Several members of the TRP (for transient receptor potential) family of ionchannels act as physiological temperature sensors in mammals, but it is not known whether the invertebrate TRP sub-families that are found in the fruitfly Drosophila and the roundworm Caenorhabditis elegans can be directly activated by temperature. The Drosophila orthologue of mammalian ANKTM1, a cold-activated ion channel in mammals, responds to a warming rather than a cooling stimulus. The thermosensing function of these channels is therefore evolutionarily conserved, and they show a surprising flexibility in their response to different temperature ranges. In mammals, four TRPVs (members of the vanilloid subfamily of TRP channels) are activated at distinct heat thresholds (33-52°C), whereas TRPM8 (of the melastatin subfamily) and ANKTM1 are activated at cold (17-25°C) temperatures. However, the molecular mechanisms that underlie thermal preference in Drosophila are not well understood (Viswanath, 2003).
To identify potential invertebrate temperature-activated ion channels, the sequences of predicted TRP channels from Drosophila and C. elegans were examined, focusing on orthologues of the mammalian thermosensitive TRPs. Orthologues were operationally defined as reciprocal best BLAST hits on a comparison of the two genomes. By this definition, mammalian TRPM8 and TRPV1-4 channels do not have invertebrate orthologues. Consistent with this, the C. elegans TRPV homologues, which have diverse sensory functions, are not directly activated by temperature. Mammalian ANKTM1 belongs to a branch of TRP channels that includes four Drosophila and two C. elegans predicted proteins. One of these proteins, Painless, is required for response to noxious thermal and mechanical stimuli in Drosophila larvae, although it is unclear whether this channel is a direct sensor (Tracey, 2003). Another of these relatives is a sequence orthologue of mouse ANKTM1 (mANK-TM1; GenBank accession number AY231177): this Drosophila channel has been termed dANKTM1 (Viswanath, 2003).
A full-length, 3.5-kilobase dANKTM1 complementary DNA was amplified from adult Drosophila RNA by using the polymerase chain reaction with reverse transcription; its behaviour as anion channel was analyzed. Cooling temperature steps did not elicit currents at 70 mV from oocytes expressing dANKTM1, but they did elicit strong inward currents in those that expressed mANKTM1 and human ANKTM1. However, transient currents were consistently activated in response to warming in oocytes expressing dANKTM1, with a threshold of 24-29°C. The heat-activated dANKTM1 current was outwardly rectifying and reversed near 30 mV, indicating that dANKTM1 is a relatively non-selective cation channel, as is mANKTM1 (Story, 2003). In calcium-imaging experiments, dANKTM1-transfected Chinese hamster ovary cells also responded to a warming stimulus, with an activation threshold of about 27°C (Viswanath, 2003).
This is the first characterization of an invertebrate temperature-activated ion channel. Given that Drosophila strongly prefers a temperature of 24°C, the activation threshold of dANKTM1 at about 24-29°C suggests that this ion channel might have a physiological role in heat sensing. Although mANKTM1 and dANKTM1 are sequence orthologues, they do not seem to be functional orthologs -- the former senses cooling, whereas the latter senses warming. The two proteins are 54% similar throughout their length, so it is not obvious which domains are crucial for the warm or cold response. But analysis of chimeric mouse and Drosophila ANKTM1 proteins should help in the mapping of temperature-activation domains of these TRP channels (Viswanath, 2003).
Antisera raised against dTRPA1 detected strong dTRPA1 protein expression in a small number of central brain neurons and in neuroendocrine cells of the corpus cardiacum. dTrpA1(RNAi) larvae had no detectable dTRPA1 expression in these regions, demonstrating that the antisera was specific for dTRPA1 and that the dTrpA1 dsRNA effectively reduced dTRPA1 protein expression. Specific dTRPA1 expression was also detected in two pairs of cells adjacent to the mouthhooks and in the developing gut. Interestingly, dTRPA1 expression was not detected in either multiple-dendritic neurons (implicated in temperature- dependent nociceptive responses) (Tracey, 2003) or chordotonal neurons, both of which show temperature- dependent calcium changes (L. Liu, 2003). The role of multiple-dendritic neurons was explored using md-Gal4:UAS-TeTxLC larvae. Consistent with no significant role of multiple-dendritic neurons in the thermotaxis assay, significant thermotaxis defects were not detected in md-Gal4:UAS-TeTxLC larvae. In addition, significant thermotaxis defects were not detected in atonal(RNAi) larvae, which lack chordotonal neurons. These data suggest that there may be differences in the neuronal circuitry required for thermotaxis in response to elevated temperature and withdrawal from a high-temperature nociceptive stimulus (Rosenzweig, 2005).
