InteractiveFly: GeneBrief

inactive: Biological Overview | References

BIOLOGICAL OVERVIEW

Animals select their optimal environmental temperature, even when faced with alternatives that differ only slightly. This behavior is critical as small differences in temperature of only several degrees can have a profound effect on the survival and rate of development of poikilothermic animals, such as the fruit fly. This study demonstrates that Drosophila larvae choose their preferred temperature of 17.5°C over slightly cooler temperatures (14-16°C) through activation of chordotonal neurons. Mutations affecting a transient receptor potential (TRP) vanilloid channel, Inactive (Iav), which is expressed specifically in chordotonal neurons, eliminated the ability to choose 17.5°C over 14°C-16°C. The impairment in selecting 17.5°C resulted from absence of an avoidance response, which is normally mediated by an increase in turns at the lower temperatures. It is concluded that the decision to select the preferred over slightly cooler temperatures requires iav and is achieved by activating chordotonal neurons, which in turn induces repulsive behaviors, due to an increase in high angle turns (Kwon, 2010).

Animals are capable of discerning small differences above and below their preferred ambient temperature, and this ability is especially important for organisms, such as insects, whose internal body temperature rapidly equilibrates with the environmental temperature. However, the molecular and cellular bases by which animals survey their thermal landscapes and decide on how to respond behaviorally to different temperature alternatives are incompletely understood (Kwon, 2010).

A key class of temperature sensors is an evolutionarily conserved set of cation channels, referred to as transient receptor potential (TRP) channels. These proteins are global mediators of sensory input and thereby control a variety of animal behaviors. In Drosophila three TRP channels participate in the responses to warm or hot temperatures in the noxious range. It has been shown that Drosophila larvae are sensitive to small deviations from their preferred temperature of ~17.5°C-18°C, and TRPA1 is critical for enabling larvae to select these optimal temperatures over slightly warmer temperatures (~24°C) (Kwon, 2008). In addition, two TRPC channels, TRP and TRPL have been reported to be involved in choosing the preferred temperature over slightly cooler temperatures (Rosenzweig, 2008; Kwon, 2010 and references therein).

A comprehensive screen was conducted for Drosophila TRP channels that enable larvae to choose their optimal temperature of 17.5°C over mildly cooler temperatures (14°C-16°C). It was found that a TRP vanilloid channel, Iav, was required in chordotonal neurons for selecting 17.5°C over 14°C-16°C. Moreover, both Iav and the chordotonal neurons were required for selecting the optimal temperatures by inducing the larvae to increase turning angles, thereby avoiding the cooler temperatures (Kwon, 2010).

To systematically address the requirements for TRP channels for selecting 17.5°C over mildly cooler temperatures, mutations were examined affecting each of the 13 Drosophila TRP channels by performing two-way choice tests. Larvae were released at the center of plates between 14° and 17.5°C zones and they were allowed to choose between the two temperatures for 15 min. If all larvae migrated to the 17.5°C side, the preference index (P.I.) would be 1.0, while a complete preference for 14°C would result in a P.I. of -1.0. The P.I. would be 0 if there were an equal number of larvae on both sides of the test plate (Kwon, 2010).

Elimination of any of the four TRPA channels, TRPM, TRPP (AMO), TRPN (NOMPC) or TRPML did not reduce the preference for 17.5°C. Mutation of the gene encoding the third TRPC channel, TRPγ, caused a slight, but statistically insignificant decrease in the P.I.. As reported previously, disruption of trpl abolished the preference for 17.5°C (Rosenzweig, 2008). However, the temperature preferences of two strong trp alleles, trp^P343 and trp⁴⁷, were indistinguishable from wild-type larvae. Nevertheless, the previous finding that trp contributed to cool sensation was not definitive since thermotaxis was normal in larvae harboring the trp mutation in trans with a deficiency that removed the trp locus (Rosenzweig, 2008; Kwon, 2010).

