InteractiveFly: GeneBrief

Ionotropic receptor 25a: Biological Overview | References

BIOLOGICAL OVERVIEW

Circadian clocks are endogenous timers adjusting behaviour and physiology with the solar day. Visual and non-visual photoreceptors are responsible for synchronizing circadian clocks to light, but clock-resetting is also achieved by alternating day and night temperatures with only 2°-4°C difference. This study shows that Drosophila Ionotropic Receptor 25a (IR25a) is required for behavioural synchronization to low-amplitude temperature cycles. This channel is expressed in sensory neurons of internal stretch receptors previously implicated in temperature synchronization of the circadian clock. IR25a is required for temperature-synchronized clock protein oscillations in subsets of central clock neurons. Extracellular leg nerve recordings reveal temperature- and IR25a-dependent sensory responses, and IR25a misexpression confers temperature-dependent firing of heterologous neurons. It is proposed that IR25a is part of an input pathway to the circadian clock that detects small temperature differences. This pathway operates in the absence of known 'hot' and 'cold' sensors in the Drosophila antenna, revealing the existence of novel periphery-to-brain temperature signalling channels (Chen, 2015).

In Drosophila, daily activity rhythms are controlled by a network of ~150 clock neurons expressing the clock genes period (per) and timeless (tim). These encode repressor proteins that negatively feedback on their own promoters resulting in 24h oscillations of clock molecules. Temperature cycles (TC) synchronize molecular clocks present in peripheral appendages in a tissue-autonomous manner, whereas synchronization of clock neurons in the brain mainly depends on peripheral temperature receptors located in the chordotonal organs (ChO) and the ChO-expressed gene no circadian temperature entrainment (nocte) (Chen, 2015).

To discover novel factors involved in temperature entrainment, Nocte-interacting proteins were identified by co-immunoprecipitation and mass-spectrometry. Focus was placed on IR25a, a member of a divergent subfamily of ionotropic glutamate receptors, and the interaction by co-immunoprecipitation was varfied after overexpressing IR25a and Nocte in all clock cells using tim-gal4. IR25a is expressed in different populations of sensory neurons, including those in the antenna and labellum. In the olfactory system IR25a acts as a co-receptor with different odour-sensing IRs (Abuin, 2011; Chen, 2015 and references therein).

To investigate if IR25a is co-expressed with nocte in ChO, IR25a expression in femur and antennal ChO was analyzed using an IR25a-gal4 line (Abuin, 2011). IR25a-gal4-driven mCD8-GFP labelled subsets of ChO neurons in the femur, overlapping substantially with nompC-QF driven QUAS-Tomato signals (using the QF binary transcriptional activation system). nompC-QF is expressed in larval ChO18 and in the adult femur ChO. Comparison of IR25a-driven mCD8-GFP and nuclear DsRed signals with those of other ChO neuron drivers suggests that IR25a is expressed in a subset of femur ChO neurons and Johnston's Organ (JO) neurons. To determine if IR25a-gal4 ChO signals reflect endogenous IR25a expression, the presence of IR25a mRNA in the femur and leg was confirmed, and the co-localization was confirmed of anti-IR25a immunofluorescence signals in femur ChO neurons. IR25a was detected in ChO neuron cell bodies and ciliated dendrites, as was an mCherry-IR25a fusion protein expressed in these cells (Chen, 2015).

As nocte¹ mutants do not synchronize to 12h-12-h 16°C:25°C temperature cycles in constant light (LL), IR25a^-/- mutants were analyzed under these conditions. Unlike nocte¹, the IR25a^-/- flies synchronized well to this regime, and similar results were obtained at warmer temperature cycles. To test whether IR25a is specifically required for synchronization to small temperature intervals, IR25^-/- flies were subjected to various temperature cycles with an amplitude of only 2°C. Surprisingly, and in contrast to wild-type, IR25^-/- mutants did not synchronize to any of the shallow temperature cycles in LL or constant darkness (DD). In LL, wild-type and IR25a rescue flies showed a clear activity peak in the second part of the warm period before and after the 6h shift of the temperature cycle. By contrast, IR25a^-/- mutants were constantly active throughout the temperature cycle, apart from a short period of reduced activity at the beginning of the warm phase of TC1. In DD, control flies slowly advanced (or delayed) their evening activity peak during phase-advanced (or delayed) temperature cycles. The phase of this activity peak was maintained in the subsequent free-running conditions (DD, constant 25°C) indicating stable re-entrainment of the circadian clock. By contrast, IR25a mutants did not shift their evening peak during the temperature cycle, keeping their original phase throughout the experiment (Chen, 2015).

To quantify entrainment in LL, the 'entrainment index' (EI) was determined, whereas for most DD experiments the phase difference of the main activity peak upon release into constant conditions between IR25a mutants and controls was calculated. In all 2°C amplitude temperature cycles tested the entrainment index of IR25a^-/- flies was significantly lower and phase calculation indicated no phase shift or a significantly reduced phase shift compared to controls. The same non-synchronization phenotype was observed in IR25-/Df(IR25a) flies, and temperature synchronization was fully restored in IR25^-/- rescue flies IR25a^-/- mutants synchronize to light and have normal free-running and temperature compensated periods. These results suggest that IR25a enables the circadian clock to sense subtle temperature changes across the entire physiological range, rather than mediating synchronization to a specific range. Increasing the temperature cycle amplitude to 4°C consistently restored temperature entrainment in IR25a^-/- flies (Chen, 2015).

Temperature receptors located in fly antennae and arista are not required for temperature-synchronized behaviour. As expected, it was found that antennal IR25a function is not required for temperature entrainment. To reveal the importance of IR25a expression in ChO neurons, tissue-specific IR25a RNA interference (RNAi) was performed using validated transgenes. IR25a RNAi in all or subsets of ChO neurons resulted in a lack of entrainment. By contrast, IR25a RNAi in multidendritic, TRPA1-expressing or clock neurons did not impair temperature entrainment. These findings are consistent with the absence of IR25a expression in clock neurons and the brain and show that IR25a functions in ChO neurons for temperature entrainment to 25°C:27°C temperature cycles in LL (Chen, 2015).