The role of dTRPA1-expressing cells in thermotaxis was further examined by expressing TeTxLC or the cell death-promoting gene Hid under control of putative dTrpA1 promoter sequences. dTrpA1-Gal4 drives GFP expression in most dTRPA1-expressing central brain neurons, but not in the other dTRPA1-expressing cell populations. dTrpA1-Gal4:UAS-Hid animals had reduced numbers of dTRPA1-expressing brain neurons (e.g., only one of the three dTPRA1-expressing cells was present), while the corpus caridiaca appeared unaffected. Consistent with the participation of dTrpA1-Gal4-expressing cells in thermosensory behavior, dTrpA1-Gal4:UAS-Hid as well as dTrpA1-Gal4:UAS-TeTxLC third instar larvae were partially, but significantly (p < 0.005), compromised in thermotactic behavior (Rosenzweig, 2005).
Interestingly, both dTrpA1-Gal4:UAS-TeTxLC and dTrpA1-Gal4:UAS-Hid larvae exhibit normal withdrawal from a high-temperature nociceptive stimulus, indicating that these animals are not defective for all thermosensory responses. Similar effects were also obtained using a second, independent dTrpA1-Gal4 insertion. These data further support the notion that there may be differences in the neuronal circuitry required for thermotaxis and high-temperature nociception (Rosenzweig, 2005).
Tests were performed to see how specific the behavioral defect of dTrpA1(RNAi) animals was for thermotaxis. (1) It was determined that the motility indices (MIs) of wild-type and dTrpA1(RNAi) larvae in the thermal gradient were indistinguishable. (The MI represents the fraction of larvae no longer in the release zone at a given time point.) Late-first instar/early-second instar wild-type larvae had MIs of 0.56 and 0.64 at 2 and 5 min after release, while dTrpA1(RNAi) larvae had MIs of 0.60 and 0.69 at these time points. Thus dTrpA1(RNAi) animals are specifically defective in thermotaxis rather than in their ability to migrate at elevated temperature. (2) Larval chemotaxis in response to an olfactory repellent (n-octyl acetate) was examined; dTrpA1(RNAi) larvae were found to respond similarly to wild type, indicating that dTrpA1(RNAi) larvae are not defective for all avoidance behavior. Finally, responsiveness of dTrpA1(RNAi) larvae to higher temperatures (55°C) was examined. Because larvae rapidly die when exposed to gradients of higher temperature, larvae were tested by using a temperature-dependent nociceptive assay (Tracey, 2003). Crawling third instar larvae rapidly and dramatically curl when touched with a hot (55°C) probe but not when touched with a 25°C or 36°C probe. dTrpA1(RNAi) larvae respond indistinguishably from wild-type larvae in this assay (Rosenzweig, 2005).
As a negative control for the assay, larvae expressing tetanus toxin light chain (TeTxLC), an inhibitor of synaptic vesicle release, were examined under md-Gal4 control (md-Gal4 is expressed in multiple-dendritic neurons and ~100 CNS neurons) (Tracey, 2003). As previously reported (Tracey, 2003), md-Gal4:UAS-TeTxLC larvae were defective in response to contact with the hot probe. The ability of dTrpA1(RNAi) larvae to chemotax and respond to other thermal stimuli suggests a specific requirement for dTrpA1 in thermotaxis (Rosenzweig, 2005).
Animals from flies to humans are able to distinguish subtle gradations in temperature and show strong temperature preferences. Animals move to environments of optimal temperature and some manipulate the temperature of their surroundings, as humans do using clothing and shelter. Despite the ubiquitous influence of environmental temperature on animal behaviour, the neural circuits and strategies through which animals select a preferred temperature remain largely unknown. This study identified small set of warmth-activated anterior cell (AC) neurons located in the Drosophila brain, the function of which is critical for preferred temperature selection. AC neuron activation occurs just above the fly's preferred temperature and depends on dTrpA1, an ion channel that functions as a molecular sensor of warmth. Flies that selectively express dTrpA1 in the AC neurons select normal temperatures, whereas flies in which dTrpA1 function is reduced or eliminated choose warmer temperatures. This internal warmth-sensing pathway promotes avoidance of slightly elevated temperatures and acts together with a distinct pathway for cold avoidance to set the fly's preferred temperature. Thus, flies select a preferred temperature by using a thermal sensing pathway tuned to trigger avoidance of temperatures that deviate even slightly from the preferred temperature. This provides a potentially general strategy for robustly selecting a narrow temperature range optimal for survival (Hamada, 2008).