Mutations that disrupted either of the TRPV genes, iav¹ or nanchung mutant nan^36a, eliminated the ability of the larvae to discriminate between 14° and 17.5°C. These results were surprising since mammalian TRPM and TRPA channels are known or implicated to function in the sensation of low temperatures, while TRPV channels participate in the responses to modestly warm temperatures, or to noxious heat (Venkatachalam, 2007; Kwon, 2010).

Since iav and nan mutants are sedentary, the impairments in thermotaxis could be due to the reduced movements rather than to defects in temperature sensation. Indeed, both iav¹ and nan^36a moved more slowly than wild-type larvae at either 14° or 17.5°C. Therefore, the effects on the P.I. were tested after changing the release zone on the temperature selection test plate. Instead of placing the larvae in the middle of the test plate, the larvae were introduced at the extreme ends of the 14°C or the 17.5°C sides. Wild-type larvae showed a preference for 17.5°C, regardless of the release site, although the P.I. was reduced slightly if they were placed at the end of the 14°C side, presumably due to slower migration at this temperature. However, the P.I. of nan^36a larvae was strongly dependent on their initial placement site on the test plate. When nan^36a larvae were introduced at the end of the 14° or 17.5°C sides, most of larvae stayed in the 14° or 17.5°C zones respectively. Consequently, it was not possible to discern whether or not nan was impaired in the selection of 17.5° over 14°C. Contrary to these results, the iav¹ larvae were randomly distributed on the test plates (P.I. values near 0), regardless of where they were placed on the test plates. These findings indicate that iav larvae were impaired in the discrimination of the optimal temperature over 14°C (Kwon, 2010).

To provide further evidence that iav was required for choosing 17.5° over 14°C, another iav allele was tested (iav³⁶²¹). In addition, both iav¹ and iav³⁶²¹ were tested in trans with a deficiency (Df) that spanned the iav locus. iav³⁶²¹, iav¹/Df and iav³⁶²¹/Df larvae all showed the same inability to select 17.5° over 14°C. Furthermore, introduction of a wild-type genomic transgene (P[iav⁺]) rescued the thermotaxis defect in the iav¹ mutant. The combination of these data demonstrated that iav was required for thermotaxis in the cool range (Kwon, 2010).

In addition to selecting 17.5° over 14°C, wild-type larvae chose 17.5°C over other cool temperatures, at least down to 12°C. Discrimination of temperatures lower than 12°C was difficult to assess due to diminishing larval migration at lower temperatures. Therefore, whether iav was required for selecting 17.5°C over 12° and 16°C was tested. It was found that the ability of iav¹ to choose 17.5° over 16°C was also eliminated, as well as the discrimination between two cool temperatures (14.5° vs 16°C). However, the mutant animals exhibited a normal preference for 17.5° over 12°C. As has been shown recently, iav¹ larvae displayed normal selection of 17.5°C over comfortably warm temperatures, such as 22° and 24°C (Kwon, 2008). These latter results were not surprising given the requirement for trpA1 for thermotactic behavior in this temperature range (Kwon, 2008). Thus, iav was required for larvae to sense the preferred temperature, 17.5°C, over moderately cool temperatures (14°C-16°C), but was dispensable for choosing 17.5°C over cooler (12°C) or comfortably warm temperatures (Kwon, 2010).

Several observations indicated that chordotonal neurons were the cells required for discriminating 17.5°C from slightly cooler temperatures (14°C-16°C), but not other temperatures. First, an iav-GAL4 line, which drove UAS-mCD8-GFP expression exclusively in chordotonal neurons, was generated, consistent with a previous report that expression of iav is specific to chordotonal neurons (Gong, 2004). To test whether the chordotonal neurons were critical for cool sensation, synaptic transmission was inhibited in these cells by combining the iav-GAL4 with UAS-TNT (tetanus toxin); this genotype prevented temperature discrimination between 14° and 17.5°C. However, there was no impairment on selection of 17.5°C over other temperatures such as 12° or 24°C. Expression of the inactive form of tetanus toxin had no effect on 17.5° versus 14°C selection. Second, the combination of UAS-iav and iav-GAL4 was sufficient to rescue the iav phenotype. Third, it was found that expression of UAS-iav under control of either of two additional chordotonal GAL4 lines (nan-GAL4 and pain-GAL4) rescued the deficit in cool temperature discrimination in iav¹ larvae. Fourth, mutations (ato^W and btv^BG01771) that impaired the normal development of chordotonal neurons caused a defect in discriminating 14° and 17.5°C. Reintroduction of UAS-iav using the only available terminal organ GAL4 (GH86-GAL4) did not rescue the iav¹ phenotype, although this result was not reliable since it was found that expression of the GH86-GAL4 alone impaired temperature selection. Nevertheless, the four lines of evidence presented in this study support the conclusion that the chordotonal neurons are critical for selection of the optimal over cool temperatures (Kwon, 2010).