To identify the neural substrates underlying the lack of behavioural synchronization, clock protein levels were quantified in wild-type, IR25a^-/-, and IR25a^-/- rescue flies exposed to a shallow temperature cycle in LL. Although TIM expression was robustly rhythmic and synchronized in all clock neuronal groups in controls, TIM was barely detectable in the Dorsal Neuron 1 (DN1) and DN2 of IR25a^-/- flies. Moreover, in the small and large ventral lateral neurons (s-LNv and l-LNv), TIM expression exhibited an additional peak during the warm phase. In the DN3, TIM declined earlier compared to controls and there was no effect on the dorsal lateral neurons (LNd). In temperature cycles and DD, TIM levels in DN1 were also blunted but oscillations in the DN2 and DN3 were similar to controls. In contrast to LL, TIM did not oscillate in any of the LN groups and was at constantly low levels, consistent with the behavioural results obtained under these conditions. The alterations of TIM expression are temperature specific, as normal oscillations were observed in LD cycles at 25°C. An increase of the temperature cycle amplitude to 4°C also restored normal TIM expression in IR25a^-/- flies, in agreement with the behavioural rescue. In summary, in low-amplitude temperature cycles, IR25a is required for normally synchronized TIM oscillations in DN1-3 and LNv in LL and in DN1 and LN clock neurons in DD (Chen, 2015).

Tests were performed to see if the clock neurons affected by the lack of IR25a are indeed involved in regulating behavioural synchronization to shallow temperature cycles by blocking synaptic transmission using tetanus-toxin (TNT). Indeed, TNT-expression in DN1 and DN2 blocked synchronization in LL, whereas in DD only DN1 blockage interfered with temperature entrainment. Consistent with the differential effect on TIM oscillations in LL and DD these results strongly suggest that IR25a is required for the synchronized output of the DN1 (LL and DD) and DN2 (LL) to control temperature-entrained behaviour (Chen, 2015).

Next, it was asked if ChO might directly sense temperature in an IR25a-dependent manner. Leg nerve activity was recorded in restrained preparations, and ChO units were identified in the compound signal. In both wild-type and IR25a^-/- flies, spontaneous leg movement changed as a function of temperature along with motor and sensory activity. Additionally, presumed ChO activity of wild-type flies also increased during periods without movement. This temperature-induced but movement-independent, ChO activity was absent in IR25^-/- flies, showing that temperature is sensed in the legs in an IR25a-dependent manner. To test if IR25a contributes directly to temperature-sensing, this channel was ectopically expressed in the physiologically well-characterized, IR25a-negative, l-LNv. As a positive control, the temperature-sensitive Drosophila TRPA1 channel was also expressed in the l-LNv. Isolated brains were exposed to a temperature ramp, and spike frequency of individual l-LNv was recorded. Control l-LNv did not show a significant temperature-dependent change in neural activity. As expected, the firing rate of TRPA1 expressing neurons drastically increased linearly with temperature, as did other cellular parameters. IR25a expression resulted in a linear and reversible temperature-dependent increase in action potential firing frequency, whereas other cellular parameters showed no difference. Increasing the temperature by only 2°-3°C also lead to a reversible increase in firing frequency in IR25a expressing l-LNv. By contrast, expression of the related, but olfactory-specific co-receptor IR8a (which is not required for temperature entrainment) did not confer temperature-sensitivity. These observations suggest that IR25a is at least part of a thermosensory receptor required for temperature entrainment (Chen, 2015).

These data indicating that IR25a contributes to temperature sensing within ChO extend the roles of IR's beyond chemoreception, reminiscent of the requirement for the 'gustatory receptor' Gr28b in warmth-avoidance (Ni, 2013). Although this study shows that IR25a-expressing leg neurons are capable of sensing temperature and mediating temperature entrainment, it is possible that this receptor has a similar role elsewhere in the peripheral nervous system. IR25a responds to small temperature changes and it is proposed that the fly continuously integrates temperature signals received from multiple ChO across the whole body for synchronization of the clock. This potential reliance on weakly responding temperature receptors might explain why the Drosophila circadian clock is insensitive to brief temperature pulses, which could help maintain synchronized clock function in natural conditions of rapid and large temperature fluctuations (Chen, 2015).

An expression atlas of variant ionotropic glutamate receptors identifies a molecular basis of carbonation sensing

Through analysis of the Drosophila ionotropic receptors (IRs), a family of variant ionotropic glutamate receptors, it was revealed that most IRs are expressed in peripheral neuron populations in diverse gustatory organs in larvae and adults. This study characterize IR56d, which defines two anatomically-distinct neuron classes in the proboscis: one responds to carbonated solutions and fatty acids while the other represents a subset of sugar- and fatty acid-sensing cells. Mutational analysis indicates that IR56d, together with the broadly-expressed co-receptors IR25a and IR76b, is essential for physiological responses to carbonation and fatty acids, but not sugars. It was further demonstrated that carbonation and fatty acids both promote IR56d-dependent attraction of flies, but through different behavioural outputs. This work provides a toolkit for investigating taste functions of IRs, defines a subset of these receptors required for carbonation sensing, and illustrates how the gustatory system uses combinatorial expression of sensory molecules in distinct neurons to coordinate behaviour (Sanchez-Alcaniz, 2018).

IRs are best-characterised in Drosophila melanogaster, which possesses 60 intact Ir genes. Of these, the most thoroughly understood are the 17 receptors expressed in the adult antenna. Thirteen of these are expressed in discrete populations of sensory neurons, and function as olfactory receptors for volatile acids, aldehydes and amines or in humidity detection. The remaining four are expressed in multiple, distinct neuron populations and function, in various combinations, as co-receptors with the selectively-expressed tuning IRs (Sanchez-Alcaniz, 2018).