Although the physiology of all cells is affected by temperature, the expression of temperature-activated members of the transient receptor potential (TRP) family (thermoTRPs) can make cell excitability highly temperature-responsive (Dhaka, 2006). ThermoTRPs are cation channels with highly temperature-dependent conductances that participate in thermosensation from insects to humans. The Drosophila TRP channel dTrpA1 promotes larval heat avoidance (Rosenzweig, 2005) and can be activated by warming in ooctyes (Viswanath, 2003). This study addressed whether dTrpA1 contributes to the selection of a preferred temperature in the adult fly. When allowed to distribute along a thermal gradient for 30 min, wild-type D. melanogaster adults prefer ~25°C, their optimal growth temperature. Compared to wild-type controls, dTrpA1 loss-of-function mutant animals showed increased accumulation in the warmest (28-32°C) regions of the gradient, but not in the coolest (18-22°C) regions. A dTrpA1 genomic minigene rescued the phenotype. Animals heterozygous for dTrpA1 loss-of-function mutations also preferred slightly elevated temperatures. Thus, dTrpA1 function is important for determining thermal preference and specifically contributes to avoidance of warm regions (Hamada, 2008).
If dTrpA1 was involved in thermotransduction, it should regulate the warmth responsiveness of thermosensors. As the identity of the adult Drosophila thermosensors was unknown, dTrpA1 protein expression was examined (using anti-dTrpA1 antisera). dTrpA1 expression was detected in three sets of previously uncharacterized cells in the brain: lateral cell (LC), ventral cell (VC) and AC neurons. dTrpA1 was also detected in the proboscis, but ablation studies detected no contribution of the proboscis to warmth avoidance. To focus on the neurons that contribute to thermal preference, where the rescuing dTrpA1 minigene restored dTrpA1 expression was examined; dTrpA1 expression was restored specifically within AC neurons, but not LC or VC neurons. This suggested that dTrpA1 expression in AC neurons (two pairs of neurons at the brain's anterior) sufficed to restore thermal preference and that AC neurons might act as thermosensors (Hamada, 2008).
Temperature responsiveness of AC neurons was examined using the fluorescent calcium indicator G-CaMP. When exposed to increasing temperature, AC neurons showed robust increases in G-CaMP fluorescence, reflecting warmth-responsive increases in intracellular calcium. Ten out of the 27 AC neurons imaged had fluorescence increases between 4% and 39%, with a mean increase over baseline among these cells of 15%. The average temperature at which fluorescence increases were initially observed was 24.9°C, compatible with AC activation as temperatures rise above preferred. In contrast, none of the 21 dTrpA1 mutant AC neurons imaged had fluorescence increases. As a control that mutant AC neurons remained physiologically active, it was confirmed that they showed robust responses on potassium chloride addition. Notably, AC responses did not depend on an intact periphery, since all G-CaMP studies were performed using isolated brains from which peripheral tissues had been removed. These observations identify AC neurons as warmth-activated, dTrpA1-dependent thermosensors (Hamada, 2008).
AC neurons project towards several brain regions, including the antennal lobe. The antennal lobe is implicated in cockroach thermosensation, but has been studied exclusively for olfaction in Drosophila. So far, 11 of the ~50 antennal lobe glomeruli remain unassociated with identified olfactory receptors. AC neurites elaborated within two such unassociated glomeruli, VL2a and VL2p. Thus the Drosophila antennal lobe contains both thermosensory and olfactory neuron processes. VL2a is also innervated by Fruitless-expressing neurons implicated in pheromone transduction, suggesting that even individual glomeruli receive multi-modal sensory information. AC processes also branched within the suboesophageal ganglion and superior lateral protocerebrum, although these target regions are less defined than in the antennal lobe. These regions have been previously implicated in processing other types of sensory input (Hamada, 2008).
As dTrpA1 expression in AC neurons seemed sufficient to restore normal thermal preference, whether such expression was also necessary was also examined. dTrpA1 was knocked down selectively in AC neurons using tissue-specific RNA interference targeting dTrpA1 controlled by dTrpA1SH-GAL4, a promoter expressed in AC but not LC or VC neurons. Consistent with the importance of dTrpA1 expression in AC neurons in thermal preference, AC knockdown increased the fraction of animals present in the 28-32°C region compared to controls. Similar results were obtained when dTrpA1 expression was knocked down using a broad neuronal promoter (Appl-GAL4). All knockdowns were assessed by dTrpA1 immunohistochemistry. dTrpA1 knockdown with the general cholinergic neuron promoter Cha(7.4)-GAL4 eliminated detectable dTrpA1 expression in AC (and LC and VC ) neurons, decreasing warmth avoidance. In contrast, dTrpA1 RNAi expressed using Cha(1.2)-GAL4 -- which is expressed in many brain cholinergic neurons but not AC neurons -- did not disrupt warmth avoidance. Taken together, these data suggest that dTrpA1 expression in AC (but not LC or VC) neurons is both necessary and sufficient for normal thermal preference behaviour. Whether LC and VC neurons participate in other warmth-activated responses is unknown (Hamada, 2008).