There are at least two mechanisms that potentially underlie the iav-dependent temperature preference behavior. The chordotonal neurons and iav could be required for attraction to the preferred temperature (17.5°C-18°C) or for avoidance of modestly cool temperatures (14°C-16°C). To distinguish between these possibilities, attempts were made to stimulate iav-expressing neurons, independent of changes in temperatures, and examined whether this induced attractive or avoidance behavior. To activate iav-expressing cells, channelrhodopsin 2 (UAS-ChR2) (Schroll, 2006) was expressed under the control of the iav-GAL4. ChR2 is a blue light-activated cation channel, so stimulation with blue but not red light leads to depolarization of neurons. Wild-type larvae express rhodopsins (Sprecher, 2007), which are classical G-protein coupled receptors. Consequently, in the absence of ChR2, wild-type larvae display an aversive response to light. To eliminate the endogenous light response, the effects were tested of introducing the iav-GAL4 and UAS-ChR2 in a norpA^P24 background, which disrupts a phospholipase C critical for the larval photoresponse (Kwon, 2010).

To determine the behavioral consequences resulting from stimulating iav-expressing neurons with ChR2, the larvae were placed in the middle of a Petri dish, half of which was kept in the dark, and the other half was exposed to light. Wild-type larvae avoided blue light, since they express rhodopsins that absorb blue light, including Rh5 (Sprecher, 2007). However, norpA^P24 larvae did not show a preference for either the dark or light zones. In contrast, expression of UAS-ChR2 under the control of iav-GAL4 restored the ability of norpA^P24 larvae to move away from the light. This behavior required blue light, consistent with the spectral sensitivity of ChR2. If the norpA ^P24;iav-GAL4/+;UAS-ChR2/+ larvae were fed on food, which was free of all-trans-retinal, they were unable to discriminate between the dark and light sides. Indistinguishable results were obtained using the nan-GAL4 to direct expression of UAS-ChR2 in a norpA^P24 background. Since stimulation of iav- or nan-expressing cells with light induced avoidance responses, these data indicated that wild-type larvae preferred 17.5° over 14°C-16°C through an avoidance response that required chordotonal neurons (Kwon, 2010).

To explore further the behavioral basis for the iav-dependent avoidance of 14°C, whether larvae displayed increased turning at the lower temperature was tested. The turning behavior of individual larvae was tracked at 17.5° and 14°C. After establishing the initial movement trajectory, the angle of deviation (theta; turning angle) was determined from the initial trajectory over the 5.1 s interval between frames. It was found that wild-type larvae showed an increase in the average turning angle at 14°C compared with 17.5°C. The percentage of lower angle turns (theta = 0°-45°) decreased while the percentage of medium angle turns (45°-90°) increased at 14°C. However, the percentage of high angle turns (theta = 90°-180°) were similar at 14° and 17.5°C Moreover, the total number of turns (theta > 45°) nearly doubled (Kwon, 2010).