By contrast, little is known about the sensory functions of the remaining, large majority of non-antennal IRs. Previous analyses described the expression of transgenic reporters for subsets of these receptors in small groups of gustatory sensory neurons (GSNs) in several different contact chemosensory structures. While these observations strongly implicate these genes as having gustatory functions, the evidence linking specific taste ligands to particular receptors, neurons and behaviours remains sparse. For example, IR52c and IR52d are expressed in sexually-dimorphic populations of leg neurons and implicated in male courtship behaviours, although their ligands are unknown. Reporters for IR60b, IR94f and IR94h are co-expressed in pharyngeal GSNs that respond to sucrose, which may limit overfeeding or monitor the state of externally digested food. IR62a is essential for behavioural avoidance of high Ca2+ concentrations, but the precise neuronal expression of this receptor is unclear. As in the olfactory system, these selectively-expressed IRs are likely to function with the IR25a and/or IR76b co-receptors, which are broadly-expressed in contact chemosensory organs, and required for detection of multiple types of tastants, including polyamines, inorganic, carboxylic and amino acid and Ca2+ (Sanchez-Alcaniz, 2018).

This study describes a set of transgenic reporters for the entire Ir repertoire. These were used to survey the expression of this receptor family in both larval and adult stages. Using this molecular map, IR56d was identified as a selectively-expressed receptor that acts with IR25a and IR76b to mediate physiological and attractive behavioural responses to carbonation, a previously orphan taste class. Furthermore, this study extends recent studies to show that IR56d is also required in sugar-sensing GR neurons to mediate distinct behavioural responses to fatty acids (Sanchez-Alcaniz, 2018).

This work describes that non-antennal IRs function to detect a myriad of chemical stimuli to evoke a variety of behavioural responses. Such properties presumably apply to the vast, divergent IR repertoires of other insect species, for example, the 455 family members in the German cockroach Blatella germanica, or the 135 IRs in the mosquito Aedes aegypti. Within Drosophila no obvious relationships were detected between IR phylogeny and stage- or organ-specific expression patterns. Phylogenetic proximity may therefore be the most indicative of functional relationships between IRs, as is the case for those expressed in the antenna. If this hypothesis is correct, the expression data presented in this study suggest that functionally-related clades of receptors act in several types of chemosensory organ (Sanchez-Alcaniz, 2018).

An important caveat to the transgenic approach used to reveal expression is the faithfulness of these reporters to the endogenous expression pattern of Ir genes. Although this strategy has been widely (and successfully) used for antennal IRs and other chemosensory receptor families, it is impossible to determine reporter fidelity without a complementary tool (e.g. receptor-specific antibodies or tagging of the endogenous genomic locus). Discrepancies were noted between the expression of some of the Ir-Gal4 lines and those described previously; many of these probably reflect differences in the length of regulatory regions used to create these distinct transgenes. Precise comparison of independently-constructed transgenic constructs may in fact be useful in informing the location of enhancer elements directing particular temporal or spatial expression patterns. Moreover, transgenic reporters provide powerful genetic tools for visualisation and manipulation of specific neuronal populations. The reagents generated in this study should therefore provide a valuable resource for further exploration of the IRs in insect gustation (Sanchez-Alcaniz, 2018).

Using the atlas, IR56d-together with the broadly-expressed co-receptors IR25a and IR76b- were identified as essential for responses of labellar taste peg (Pit-like sensilla) neurons to carbonation. Such observations implicate IR56d as the previously unknown tuning receptor for this stimulus. However, these IRs do not appear to be sufficient for carbonation detection, as their misexpression in other neurons failed to confer sensitivity to carbonated stimuli. This observation suggests that additional molecules or cellular specialisations are required. Such a factor may be rather specific to taste pegs, given the minimal/absent responses of Ir56d-expressing taste bristle/leg neurons to carbonation, but does not appear to be another IR, as no other IR reporters were detected that expressed in this population of cells (Sanchez-Alcaniz, 2018).

While precise mechanistic insights into carbonation sensing will require the ability to reconstitute IR56d-dependent carbonation responses in heterologous systems, it is interesting to compare how insects and mammals detect this stimulus. The main mammalian gustatory carbonation sensor, the carbonic anhydrase Car4 is an enzyme tethered to the extracellular surface of sour (acid) taste receptor cells in lingual taste buds, where it is thought to catalyse the conversion of aqueous CO2 into hydrogencarbonate (bicarbonate) ions (HCO3-) and protons (H+). The resulting free protons, but not hydrogencarbonate ions, provide a relevant signal for the sour-sensing cells. By contrast, IR56d neurons are not responsive to low pH, suggesting a different chemical mechanism of carbonation detection. The observation that IR56d is also essential for sensitivity to hexanoic acid suggests that IR56d could recognise the common carboxyl group of hydrogencarbonate and fatty acid ligands. However, IR56d neurons are not responsive to all organic acids, indicating that this cannot be the only determinant of ligand recognition (Sanchez-Alcaniz, 2018).

Characterisation of IR56d neurons extends previous reports to reveal an unexpected complexity in the molecular and neuronal basis by which attractive taste stimuli are encoded. The taste bristle population of IR56d neurons represents a subset of sugar-sensing cells that are also responsive to fatty acids, glycerol and, minimally, to carbonation. Although activation of these neurons promotes PER, it was found that carbonation-evoked stimulation is insufficient to trigger this behaviour, which suggests that taste bristles are not a relevant sensory channel for this stimulus. While members of a specific clade of GRs are well-established to mediate responses to sugars and glycerol, the detection mechanisms of fatty acids appear to be more complex. Earlier work demonstrated an important role of a phospholipase C homologue (encoded by norpA) in labellar fatty acid responses. More recently, GR64e was implicated as a key transducer of fatty acid-dependent signals, but suggested to act downstream of NorpA, rather than as a direct fatty acid receptor. By contrast, an independent study of the legs showed that all sugar-sensing Gr genes (including Gr64e) were dispensable for fatty acid detection and provided evidence instead for an important role of IR25a and IR76b in these responses. Analysis of Ir56d mutants indicates an IR-dependent fatty acid-detection mechanism also exists in the labellum; future work will be needed to relate this to the roles of GR64e and NorpA (Sanchez-Alcaniz, 2018).

