The Interactive Fly

Genes involved in tissue and organ development

Antenna, auditory apparatus, and sound sensation



Schematic view of the antenna and Johnston's Organ
Cellular mechanisms in the development of the arista
Auditory apparatus of Drosophila
Distinct sensory representations of wind and near-field sound in the Drosophila brain
Functional maps of mechanosensory features in the Drosophila brain
Fast intensity adaptation enhances the encoding of sound in Drosophila
NompC TRP channel is essential for Drosophila sound receptor function
Prestin is an anion transporter dispensable for mechanical feedback amplification in Drosophila hearing
Flying Drosophila stabilize their vision-based velocity controller by sensing wind with their antennae
Engrailed alters the specificity of synaptic connections of Drosophila auditory neurons with the giant fiber
Cell-type-specific roles of Na+/K+ ATPase subunits in Drosophila auditory mechanosensation
Diverse roles of axonemal dyneins in Drosophila auditory neuron function and mechanical amplification in hearing
Ciliary phosphoinositide regulates ciliary protein trafficking in Drosophila
The organization of projection neurons and local neurons of the primary auditory center in the fruit fly Drosophila melanogaster
Shaking B mediates synaptic coupling between auditory sensory neurons and the giant fiber of Drosophila melanogaster
Auditory efferent system modulates mosquito hearing
A mechanosensory circuit that mixes opponent channels to produce selectivity for complex stimulus features
Active mechanisms of vibration encoding and frequency filtering in central mechanosensory neurons


A set of structural features defines the cis-regulatory modules of antenna-expressed genes in Drosophila melanogaster
Antennal mechanosensory neurons mediate wing motor reflexes in flying Drosophila
Humidity sensing in Drosophila
Antennae sense heat stress to inhibit mating and promote escaping in Drosophila females
Odorant receptors and odor sensation
Genes involved in antennal differentiation



Cellular mechanisms in the development of the arista

Epidermal cells of Drosophila form a variety of polarized structures during their differentiation. These polarized structures include epidermal hairs, the shafts of sensory bristles, larval denticles and the arista laterals. The arista is the terminal segment of the antenna and consists of a central core and a series of lateral extensions. The development of the arista is a complex process that involves coordinated cell shape changes, elongation of the central core, apoptosis, nuclear migration, the formation of polyploid cells and the outgrowth of the laterals. This developmental program is highly conserved in the development of the arista in the housefly (Musca domestica). Altering arista cell number in Drosophila by stimulating or inhibiting apoptosis results in an altered number of laterals. Interestingly, the increased number of laterals that result from the inhibition of apoptosis in Drosophila results in an arista whose morphology is reminiscent of the Musca arista. Both the actin and microtubule cytoskeletons have been shown to have important functions in the cellular morphogenesis of hairs and bristles. This is also the case for the formation of the arista laterals, arguing that the actin and microtubule cytoskeletons have similar functions in the morphogenesis of all of these cell types. It is concluded that the arista laterals are a valuable complementary cell type system for studying the morphogenesis of polarized cellular extensions in Drosophila (He, 2001).

The adult Drosophila arista consists of a central core that is typically around 300 mm long and 10-20 mm in diameter. Laterals extend both anteriorly and posteriorly off of the central core. There are 3-4 long laterals on the anterior side and 5-7 on the posterior side of a typical Drosophila antenna. The length of the long laterals is approximately 140 mm. In addition to the long laterals, 5-7 smaller laterals (about 20-30 mm long) are found on the dorsal side of the central core shaft. A comparative study was carried out of arista development in the larger dipteran Musca domestica . The adult housefly arista differs in several respects from the Drosophila arista. The house fly arista (over 650 mm) is approximately twice as long and the central core is shaped somewhat differently being much wider at its proximal end (diameter about 80 mm). The Musca arista contains a larger number of both long and short laterals extending off of the central core and large laterals are absent near the distal end of the arista (He, 2001).

The arista are composed of a series of long thin laterals. This seems likely to be of functional importance. Evidence suggests that the arista functions in the detection of sound. It is thought that the arista is deflected by sound vibrations and this deflection results in the movement of the third antennal segment and this is detected by sensory neurons in the second antennal segment. There are also six neurons present in the central core and these send processes distally through the hemolymph-filled lumen of the thin central core. Based on morphology it has been suggested that three of these function as thermoreceptors. For sound and temperature sensing functions, the long thin morphology of the arista seems appropriate, although further research will be necessary to obtain rigorous evidence on this point (He, 2001 and references therein).

The dramatic morphology of the arista is likely to have put constraints on the developmental mechanisms involved in its morphogenesis. The development of the epidermal arista involves a number of different types of cellular processes. The shaping of the central core appears to take place in two steps: extension and thinning. The arista is first seen as an outpocketing of cells from the third antennal segment. This outpocketing elongates over about a 12-h period to reach its maximal length. No experimental evidence has been seen for cell division in the elongating arista suggesting that cell number is constant during this process. The elongation results in a greatly reduced number of cells along the circumference at all locations along the proximal distal axis of the central core. Hence, arista elongation appears to be an example of convergent extension. The elongation of the arista differs from the eversion of the appendages such as the leg and wing in that it happens in the pupae and not the prepupae (He, 2001).