In contrast to wild-type, in iav¹ mutant larvae, the total number of turns (> 45°) and the average turning angles did not increase at 14°C. At 17.5°C, iav¹ larvae displayed average turning angles similar to wild-type larvae. This value did not increase at 14°C, and the percentages of low, medium and high angle turns were unchanged at the two temperatures. Even though iav¹ larvae moved slower than wild-type larvae, the vectoral distance between the starting and ending points was similar to that of wild type at 14°C, due to the lower turning angles. Thus, iav¹ larvae traveled as far as wild-type larvae and were able to survey as large a proportion of the thermal environment as the wild type. This is in contrast to nan^36a mutant larvae, which traveled half the vectoral distance of wild-type and iav¹ larvae due to a combination of slow movement and high turning at both 14°C and 17.5°C. Introduction of the wild-type iav⁺ transgene in iav¹ restored the wild-type increase in the number of turns and average turning angle at 14°C. Based on these analyses, it is suggested that iav is required for choosing the preferred temperature (17.5°C) over the cool temperature (14°C) through avoidance of 14°C, and this behavior is mediated by increases in the number of turns and turning angles at this lower temperature (Kwon, 2010).

A key goal in behavioral neurobiology is to define the cellular and molecular determinants that enable animals to decide between two or more alternative actions based on environmental cues. Thermotactic discrimination in Drosophila larvae represents a simple model to address the interplay between sensory input and choice selection. The current study defined the cellular and molecular requirements for selection of slightly cool over the optimal temperature, and demonstrated how the cells and TRP channels contribute to thermotactic behavior. Specifically, it was found that the chordotonal neurons functioned in the discrimination of 17.5°C and slightly cooler temperatures, and this required a TRPV channel, Iav. In support of a requirement for the chordotonal neurons, the effect due to loss of iav was reversed by introduction of the wild-type gene in chordotonal neurons. Moreover, expression of tetanus toxin in chordotonal neurons suppressed the ability to select 17.5 over 14°C, consistent with a requirement for synaptic transmission from the chordotonal neurons for cool temperature discrimination (Kwon, 2010).

It has been reported previously that the terminal organs are involved in selecting 18°C over 11°C. This study found that the chordotonal neurons and iav were not required for selecting the optimal temperature over temperatures cooler than 14°C. It is proposed that the chordotonal organ functions in the discrimination of 17.5°C versus slightly cooler temperatures (14°C-16°C), while the terminal organ functions in the selection of 17.5°C over 12°C and cooler temperatures (Kwon, 2010).

The finding that Iav was required for choosing the optimal temperatures over slightly cool temperatures underscores the broad evolutionary role for TRPV channels in temperature selection. However, the requirement for Iav is distinct from mammalian TRPVs, several of which function in the responses to warm or hot temperatures (Bandell, 2007; Venkatachalam, 2007). Whether the other Drosophila TRPV, Nan, also contributes to temperature selection is unclear, since the thermotaxis assay was complicated by the greater sedentary behavior than iav, and the abnormally high turning even at 17.5°C. As a result, the nan larvae traveled small distances and remained near the initial site in which they were placed. Nevertheless, as is the case for Iav, the Nan channel is also expressed in chordotonal neurons, and therefore could potentially function in cool sensation (Gong, 2004). However, at least one other TRP channel, Pain, is expressed in chordotonal neurons, but is not required for choosing 17.5°C over mildly cool temperatures (Kwon, 2010).

In addition to Iav, another TRP channel, TRPL, functions in cool sensation (Rosenzweig, 2008). As was previously reported for TRPL (Rosenzweig, 2008), expression of Iav in oocytes did not elicit a cool-activated current. Thus, it is not clear whether activation of either of these channels by thermal cool is direct or indirect. In contrast to iav and trpl, no requirement was detected for trp in temperature discrimination in the cool range, which differs from a previous report (Rosenzweig, 2008). However, a role for trp was less clear, given the lack of phenotype when the trp mutation was placed in trans with a deficiency, which uncovered the gene (Rosenzweig, 2008). Nevertheless, given that the previous and current studies focused on early and late stage larvae respectively, it cannot be excluded that there are developmental differences in requirements for the TRP channel for sensing mildly cool temperatures (Kwon, 2010).