The IR56d taste peg population is, by contrast, sensitive to carbonation and fatty acids (but not sugars or glycerol), and these responses can be ascribed to IR56d (a Gr64eLexA reporter is not expressed in taste peg neurons). Although these neurons mediate taste-acceptance behaviour, they do not appear to promote proboscis extension or food ingestion. Recent work using optogenetic neuronal silencing experiments provided evidence that taste peg neuron activity is important for sustaining, rather than initiating, feeding on yeast, by controlling the number of sips an animal makes after proboscis extension. These observations are concordant with the internal location of taste pegs on the labellum, as they will not come into contact with food until the proboscis has been extended, and could explain the positional preference for carbonated substrates that were observed. This study has attempted to determine whether carbonation can influence sipping behaviour using flyPAD. Although these experiments did not reveal a statistically-significant effect, interpretation is complicated by the difficulty of providing and maintaining carbonation stimuli in the solid medium used in flyPAD assays. Future development of other approaches to provide this stimulus in feeding assays will be necessary. Nevertheless, the data strengthen the view that carbonation, a non-nutritious microbial fermentation product, regulates-via activation of IR56d taste peg neurons-a distinct motor programme to PER as part of a multicomponent behavioural response (Sanchez-Alcaniz, 2018).

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).

Molecular basis of fatty acid taste in Drosophila

Behavioral studies have established that Drosophila appetitive taste responses towards fatty acids are mediated by sweet sensing Gustatory Receptor Neurons (GRNs). This study shows that sweet GRN activation requires the function of the Ionotropic Receptor genes IR25a, IR76b and IR56d. The former two IR genes are expressed in several neurons per sensillum, while IR56d expression is restricted to sweet GRNs. Importantly, loss of appetitive behavioral responses to fatty acids in IR25a and IR76b mutant flies can be completely rescued by expression of respective transgenes in sweet GRNs. Interestingly, appetitive behavioral responses of wild type flies to hexanoic acid reach a plateau at ~1%, but decrease with higher concentration, a property mediated through IR25a/IR76b independent activation of bitter GRNs. With previous report on sour taste, these studies suggest that IR-based receptors mediate different taste qualities through cell-type specific IR subunits (Ahn, 2017).

IR genes have emerged as a second large gene family encoding chemoreceptors in insects. In the Drosophila olfactory system, IRs function as multimeric receptors in coeloconic olfactory sensory neurons (OSN) and are thought to sense volatile carboxylic acids, amines and aldehydes. Expression analyses have shown that each coeloconic OSN expresses up to four IR genes, including high levels of either IR8a or IR25a. IR25a and IR8a are distinct from other IRs in that they are more conserved to each other and iGluRs, and they share a long amino terminal domain absent in all other IRs. These observations, along with functional analyses of basiconic olfactory neurons that express combinations of IR genes, led to a model in which IR based olfactory receptors are tetrameric complexes thought to consist of up to three different subunits that contain at least one core unit (IR8a or IR25a) and two additional IRs that determine ligand binding specificity. The findings presented in this paper expand this concept to taste receptors that sense fatty acids through the sweet GRNs found in tarsal taste sensilla (Ahn, 2017).

This analysis extends the multimodal role of IR25a and IR76b to the taste systems. Consistent with gene expression arrays, this paper shows that up to three GRNs, including many sweet and bitter GRNs, co-express IR25a and IR76b. Functional studies have established a novel role for these two IR proteins in fatty acid taste, which revealed that these two subunits are not only critically important to elicit Proboscis Extension Reflex (PER) responses in flies when challenged with fatty acids, but are also necessary for fatty acid induced Ca2+ increases in tarsal sweet GRNs. Based on these findings and with consideration of their established role in other sensory systems, it is proposed that IR25a and IR76b play central roles in sweet GRNs in a multimeric receptor complex for initiating appetitive taste behavior to these chemicals. Intriguingly, both genes are also co-expressed in two other GRNs of most tarsal taste sensilla, strongly arguing for additional taste functions. While the subset of tarsal bitter GRNs activated by hexanoic acid does not require either gene, the third GRN (the sour GRN) is narrowly tuned to acids in an IR25a/IR76b dependent manner. These observations suggest that modality specific IRs are likely expressed in a cell-type specific fashion whereby they complement IR25a/IR76b to function as either a fatty acid or a sour taste receptor. Indeed, the screen identified IR56d, a gene that is expressed in sweet GRNs of tarsal taste sensilla, as a likely candidate encoding an IR subunit specific for a fatty acid taste receptor. It remains to be seen whether IR25a, IR76b and IR56d comprise all subunits that constitute this receptor or whether yet additional IRs are necessary to mediate responses to these chemicals (Ahn, 2017).

The fact that different food chemicals can activate a single class of neurons raises the question how flies discriminate between sugars and fatty acids. First, the difference is noted in sensitivity of appetitive GRNs to sugars and fatty acids, respectively: The most responsive GRN for sugars is the one associated with the 5v sensilla, followed by that with the 5s and finally the 5b sensilla, while the responsiveness for fatty acids is the reverse (5b > 5s > 5v). Second, fatty acids induces weaker PER responses from stimulation of the labial palps as opposed to tarsi, while sugars induce equally strong PER responses from stimulation of either taste organ. Third, at least some fatty acids activate bitter GRNs, and hence, generate more complex activation patterns in the brain than sugars, which are not known to activate neurons other than sweet GRNs. These properties may provide a rationale for differential coding of these two classes of chemicals in the brain. Finally, sugars but not fatty acids are soluble in water, and hence, the specific solvents in which these chemicals are presented provides different textural quality, which was recently shown to play a role in taste perception (Ahn, 2017).

NorpA, which encodes a phospholipase C (PLC), plays a critical role in sweet GRNs for appetitive feeding responses to fatty acids, but it is dispensable for behavioral responses to sugars. This study found that its absence also selectively abolishes Ca2+ responses to fatty acids, but not sugars, in sweet cells. NorpA is known for its role as downstream effector of G-protein coupled receptors in the fly's visual system, but interestingly it is also required for olfactory responses in neurons of the maxillary palps, which express ORs that are thought to function as ligand-gated ion channels. It is noted that fatty acid taste in mammals is in part mediated by two G-protein coupled receptors, GPR40 and GPR120, and that one of these (GPR120) was found to signal through a phospholipase C. Thus, future studies will be necessary to gain insights for how PLC mediates chemosensory responses through ORs and the phylogenetically unrelated IRs (Ahn, 2017).