The identification of an internal sensor controlling temperature preference conflicts with the established view that Drosophila sense moderate warming using thermosensors in the third antennal segment. The effects were tested of surgically removing either one third antennal segment and arista (unilateral ablation) or both (bilateral ablation). Both unilateral and bilateral ablation increased the fraction of animals in cool (18-22°C), but not warm (28-32°C), regions. Thus these tissues were dispensable for warmth avoidance, but essential for cool avoidance. When dTrpA1 mutants were subjected to bilateral ablation, they accumulated in both cool and warm regions: the fraction between 18-22°C did not differ from wild-type ablation animals; the fraction between 28-32°C did not differ from non-ablated dTrpA1 mutants. Thus dTrpA1-expressing cells and antennal cells function additively to set preferred temperature, promoting avoidance of elevated and reduced temperatures, respectively (Hamada, 2008).
These data are consistent with warmth activation of dTrpA1 serving as the molecular basis of warmth sensing by AC neurons. As thermal activation of mammalian TRPA1 proteins is controversial, whether dTrpA1 could act as a molecular sensor of warming in the fly was tested. Indeed, misexpression of dTrpA1 throughout the fly nervous system (using C155-GAL4) caused a dramatic phenotype not observed in controls: heating these flies to 35°C for 60 s caused incapacitation, an effect reversed on return to 23°C. Similar effects were observed using electrophysiology, with moderate warming (above ~25°C) triggering a barrage of excitatory junction potentials at the neuromuscular junction. These data strongly support dTrpA1 acting as a molecular sensor of warming. The ability of dTrpA1 mis-expression to confer warmth activation also suggests that dTrpA1 can be used as a genetically encoded tool for cell-specific, inducible neuronal activation. dTrpA1 might be particularly useful in tissues such as the fly brain where thermal stimulation is easier to deliver than the chemical or optical stimulation that controls other tools for modulating neuronal activity (Hamada, 2008).
To test whether warmth activation is a property of other insect TrpA1s, the malaria mosquito Anopheles gambiae TrpA1 (agTrpA1) was examined. dTrpA1 is warmth-activated when expressed in Xenopus laevis oocytes. agTrpA1 also showed robust warmth activation. These currents were specific, they were not observed in uninjected oocytes and were inhibited by ruthenium red (which antagonizes other TRPs). Similar to mammalian thermoTRPs, both dTrpA1 and agTrpA1 showed outward rectification. Closely related TrpA1s are present in the flour beetle Tribolium castaneum and in disease vectors such as Pediculus humanus corporis (body lice), Culex pipiens (common house mosquito) and Aedes aegypti (yellow and dengue fever mosquito) which use warmth-sensing for host location and habitat selection. Such insect TrpA1s constitute potential targets for disrupting thermal preference and other thermosensory behaviours in agricultural pests and disease vectors (Hamada, 2008).
Environmental temperature affects the physiology of all animals. Increasing temperatures associated with climate change are linked to poleward redistributions of hundreds of species including insects, fish, birds and mammals, AC neurons are internal. As a ~1 mg fly is readily penetrated by ambient temperature variations, such an internal sensor should monitor environmental temperature effectively. dTrpA1 activation seems to be critical for AC neuron activation, suggesting that dTrpA1 threshold and expression changes could modulate thermal preferences. More speculatively, changes in insect TrpA1 function and expression could facilitate movements into novel environments or development of novel behaviours such as host seeking (Hamada, 2008).
Although effects of environmental temperature on behaviour are ubiquitous, the mechanisms animals use to seek out optimal temperatures are largely unknown. AC neurons become active as temperatures rise above the preferred temperature, suggesting that they may function as 'discomfort' receptors that, together with putative antennal cool receptors (similar to those described in other insect antennae), repel the fly from all but the most optimal temperatures. Notably, mice lacking the cool-activated channel TRPM8 prefer abnormally cool temperatures, whereas mice lacking heat-activated TRPV4 prefer warmer temperatures, indicating that similar strategies may be used in mammals (Hamada, 2
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date revised: 20 February 2005
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