Even though wild-type third instar larvae prefer 17.5°C over any other temperature, it was found that the P.I. increased significantly in proportion to the alternative temperature (~0.2, 12°C; ~0.35, 14°C; ~0.7, 24°C). It is suggested that these differences are only partially due to the slower movements at the lower temperatures. The variations in the average P.I. values may reflect differences in the molecules, mechanisms and cell types involved in discriminating the optimal temperature (17.5°C) from mildly cool (14°C-16°C), cool (12°C) and comfortably warm (22°C-24°C) temperatures. In support of this proposal, inhibition of neurotransmission in chordotonal neurons specifically impaired the ability to discriminate between the preferred temperature over 14°C, and did not reduce selection of 17.5° over either 12°C or 24°C. Furthermore, a Gq/phospholipase C/TRPA1 thermosensory signaling cascade participates in choosing the optimal temperature over other temperatures in the comfortable range (20°C-24°C) (Kwon, 2008), while Iav and TRPL are required for opting for 17.5°C over mildly cool temperatures (Kwon, 2010).

A critical question is how the thermotactic selection of the optimal over mildly cooler temperatures is accomplished via the chordotonal neurons and iav. The following three observations lead to proposal of a model. First, iav mutant animals retain the ability to select the optimal temperature over very cool and comfortably warm temperatures. These results argue against a requirement for iav for positive selection of 17.5°C only, since this optimal temperature is still selected versus some temperatures. Second, stimulation of iav-expressing neurons with channelrhodopsin induces an avoidance response to light. Third, the modestly cooler temperature increased the average turning angles and the total number of turns (theta > 45°) relative to wild type. Thus, it is concluded that iav-dependent decision to choose the optimal over slightly cooler temperatures is mediated by increased activity of chordotonal neurons, which stimulates an avoidance response by increasing the number and magnitude of turning angles (Kwon, 2010).

A TRPV channel in Drosophila motor neurons regulates presynaptic resting Ca(2+) levels, synapse growth, and synaptic transmission

Presynaptic resting Ca²⁺ influences synaptic vesicle (SV) release probability. This study reports that a TRPV channel, Inactive (Iav), maintains presynaptic resting [Ca²⁺] by promoting Ca²⁺ release from the endoplasmic reticulum in Drosophila motor neurons, and is required for both synapse development and neurotransmission. Iav activates the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin, which is essential for presynaptic microtubule stabilization at the neuromuscular junction. Thus, loss of Iav induces destabilization of presynaptic microtubules, resulting in diminished synaptic growth. Interestingly, expression of human TRPV1 in Iav-deficient motor neurons rescues these defects. The absence of Iav causes lower SV release probability and diminished synaptic transmission, whereas Iav overexpression elevates these synaptic parameters. Together, these findings indicate that Iav acts as a key regulator of synaptic development and function by influencing presynaptic resting [²⁺] (Sullivan, 2014).

Sound response mediated by the TRP channels NOMPC, NANCHUNG, and INACTIVE in chordotonal organs of Drosophila larvae

Mechanical stimuli, including tactile and sound signals, convey a variety of information important for animals to navigate the environment and avoid predators. Recent studies have revealed that Drosophila larvae can sense harsh or gentle touch with dendritic arborization (da) neurons in the body wall and can detect vibration with chordotonal organs (Cho). Whether they can also detect and respond to vibration or sound from their predators remains an open question. This study reports that larvae respond to sound of wasps and yellow jackets, as well as to pure tones of frequencies that are represented in such natural sounds, with startle and burrowing behaviors. The larval response to sound/vibration requires Cho neurons and, to a lesser extent, class IV da neurons. Calcium imaging and electrophysiological experiments reveal that Cho neurons, but not class IV da neurons, are excited by natural sounds or pure tones, with tuning curves and intensity dependence appropriate for the behavioral responses. Furthermore, this study implicates the transient receptor potential (TRP) channels NOMPC, NANCHUNG, and INACTIVE, but not the dmPIEZO channel, in the mechanotransduction and/or signal amplification for the detection of sound by the larval Cho neurons. These findings indicate that larval Cho, like their counterparts in the adult fly, use some of the same mechanotransduction channels to detect sound waves and mediate the sensation akin to hearing in Drosophila larvae, allowing them to respond to the appearance of predators or other environmental cues at a distance with behaviors crucial for survival (Zhang, 2013).