Multimeric IR based receptors were recently shown to be required in non-chemosensory processes. Specifically, Dorsal Organ Cool Cells (DOCCs) located in the larval brain, express and require the function of three IRs (IR21a IR25a and IR93a), thereby allowing larvae to avoid temperatures below ~20°C. Similarly, two sets of cells in the antennal sacculus of adult flies, requiring the functions of IR25a and IR93a and either IR40a or IR68a, were shown to mediate a fly's preferred humidity environment, which is generally in the dry range, but is also dependent on the fly's hydration state. Intriguingly, these non-chemosensory IR complexes share a common theme with the fatty acid and carboxylic acid taste receptors in that they all require a core unit (IR25a) and two additional IRs that mediate specificity for a particular stimulus type (i.e., temperature, humidity, fat, acid) (Ahn, 2017).

An IR76b based sodium channel and an IR76b based amino acid receptor appear to lack an obligate core unit (IR25a or IR8a) found in olfactory receptors or fatty acid and carboxylic acid taste receptors. The IR76b sodium channel mediates salt responses in a heterologous systems independently of any other IRs, while a proposed multimeric IR76b containing receptor mediates amino acids taste in wild type and IR25a mutant flies (IR8a is not expressed in taste neurons). It will be interesting to elucidate the compositions of complete IR based amino acid and sour taste receptors, and (with regard of amino acid receptors) to identify the neurons that mediate this taste modality (Ahn, 2017).

Distinct activity-gated pathways mediate attraction and aversion to CO2 in Drosophila

Carbon dioxide is produced by many organic processes and is a convenient volatile cue for insects that are searching for blood hosts, flowers, communal nests, fruit and wildfires. Although Drosophila melanogaster feed on yeast that produce CO2 and ethanol during fermentation, laboratory experiments suggest that walking flies avoid CO2. This study resolved this paradox by showing that both flying and walking Drosophila find CO2 attractive, but only when they are in an active state associated with foraging. Their aversion to CO2 at low-activity levels may be an adaptation to avoid parasites that seek CO2, or to avoid succumbing to respiratory acidosis in the presence of high concentrations of CO2 that exist in nature. In contrast to CO2, flies are attracted to ethanol in all behavioural states, and invest twice the time searching near ethanol compared to CO2. These behavioural differences reflect the fact that ethanol is a unique signature of yeast fermentation, whereas CO2 is generated by many natural processes. Using genetic tools, it was determined that the evolutionarily conserved ionotropic co-receptor IR25a is required for CO2 attraction, and that the receptors necessary for CO2 avoidance are not involved in this attraction. This study lays the foundation for future research to determine the neural circuits that underlie both state- and odorant-dependent decision-making in Drosophila (van Breugel, 2018).

D. melanogaster feed, mate and deposit eggs on rotting fruit. Between 10 and 14 days later, the next generation of flies must locate a fresh ferment. Because of the high volatility of CO2, the emission of CO2 is greatest near the start of fermentation, whereas ethanol emission increases more slowly. Other odours associated with fermentation (for example, acetic acid and ethyl acetate) form later, when bacteria break down ethanol. In trap assays, Drosophila show a preference for two-day-old apple juice ferments compared to older solutions, which suggests that they might be attracted to CO2. Although it is difficult to estimate concentrations of CO2 in wild ferments, the CO2 concentration in bottles commonly used to rear flies has been determined to be 0.5-1% (van Breugel, 2018).

This evidence that CO2 might attract Drosophila contradicts previous studies conducted using small chambers. To study how flies respond to odours under more-ethological conditions, the flight trajectories was recorded of flies in a wind tunnel that contained a landing platform, which was programmed to periodically release plumes of CO2 or ethanol. Both odours elicited approaches, landings and explorations of a conspicuous visual feature, which is consistent with previous experiments with flies and mosquitoes. Flies were more likely to approach the platform or dark spot in the presence of ethanol compared to CO2, but were equally likely to land in response to either odour (van Breugel, 2018).

To quantify the behaviour of flies after they land, a platform was designed that is suitable for automated tracking. At a flow rate of 60 ml min-1 CO2, the CO2 concentration near the surface of the platform was approximately 3%. After landing near a source of CO2, ethanol or apple cider vinegar, flies exhibited a local search behaviour that was similar to so-called 'dances'. Flies spent twice the amount of time exploring platforms that emitted ethanol compared to CO2 or vinegar. Flies approached a source that emitted both ethanol and CO2 more frequently than they approached vinegar, or either odour alone. Vinegar elicited smaller local searches and slightly fewer approaches compared to CO2, consistent with the hypothesis that vinegar might indicate a less favourable, late-stage ferment. Flies spent significantly less time standing still on the platform in the presence of CO2 compared to any other odour, with a mean walking speed > 2 mm s-1 (van Breugel, 2018).

One previous study showed that Drosophila are attracted to CO2 while flying on a tether. The current results confirm this observation in freely flying flies; however, it was also found that flies remain attracted to CO2 after they land, which contradicts previous studies. One potential explanation is that flies in constrained walking chambers might behave differently to those that arrived on the open wind tunnel platform after tracking the odour plume and landing. To test this hypothesis, an enclosed arena was built in which flies were unable to fly, and they were presented with pulses of 5% CO2. Groups of 10 starved flies presented with CO2 after acclimating to the arena for 10 min exhibited aversion, as previously reported. However, if allowed to acclimate in the chamber for two hours, the flies exhibited attraction to CO2 (van Breugel, 2018).

To study the response of these flies in more detail, the behaviour of flies was recorded for 20 h, while providing 10-min presentations of CO2 from alternating sides of the arena every 40 min. To control for humidity, 20 ml min-1 of H2O-saturated air was continuously pumped through the odour ports on both sides of the chamber. The flies exhibited a clear circadian rhythm within the chamber, as indicated by their mean walking speed. At times of peak activity-near dusk and dawn-flies showed a strong initial attraction to CO2, which decayed stereotypically during the 10-min presentation. At times of low activity-at mid-day and during the night-flies exhibited a mild aversion to CO2. Starving flies for 24 h before the experiment changed their activity profile, resulting in a slightly elevated attraction during the night. Ethanol, by contrast, elicited sustained attraction regardless of baseline activity (van Breugel, 2018).

