli mediated by these receptor combinations to cool temperatures. Interestingly, IR21a- and IR25a-dependent cool sensation appears independent of Brivido 1 and Brivido 2, two TRP channels implicated in cool sensing in the adult (Gallio, 2011; Ni, 2016 and references therein).

The precise nature of the molecular complexes that IRs form is not well understood. IR25a has been shown to act with other IRs in the formation of chemoreceptors, potentially as heteromers (Rytz, 2013). This precedent raises the appealing possibility that IR25a might form heteromeric thermoreceptors in combination with IR21a. However, an inability to readily reconstitute temperature-responsive receptor complexes in heterologous cells suggests that the mechanism by which these receptors contribute to cool responsiveness is likely to involve additional molecular cofactors. It is interesting to note that the range of cell types in which ectopic IR21a expression confers cool-sensitivity is so far restricted to neurons that already respond to temperature. This observation suggests the existence of additional co-factors or structures in these thermosensory cells that are critical for IR21a and IR25a to mediate responses to temperature. All studies to date implicate IRs as receptors for sensory stimuli (Rytz, 2013), and misexpression studies are consistent with a similar role for Ir21a and IR25a in cool sensation. However, the possibility cannot be fully excluded that they could have indirect, and possibly separate, functions in this process, for example, in regulating the expression or function of an unidentified cool receptor. Interestingly, IR25a was recently implicated in warmth-responsive resetting of the circadian clock, and suggested to confer warmth-sensitivity on its own, without the co-expression of other IRs (Chen, 2015). The ability of IR25a to serve as a warmth receptor on its own would be a surprise given both its broad expression and its established role as an IR co-receptor (Abuin, 2011). As IR25a misexpression only slightly enhanced the thermosensitivity of an already warmth-responsive neuron (Chen, 2015), this raises the alternative possibility that (analogous to cool-sensing) IR25a acts not on its own, but rather as a co-receptor with other IRs involved in warmth-sensing (Ni, 2016).

While the present study focuses on the role of IR21a and IR25a in larval thermosensation, it is interesting to note that the expression of both IR21a and IR25a has been detected in the thermoreceptors of the adult arista (Benton, 2009). Thus, related mechanisms could contribute to thermosensory responses not only in the DOCCs, but also in other cellular contexts and life stages. Moreover, the presence of orthologs of IR21a and IR25a across a range of insects (Croset, 2010) raises the possibility that these IRs, along other members of the IR family, constitute a family of deeply-conserved thermosensors (Ni, 2016).

Distinct combinations of variant ionotropic glutamate receptors mediate thermosensation and hygrosensation in Drosophila

Ionotropic Receptors (IRs) are a large subfamily of variant ionotropic glutamate receptors present across Protostomia. While these receptors are most extensively studied for their roles in chemosensory detection, recent work has implicated two family members, IR21a and IR25a, in thermosensation in Drosophila. This study characterized one of the most evolutionarily deeply conserved receptors, IR93a, and shows that it is co-expressed and functions with IR21a and IR25a to mediate physiological and behavioral responses to cool temperatures. IR93a is also co-expressed with IR25a and a distinct receptor, IR40a, in a discrete population of sensory neurons in the sacculus, a multi-chambered pocket within the antenna. This combination of receptors was demonstrated to be required for neuronal responses to dry air and behavioral discrimination of humidity differences. These results identify IR93a as a common component of molecularly and cellularly distinct IR pathways important for thermosensation and hygrosensation in insects (Knecht, 2016).


Search PubMed for articles about Drosophila Ir21a

Abuin, L., Bargeton, B., Ulbrich, M. H., Isacoff, E. Y., Kellenberger, S. and Benton, R. (2011). Functional architecture of olfactory ionotropic glutamate receptors. Neuron 69: 44-60. PubMed ID: 21220098

Benton, R., Vannice, K. S., Gomez-Diaz, C. and Vosshall, L. B. (2009). Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila. Cell 136: 149-162. PubMed ID: 19135896

Budelli, G., Ni, L., Berciu, C., van Giesen, L., Knecht, Z. A., Chang, E. C., Kaminski, B., Silbering, A. F., Samuel, A., Klein, M., Benton, R., Nicastro, D. and Garrity, P. A. (2019). Ionotropic receptors specify the morphogenesis of phasic sensors controlling rapid thermal preference in Drosophila. Neuron 101(4): 738-747. PubMed ID: 30654923

Chen, C., Buhl, E., Xu, M., Croset, V., Rees, J. S., Lilley, K. S., Benton, R., Hodge, J. J. and Stanewsky, R. (2015). Drosophila Ionotropic Receptor 25a mediates circadian clock resetting by temperature. Nature 527: 516-520. PubMed ID: 26580016

Croset, V., Rytz, R., Cummins, S. F., Budd, A., Brawand, D., Kaessmann, H., Gibson, T. J. and Benton, R. (2010). Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction. PLoS Genet 6: e1001064. PubMed ID: 20808886

Gallio, M., Ofstad, T. A., Macpherson, L. J., Wang, J. W. and Zuker, C. S. (2011). The coding of temperature in the Drosophila brain. Cell 144: 614-624. PubMed ID: 21335241

Greppi, C., Laursen, W. J., Budelli, G., Chang, E. C., Daniels, A. M., van Giesen, L., Smidler, A. L., Catteruccia, F. and Garrity, P. A. (2020). Mosquito heat seeking is driven by an ancestral cooling receptor. Science 367(6478): 681-684. PubMed ID: 32029627

Hernandez-Nunez, L., Chen, A., Budelli, G., Berck, M. E., Richter, V., Rist, A., Thum, A. S., Cardona, A., Klein, M., Garrity, P. and Samuel, A. D. T. (2021). Synchronous and opponent thermosensors use flexible cross-inhibition to orchestrate thermal homeostasis. Sci Adv 7(35). PubMed ID: 34452914

Knecht, Z. A., Silbering, A. F., Ni, L., Klein, M., Budelli, G., Bell, R., Abuin, L., Ferrer, A. J., Samuel, A. D., Benton, R. and Garrity, P. A. (2016). Distinct combinations of variant ionotropic glutamate receptors mediate thermosensation and hygrosensation in Drosophila. Elife 5. PubMed ID: 27656904

Ni, L., Klein, M., Svec, K. V., Budelli, G., Chang, E. C., Ferrer, A. J., Benton, R., Samuel, A. D. and Garrity, P. A. (2016). The ionotropic receptors IR21a and IR25a mediate cool sensing in Drosophila. Elife 5. PubMed ID: 27126188

Rytz, R., Croset, V. and Benton, R. (2013). Ionotropic receptors (IRs): chemosensory ionotropic glutamate receptors in Drosophila and beyond. Insect Biochem Mol Biol 43: 888-897. PubMed ID: 23459169

Biological Overview

date revised: 26 December 2021

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