The ability to sense mechanical stimuli that indicate potential harm is important for survival. Drosophila larvae use their mechanosensory neurons to sense the mechanical pain caused by a predator attack. The da neurons on the body wall are capable of sensing gentle and harsh touch, allowing larvae to move away from harm. Their survival could be further enhanced if larvae could detect signals such as sound from predators at a distance. The results show that Drosophila larvae exhibit startle behavior in response to certain frequencies of sound, including the sound from predators such as wasps and yellow jackets. This startle behavior and ensuing escape or avoidance behavior may increase a larva's chance of survival. Interestingly, Drosophila larvae are highly sensitive to low-frequency sounds but not to high-frequency sounds, unlike some other insects that can detect high-frequency sounds including ultrasonic sounds. This diversity in hearing might reflect evolutionary adaptation to different predators for organisms ranging from insects to bats, and might entail interspecies differences at both structural and molecular levels (Zhang, 2013).

Although both Cho neurons and class IV da neurons are involved in the sound-triggered startle response, only Cho neurons are sensitive to sound. Class IV da neurons may have modulatory effects on the neural circuits activated by the Cho neuronal response to sound -- a likely scenario, considering that class IV da neurons mediate avoidance behaviors to several noxious stimuli. The startle response and avoidance of sound also may depend on this neural circuit for avoidance behaviors. Alternatively, class IV da neurons may contribute to the behavioral response through their involvement in peristalsis (Zhang, 2013).

Several TRP channels have been implicated in hearing and touch sensation in Drosophila, although the roles of these channels in mechanotransduction may differ in different sensory neurons. For example, NOMPC is critical for touch sensation but IAV and NAN are not, whereas IAV and NAN are important for adult hearing. With respect to larval Cho neurons, it appears that IAV and NAN are required for sound transduction, whereas NOMPC function is important, but not essential, for the detection of loud sound. A possible model is one in which NOMPC serves as one of the primary sensors for sound and enhances the movement of the Cho neuronal cilium to activate IAV and NAN, which may be able to sense loud sound on their own in the absence of NOMPC. An alternative model has been suggested for the adult Johnston organs, which may use IAV and NAN rather that NOMPC as the primary sensor (Zhang, 2013).

Given that the cytoplasmic calcium indicator G-CaMP5 might not be localized to the small structure within the tip of the cilium, the Ca2+ imaging method in these experiments might not be sufficiently sensitive to detect Ca2+ influx at the site of mechanotransduction. Thus, the absence of a Ca2+ signal in Cho neurons might be attributed to the lack of downstream amplification. dmPIEZO, one of the first mechanotransduction channels identified for mechanical nociception in Drosophila larvae, appears to have no involvement in hearing, suggesting that larvae make use of different channels for different modalities of mechanosensation (Zhang, 2013).

Recent microarray studies have identified hundreds of genes implicated in the hearing of adult flies. Many of these genes also have been implicated in other sensory modalities besides hearing. A major challenge is the difficulty of recording from a single neuron in the adult antenna. The larval Cho neurons are accessible to electrophysiological recording at single-cell resolution. Moreover, the entire structure of a Cho neuron can be imaged simultaneously in vivo. In conjunction with the extensive genetic resources available, larval Cho neurons lend themselves to mechanistic studies of mechanotransduction for hearing in Drosophila (Zhang, 2013).

Two interdependent TRPV channel subunits, Inactive and Nanchung, mediate hearing in Drosophila

Hearing in Drosophila depends on the transduction of antennal vibration into receptor potentials by ciliated sensory neurons in Johnston's organ, the antennal chordotonal organ. A protein in the vanilloid receptor subfamily (TRPV) channel subunit, Nanchung (NAN), is localized to the chordotonal cilia and required to generate sound-evoked potentials (Kim, 2003). This study shows that the only other Drosophila TRPV protein is mutated in the behavioral mutant inactive (iav). The IAV protein forms a hypotonically activated channel when expressed in cultured cells; in flies, it is specifically expressed in the chordotonal neurons, localized to their cilia and required for hearing. IAV and NAN are each undetectable in cilia of mutants lacking the other protein, indicating that they both contribute to a heteromultimeric transduction channel in vivo. A functional green fluorescence protein-IAV fusion protein shows that the channel is restricted to the proximal cilium, constraining models for channel activation (Gong, 2004).