To probe this relationship between activity and CO2 attraction, the temperature was increased and the wind speed-manipulations that are known to elevate and depress activity were elevated, respectively. When wthe bulk-flow rate was increased to 100 ml min-1, flies exhibited a peak walking speed of about 1.5 mm s-1 at dusk-nearly half the speed measured at a flow rate of 20 ml min-1. Instead of showing attraction, these flies exhibited aversion to 5% CO2, although they were still attracted to ethanol. This result helps to explain why previous studies that used higher flows (100-1,000 ml min-1) to present CO2 observed aversion. To further explore the effect of wind, the aristae of the flies, which destroys their primary means of detecting airflow but does not interfere with the detection of odours, were clipped. The flies without aristae exhibited the same walking speed and attraction to CO2 at the high flow rate as was exhibited by normal flies at the low flow rate. Warming flies with intact aristae to 32°C also increased their baseline activity and recovered their attraction to CO2 at the higher flow rate. Pooling data across all experimental conditions, it was found that flies were attracted to CO2 when they had a baseline walking speed that was above about 2.4 mm s-1. This value is similar to the walking speed that was observed in the wind tunnel assay, which was higher for CO2 than the other odours. To confirm that activity-dependent attraction to CO2 is not a function of social interactions, 29 single flies, which behaved similarly to the cohorts of 10, were tested. Three concentrations of CO2 (1.7%, 5% and 15%) were also tested and found that the 5% concentration elicited the strongest response, consistent with wind tunnel experiments (van Breugel, 2018).

Although the responses of flies to ethanol and CO2 were similar at stimulus onset, attraction to ethanol was more sustained. The time course of behaviour was notably similar in the walking arena and wind tunnel, which suggests that the behavioural dynamics of olfactory attraction are robust to the stimulus environment and may represent an adaptation for using information that broad (CO2) and more specific (ethanol) odorants provide (van Breugel, 2018).

Previous research shows that CO2 aversion is mediated by Gr63a and Gr21a receptors; high concentrations of CO2 are also detected by an acid-sensitive ionotropic receptor, IR64a10. In the current assay, mutant flies that lack the IR64a receptor showed no significant change in their behaviour compared to wild type. Consistent with previous work, mutants that lack the Gr63a receptor exhibited no aversion to CO2; however, they were still attracted to CO2 when active. Mutant flies that are homozygous for both Gr63a and IR64a behaved similarly to the Gr63a mutants. It is noteworthy that the characteristic decaying time course of attraction was unaffected in Gr63a mutants, even though these flies showed no aversion. Thus, the decay in attraction to CO2 is not caused by an increase in aversion over time (van Breugel, 2018).

Given that CO2 attraction is not mediated by Gr63a, Gr21a or IR64a, it was of interest to confirm that the attraction is indeed a chemosensory response. To determine whether CO2 attraction is mediated by either an olfactory or ionotropic receptor, a mutant was tested that lacks the olfactory and ionotropic co-receptors (Orco, IR25a and IR8a) as well as Gr63a. These near-anosmic mutants exhibited no detectable behavioural response to CO2. Flies in which the third antennal segment was surgically removed showed no response to CO2, despite normal levels of activity. Together with the arista ablations, these experiments show that CO2 attraction is mediated by receptors on the third antennal segment. To further confirm this, each co-receptor mutant was tested individually, and it was found that mutants that lack IR25a did not exhibit wild-type CO2 attraction, whereas Orco and IR8a mutants did. Mutant flies that lack Orco, IR8a and Gr63a also exhibit wild-type attraction to CO2, confirming that the only required co-receptor is IR25a. IR25a has previously been implicated in a wide range of behaviours, including temperature and humidity sensation. The temperature in the arena near the CO2 port was measured, and no change was found in temperature as a result of the stimulus. To eliminate the possibility of a humidity artifact, an IR40a mutant, which still exhibited attraction to CO2, was tested. In summary, these experiments show that CO2 attraction is mediated by a separate chemosensory pathway from that which governs aversion, and that CO2 attraction requires the IR25a co-receptor. IR25a is the most highly conserved olfactory receptor among insects. It is possible that other insect species that lack Gr63a26 but that still respond to CO2 use the same IR25a-dependent pathway. Unfortunately, the GAL4 driver for the IR25a promoter is expressed only in about half of the endogenous IR25a-expressing neurons, which makes imaging experiments that aim to identify which glomerulus is involved difficult at this time (van Breugel, 2018).

The finding that active flies are attracted to CO2 makes ethological sense, given that CO2 is generated by yeast-the preferred food of these flies. Why it might be that Drosophila avoid CO2 when in a low-activity state was considered. Flies do not exhibit this state-dependent reaction to ethanol and vinegar; perhaps the aversion to CO2 at low activity is an adaptation that minimizes encounters with parasites that seek CO2. Alternatively, the behaviour may help flies to avoid respiratory acidosis when near high concentrations of CO2 within the environment. Previous studies have suggested that CO2 serves as an aversive pheromone by which stressed flies signal others to flee a local environment. However, an alternative explanation is that agitated flies release CO2 not as a social signal but simply because it is present in their tracheal system owing to their process of discontinuous respiration. Further work on this state-dependent reaction to CO2 will require experiments that carefully consider the natural ethology of the flies (van Breugel, 2018).

Functional architecture of olfactory ionotropic glutamate receptors

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate chemical communication between neurons at synapses. A variant iGluR subfamily, the Ionotropic Receptors (IRs), was recently proposed to detect environmental volatile chemicals in olfactory cilia. This study elucidate how these peripheral chemosensors have evolved mechanistically from their iGluR ancestors. Using a Drosophila model, IRs were demonstrated act in combinations of up to three subunits, comprising individual odor-specific receptors and one or two broadly expressed coreceptors. Heteromeric IR complex formation is necessary and sufficient for trafficking to cilia and mediating odor-evoked electrophysiological responses in vivo and in vitro. IRs display heterogeneous ion conduction specificities related to their variable pore sequences, and divergent ligand-binding domains function in odor recognition and cilia localization. These results provide insights into the conserved and distinct architecture of these olfactory and synaptic ion channels and offer perspectives into the use of IRs as genetically encoded chemical sensors (Abuin, 2011).