Previously, it was shown that the Drosophila TRPV channel NAN is required for hearing (Kim, 2003). This study shows that the only other Drosophila TRPV channel protein is encoded by the iav gene. Like NAN, IAV is gated by hypotonic stress in vitro; the endogenous protein is specifically expressed in chordotonal neurons and localized to their cilia and is required for auditory transduction. Interestingly, localization of either NAN or IAV in the cilia is in each case dependent on the presence of the other protein, indicating that NAN-IAV interactions are required for channel stability and/or localization. Finally, it was shown that IAV is restricted to the proximal part of the cilia, suggesting that activating forces are transmitted to the IAV-NAN channels via the ciliary membrane or cytoskeleton (Gong, 2004).

The interdependence of IAV and NAN is consistent with the complete absence of transduction in nan and iav mutants but contrasts with the ability of each protein to promote a hypotonically activated current when individually expressed in cell culture. This suggests that either endogenous TRPV subunits in the cultured cells can heteromultimerize with the expressed TRPV channels, or that homomultimeric channels are more stable in cultured cells than in chordotonal neurons. These low-abundance channels may only be detectable if concentrated in the cilium; homomultimers may not be visible if they are excluded from the cilium (Gong, 2004).

These data are comparable with the functional interdependence of OSM-9 and OCR channel proteins in Caenorhabditis (Tobin, 2002). OSM-9, the nematode protein most similar to IAV, is expressed in diverse sensory neurons, interneurons, and rectal gland cells, whereas the four different OCR proteins are expressed in more restricted subsets of sensory neurons or in rectal gland cells. Coexpression of OSM-9 and OCR-2 in the ciliated ASH neuron is required for their localization to the cilium and for responses to aversive odorants, hyperosmotic stimuli, and touch. OCR-4, the nematode protein most similar to NAN, is expressed together with OSM-9 only in the mechanosensitive OLQ neurons. These cells, which, like the Drosophila chordotonal neurons, have a differentiated cilium and extended ciliary rootlet, could be the nematode version of chordotonal organs (Gong, 2004).

How is the NAN/IAV channel gated? TRPV channels can be activated by diverse physical factors including temperature and hypotonic stress, whereas OSM-9, the founding member of the family, was first identified by its requirement in the transduction of hyperosmotic stimuli and nose touch. However, the structural basis for TRPV channel gating is not yet known. Several TRPVs open in response to hypotonic stress or cell swelling, but this is not necessarily because of direct gating by membrane tension; TRPV4 channels in cell-attached patches could not be opened by applied pressure. Divergent evidence implicates either phosphorylation or arachidonic acid signaling in the indirect gating of mammalian TRPV4 by hypo-osmotic stimuli, but neither pathway is fast enough to account for the speed of acoustic transduction. The Drosophila Johnston's organ can transduce signals up to 500 Hz with millisecond latencies (Gong, 2004 and references therein).

A prevailing conceptual model for direct mechanogating, based on studies of vertebrate hair cells and C. elegans mutants defective in body touch, posits a transcellular complex in which the gated channel is anchored to both the cytoskeleton and to an extracellular link or matrix; relative movement of the intracellular and extracellular structures opens the channel. These elements are indeed present in chordotonal organs. The ciliary axoneme provides an extended cytoskeleton to which the TRPV channels could be anchored, whereas a specially shaped extracellular matrix, the dendritic cap, attaches to the distal tip of the cilium. In mutants lacking NOMPA, a ZP-domain cap protein, the cap is disorganized and detached from the cilia, and transduction is eliminated. However, the restricted localization of IAV in the proximal part of the cilium means that the IAV-NAN channel cannot interact directly with cap components. If NAN and IAV are mechanically gated, the forces that gate the IAV/NAN channel may be transmitted down the axoneme through the ciliary membrane or via other extracellular material in the scolopale space that encloses the cilium (Gong, 2004).