'Chemosensory synapses' between the environment and sensory neurons have been proposed as novel models to characterize mechanisms of neuronal activation and regulation by external stimuli. The IRs provide an intriguing example of molecular homology between peripheral sensory and postsynaptic receptors, and suggested the comparison of the conserved and divergent properties of these olfactory receptors to their iGluR ancestors (Abuin, 2011).

Cross-species analyses have demonstrated that IR25a is the 'ancestral' IR, as orthologs of this gene are expressed in chemosensory neurons in insects, nematode worms, and mollusks (Croset, 2010). By contrast, IR8a is a recently evolved, insect-specific duplicate of IR25a, although it retains a similar domain organization and sequence identity to iGluRs (Croset, 2010). The chemosensory role of IR25a in the common protostome ancestor is unknown, but it is attractive to suggest that it initially retained function as a glutamate-sensing receptor in the distal dendritic membranes of peripheral sensory neurons, analogous to the role of iGluRs in postsynaptic membranes of interneurons. Subsequent expansion of the IR repertoire may have allowed specialization of IR8a and IR25a as coreceptors acting in conjunction with more divergent odor-specific IRs. The dedication of these relatively slowly evolving members of the IR repertoire as a structural core of heteromeric IR complexes may help maintain the central function of these receptors as ligand-gated cation channels (Abuin, 2011).

Analysis of IR8a suggests that one specific function of the coreceptors may be to link IR complexes to the cilia transport pathway through their intracellular cytoplasmic tail, similar to the role of this region in coupling iGluRs to the postsynaptic transport machinery. Conserved motifs for subcellular targeting are not apparent between iGluRs and IR8a or IR25a, perhaps reflecting the novel signals required to localize IRs to specialized sensory cilia membranes. The maintenance of LBDs in coreceptor IRs raises the possibility that these proteins still bind ligands. Mutational analysis of IR8a argues that glutamate is very unlikely to be recognized by its LBD and suggests that this domain serves in complex localization rather than peripheral ligand responses. Notably, LBD mutations in certain Kainate receptors also reduce cell-surface expression in cultured cells. While IR8a (and IR25a) may associate with unknown ligands important for trafficking, a model is favored in which the conformation of coreceptor LBDs contributes to a scaffold for correct assembly of an IR complex to ensure only functional heteromers reach sensory cilia (Abuin, 2011).

In contrast to IR8a and IR25a, evolution of odor-specific IRs, such as IR84a and IR75a, was accompanied by a significant reduction in the structural complexity, as these proteins lack an ATD and bear only short, and apparently dispensable, cytosolic C termini. Divergent LBDs and pore filters in these proteins appear to confer specificity of odor recognition and ion conduction properties of IR-receptor complexes, respectively. Traces of ancestral glutamate-binding mechanisms are detectable, however, as it was shown that a glutamate-conjugating arginine is conserved and essential in IR84a for recognition of its odor ligand, phenylacetaldehyde. Odor-specific IR sequences may provide a valuable source of natural (and functional) 'site-directed mutants' to understand how the ion conduction and other properties of these ligand-gated ion channels are specified at the molecular level (Abuin, 2011).

Reconstitution of olfactory responses using a combination of three distinct IRs (IR25a, IR76a, and IR76b) highlights a further level of sophistication in how these proteins assemble into functional odor-sensing complexes. While IR76a is very likely to define ligand-specificity, the precise contributions of IR25a and a second putative coreceptor, IR76b, have not yet been resolved. It is possible that IR76b, which is more closely related to odor-specific IRs than to IR25a or IR8a, recognizes an unknown chemical ligand, whose copresence with phenylethyl amine in an odor blend could lead to synergistic or diminished neuronal responsiveness. Further variations in IR complexes are apparent. For example, IR25a is likely to have IR76b-independent roles as a coreceptor for sacculus and aristal odor-specific IRs, as the latter receptor is not expressed in these structures (Benton, 2009). Moreover, the ammonia receptor in ac1 is independent of both IR8a and IR25a. Thus, while OR-expressing neurons in vertebrates and insects encode odor stimuli through the activity of singularly expressed odor-specific receptors, the IRs appear to function in 'combinatorial codes' within individual OSNs. These may define unique ligand sensitivities and signaling dynamics akin to the heteromer-specific properties of iGluRs in synaptic localization and signaling. In contrast to iGluRs, however, IRs do not appear to depend upon additional accessory proteins, such as TARPs, for cell surface expression or function (Abuin, 2011).

Olfactory receptor repertoires have long attracted the attention of molecular, structural, and evolutionary biologists interested in the outstanding problems of odor recognition specificity and functional adaptability of these rapidly evolving proteins. While ever-expanding numbers of OR genes are being identified in genome sequences, progress in understanding of the functional properties of the corresponding proteins has been relatively slow. Vertebrate ORs are notoriously difficult to express in experimentally amenable heterologous systems, although recent identification in mammals of accessory factors that enhance their expression and/or function have begun facilitating the matching of odors to receptors. More challengingly, their seven transmembrane domain organization has eluded crystallization, obliging experimental probing of the odor-binding site to be guided by bioinformatic and modeling approaches (Abuin, 2011).

In insects, in vivo analyses of ORs have assigned ligands to a large fraction of this repertoire. Similar to IRs, odor-specific ORs function with a common coreceptor OR83b, which has an essential role in cilia targeting in vivo. Detailed understanding of insect ORs has, however, been hampered by the lack of homology of these polytopic membrane proteins to known receptors. Although initially assumed to be GPCRs, more recent analyses suggest these receptors function at least in part as odor-gated ion channels (Abuin, 2011).

In the face of these challenges, it is proposed that this comprehensive functional analysis of the IRs now establishes these proteins as an attractive model olfactory receptor repertoire to determine how diverse molecular recognition and signaling properties have evolved and contribute to odor perception in vivo. The clear modular organization of the IRs offers the possibility to selectively manipulate the localization, ligand recognition, and signaling properties of these receptors. Perhaps most significantly, the amenability of the iGluR LBD to crystallographic analysis suggests that atomic-resolution visualization of odor/IR interactions will also be feasible, which would provide important insights into how olfactory receptors achieve their diverse ligand specificity (Abuin, 2011).

Finally, definition of the molecular constituents of functional IR complexes in heterologous cells lays the foundation for the use of these receptors as unique types of genetically encoded chemical sensors. Although the LBDs of iGluRs are well described and their potential as targets for directed modifications already demonstrated through the generation of a light-activated iGluR, this class of ion channel has been surprisingly underexploited as a tool to couple recognition of different types of chemicals with cellular physiological responses. The existence of many hundreds of divergent IRs of presumed distinct specificity reveals a natural exploitation of this ligand-gated ion channel for chemical sensing. The molecular properties of IRs uncovered in this study provides a basis for their rational modification to generate custom-designed chemoreceptors of desired specificity. Such sensors could offer invaluable tools as genetically encoded neuronal activators or inhibitors as well as have broad practical applications, for example, in environmental pollutant detection or clinical diagnosis (Abuin, 2011).

Humidity sensing in Drosophila

Environmental humidity influences the fitness and geographic distribution of all animals. Insects in particular use humidity cues to navigate the environment, and previous work suggests the existence of specific sensory mechanisms to detect favorable humidity ranges. Yet, the molecular and cellular basis of humidity sensing (hygrosensation) remains poorly understood. This study describes genes and neurons necessary for hygrosensation in the vinegar fly Drosophila melanogaster. It was found that members of the Drosophila genus display species-specific humidity preferences related to conditions in their native habitats. Using a simple behavioral assay, it was found that the ionotropic receptors IR40a, IR93a, and IR25a are all required for humidity preference in D. melanogaster. Yet, whereas IR40a is selectively required for hygrosensory responses, IR93a and IR25a mediate both humidity and temperature preference. Consistent with this, the expression of IR93a and IR25a includes thermosensory neurons of the arista. In contrast, IR40a is excluded from the arista but is expressed (and required) in specialized neurons innervating pore-less sensilla of the sacculus, a unique invagination of the third antennal segment. Indeed, calcium imaging showed that IR40a neurons directly respond to changes in humidity, and IR40a knockdown or IR93a mutation reduces their responses to stimuli. Taken together, these results suggest that the preference for a specific humidity range depends on specialized sacculus neurons, and that the processing of environmental humidity can happen largely in parallel to that of temperature (Enjin, 2016).

Due to their small size and low heat capacity, insects are at constant risk of desiccation. As a result, they have evolved uniquely sensitive receptor systems to sense and respond to changes in the amount of water vapor in the air. This study describes genes and neurons necessary for hygrosensory responses in Drosophila. The work identifies sacculus neurons innervating chambers I and II as essential players in the behavioral responses to environmental humidity. These neurons appear to co-express IR25a, IR93a, and IR40a but, whereas IR25a and IR93a are also required for thermal preference, IR40a is uniquely important for hygrosensory responses. Genetic labeling of IR40a-expressing neurons also allowed tracking of their projections to the brain and identification of a unique glomerular structure (the Arm) that responds to specific changes in external humidity, i.e., 'dry-air' stimuli (Enjin, 2016).

Work in other insects suggests that the neural response to dry air could be mediated by evaporative cooling (as in man-made evaporation detectors, or psychrometers). Yet, the poor thermal sensitivity of IR40a neurons targeting the Arm (and their unchanged responses to cooling in IR40aRNAi) seems to disfavor this model. Furthermore, sensilla responding to changes in humidity have been electrophysiologically characterized in a number of insects, and typically consist of a 'dry cell' and a 'moist cell' (i.e., activated by humid air) housed in the same sensillum together with a 'cold cell.' This study identifies a dry-cell type associated with a cold-responding one in the sacculus. This indicates that hygrosensilla may share a common organization across insect groups (Enjin, 2016).

These results reveal some of the cellular substrates and molecular transducers that allow flies to detect changes in humidity. Interestingly, some of the key molecules described seem to be shared between thermosensory and hygrosensory neurons, and yet thermal preference and humidity preference are mediated by independent cellular substrates. Hence, the two sensory systems can function largely in parallel in Drosophila, perhaps providing a mechanism to independently modulate the behavioral responses to each of these two key environmental parameters (Enjin, 2016).

Search PubMed for articles about Drosophila Ir25a

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

Ahn, J. E., Chen, Y. and Amrein, H. (2017). Molecular basis of fatty acid taste in Drosophila. Elife 6. PubMed ID: 29231818

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

Enjin, A., Zaharieva, E.E., Frank, D.D., Mansourian, S., Suh, G.S., Gallio, M. and Stensmyr, M.C. (2016). Humidity sensing in Drosophila. Curr Biol [Epub ahead of print]. PubMed ID: 27161501

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

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

Ni, L., Bronk, P., Chang, E. C., Lowell, A. M., Flam, J. O., Panzano, V. C., Theobald, D. L., Griffith, L. C. and Garrity, P. A. (2013). A gustatory receptor paralogue controls rapid warmth avoidance in Drosophila. Nature 500: 580-584. PubMed ID: 23925112

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

Sanchez-Alcaniz, J. A., Silbering, A. F., Croset, V., Zappia, G., Sivasubramaniam, A. K., Abuin, L., Sahai, S. Y., Munch, D., Steck, K., Auer, T. O., Cruchet, S., Neagu-Maier, G. L., Sprecher, S. G., Ribeiro, C., Yapici, N. and Benton, R. (2018). An expression atlas of variant ionotropic glutamate receptors identifies a molecular basis of carbonation sensing. Nat Commun 9(1): 4252. PubMed ID: 30315166

van Breugel, F., Huda, A. and Dickinson, M. H. (2018). Distinct activity-gated pathways mediate attraction and aversion to CO2 in Drosophila. Nature 564(7736):420-424. PubMed ID: 30464346

date revised: 1 January 2024

Home page: The Interactive Fly © 2011 Thomas Brody, Ph.D.