The involvement in transduction of a more distally located channel cannot be excluded. One candidate is NOMPC, the TRPN channel that mediates the mechanoreceptor current in mechanosensory bristles. Sound-evoked potentials, which reflect the aggregate activity of many individual chordotonal neurons, are reduced by approximately half in nompC null mutants, suggesting either an absolute requirement for NOMPC in some chordotonal neurons or a partial contribution to transduction in all of them. In contrast to the limited area of cap/cilium contact in chordotonal organs, the entire ciliary outer segment in a bristle neuron is ensheathed by the dendritic cap. Although NOMPC has never been definitively localized, its modest contribution to chordotonal transduction could reflect a NOMPC-cap interaction restricted to the distal cilium. Because nompC null mutants retain substantial sound-evoked potentials but iav and nan mutants eliminate them completely, NOMPC activity, even if present, is not required to activate the IAV/NAN channels. Conversely, the TRPV channels are not expressed in bristles or other external sensory organs, and thus are not the source of the residual, nonadapting, bristle mechanoreceptor current in nompC mutants (Gong, 2004).

Previously described phenotypes of iav¹ mutants include locomotor inactivity, courtship abnormalities, and altered responses to cocaine (Kaplan, 1977; O'Dell, 1987; O'Dell, 1993; O'Dell, 1994; McClung, 1999). In addition, reduced levels of octopamine and tyramine have been reported in iav (O'Dell, 1987; McClung, 1999), but these observations could not be repeated using more advanced analytical methods. Rearing iav mutants on tyramine-supplemented medium did not restore sound-evoked potentials (Gong, 2004).

Does the defect in chordotonal transduction underlie the full range of phenotypes seen in iav mutants? Both nan (Kim, 2003) and other deaf mutants also show sedentary behavior, as does the iav³⁶²¹ allele described in this study, and similarly, antennal amputation also causes sedentary behavior. These observations imply that chordotonal sensory input is needed for normal levels of locomotor activity. However, iav¹ mutants are less active than either nan or iav³⁶²¹ mutants, although all three mutants completely lack sound-evoked potentials. This suggests that another IAV function, undetectable by auditory recording, is retained in the iav³⁶²¹ and nan mutants but not in iav¹ perhaps at an extraciliary site in chordotonal neurons or elsewhere in the nervous system. Precedent for a nontransducing role for IAV may be found in the OCR-independent expression of OSM-9 in C. elegans AWC neurons, where OSM-9 is located in the cell body and required for olfactory adaptation (Tobin, 2002). The possibility cannot be ruled out that the iav¹ mutant chromosomes carry a second-site mutation that contributes independently to inactivity, because this phenotype is fully rescued by iav transgenes, but a linked enhancer of the iav phenotype remains a possibility. Targeting expression of iav⁺ specifically to chordotonal organs and additional testing of neurotransmitter levels and drug interactions in iav³⁶²¹, nan, and other deaf mutants and transgenic animals may help to clarify this issue (Gong, 2004).

The alterations in cocaine responses observed in iav¹ (McClung, 1999) and iav³⁶²¹ cannot yet be interpreted in terms of TRPV channel activity, because the iav⁺ transgene insertions that restore evoked auditory potentials and locomotor activity do not appear to rescue the cocaine response phenotypes seen in iav¹ or iav³⁶²¹ (Z. Gong, J. Young, and Hirsh, unpublished observations cited in Gong, 2003). A further indication that the cocaine responses are independent of auditory transduction comes from the observation that nan flies show normal cocaine responses as do flies made deaf by amputation of their antennae. The favored interpretation of these findings is that there is a secondary site of low level but functionally important IAV expression within the nervous system that is not restored by the regulatory elements included in the iav⁺ transgenes used in this study. In any case, these observations point to the possibility of a divergent role for the IAV TRPV channel that may be independent of NAN and independent of the role of IAV in auditory transduction (Gong, 2004).