The elongation of the arista laterals starts after the central core reaches its final length. At this time nuclei start to disappear from the distal half of the arista. From time-lapse observations it is argued that this is due in large part (or entirely) to the proximal migration of nuclei. During this time period the cells become elongated at their apical surface and the extent of nuclear movement suggests that the elongation might be more extreme basally. The migration of the nuclei allows the central core of the arista to become much thinner and it is suggested that this is important for the physiological function of the arista. Consistent with this hypothesis nuclear movement is inhibited by the injection of microtubule antagonists, which results in a thickened central core. Evidence for nuclear migration is also seen during the development of the house fly arista. Starting shortly before the beginning of lateral outgrowth, apoptosis is seen in the central core. This results in a decrease in the number of cells in the arista and the data argue that this is important for arista morphogenesis. Cell number was manipulated by genetic means and it was found that increasing cell number (by decreasing apoptosis) results in the formation of ectopic laterals, while decreasing cell number (presumably by increasing apoptosis) results in a loss of laterals. How cell number alters the cellular decision to make or not make a lateral is unclear, but it is likely that there is an intercellular signaling system that allows cells to assess arista number or whether their neighbors have made a lateral. This system appears to be one that evolution has used to select for altered arista morphology. The house fly arista central core is thicker than its Drosophila homolog and it is decorated with a large number of short laterals that are reminiscent of those seen when apoptosis is inhibited in Drosophila. It is not clear why dipterans have evolved a developmental program that produces extra arista cells that are lost due to apoptosis. One possibility is that it is a consequence of the very long and thin morphology of the arista. Perhaps the extra cells are needed to allow convergent extension to produce an arista of sufficient length. The subsequent apoptosis, nuclear migration and proximal/distal elongation of the remaining cells could all function to allow the central core to achieve its long thin shape (He, 2001).

The development of the arista and in particular the laterals involves dramatic shape changes in cells. Not surprisingly the developing laterals stain very strongly for F-actin and also contain microtubules. The actin and microtubule cytoskeletons appear to have overlapping but distinct functions in the development of the laterals. Antagonism of either cytoskeleton by injecting inhibitors into pupae results in delayed initiation of lateral outgrowth, slowed lateral elongation and split laterals. These effects are similar to the effects that the same inhibitors have on the morphogenesis of hairs and bristles, which are other examples of Drosophila epidermal cells that form highly polarized outgrowths. Some differences were seen in the effects of actin versus microtubule inhibitors. The actin cytoskeleton antagonists are more potent at delaying lateral initiation and in causing split laterals. Microtubule antagonists are relatively more potent at inhibiting lateral extension and the thinning of the central core. Several of these observations are reminiscent of observations made previously on bristles and hairs. Inhibitors of actin polymerization are potent at causing split bristles and hairs, while this is a minor effect of microtubule cytoskeleton antagonists. Similarly, actin polymerization inhibitors are potent at delaying prehair and prebristle initiation, while microtubule antagonists are not. Finally, microtubule antagonists are particularly potent at inhibiting the elongation of bristles. These similarities in the effects of cytoskeleton inhibitors suggest parallel functions for the actin and microtubule cytoskeletons in the morphogenesis of all three of these polarized cells. The strikingly similar arrangement of actin bundles and microtubules in bristles and laterals also points to equivalent functions for the cytoskeleton in the morphogenesis of these cell types. These commonalties are also supported by analogous phenotypes produced in all of these cell types by mutations in some genes (e.g. tricornered, which codes for a protein kinase) (He, 2001).

Auditory apparatus of Drosophila

The Drosophila auditory system is presented as a powerful new genetic model system for understanding the molecular aspects of development and physiology of hearing organs. The fly's ear resides in the antenna, with Johnston's organ serving as the mechanoreceptor. New approaches using electrophysiology and laser vibrometry have provided useful tools to apply to the study of mutations that disrupt hearing. The fundamental developmental processes that generate the peripheral nervous system are fairly well understood, although specific variations of these processes for chordotonal organs (CHO) and especially for Johnston's organ require more scrutiny. In contrast, even the fundamental physiologic workings of mechanosensitive systems are still poorly understood, but rapid recent progress is beginning to shed light. The identification and analysis of mutations that affect auditory function are summarized here, along with a discussion of how analysis of the role of the Drosophila auditory system will further an understanding of both insect and vertebrate hearing (Caldwell, 2002).

The antenna of Drosophila is an asymmetric, flagellar structure composed of three segments [a1 (antennal segment) or scape, a2 or pedicelus, and a3 or funiculus] and a feathery arista extending from the distal-most segment. The arista resonates in the presence of the species-specific courtship song and twists a3 relative to a2. The feather-like arista (antennal segments 4, 5, and 6) extends anterolaterally from a3 and slightly downward. The arista is innervated with three sensilla; these are not physiologically involved in hearing, but rather likely in thermosensation. For hearing, the arista is a component of the mechanical operation of the antenna (Caldwell, 2002).

The only known acoustic stimulus to which Drosophila responds is the courtship song, produced by the courting male. The D. melanogaster species-specific 'love song' is composed of pulse and sine components. The sine song is thought to 'woo' females prior to courtship and is, on average, a 160-Hz sinusoidal sound wave, although there is considerable variation between males. The pulse song is composed of trains of pulses with characteristic 30- to 45-ms interpulse intervals between 5- to 10-ms pulses. The interpulse interval oscillates rhythmically with a period of 50-65 s. Interestingly, this has been described as the most relevant song feature in increasing female receptivity and stimulating both partners in the courtship to expedite copulation (Caldwell, 2002).

Sound intensity is expressed in a simple relationship of the product of pressure and particle velocity. The male stays less than 5 mm away from the female during courtship, less than the wavelength of the sound being produced. At this distance, the acoustic energy is almost entirely in particle velocity rather than in pressure. When the male is positioned within one wavelength of the female, energy dissipation is low and the near-field amplitude is 80-95 dB. In Drosophila, therefore, it is advantageous to use a displacement receptor like the arista rather than a pressure receptor. Interestingly, the male also detects, and responds to the courtship song. This feature has been exploited in the design of a mutagenesis screen for deaf mutants. The roles of courtship stimuli for male courtship are poorly understood; they may be auto-stimulatory or they may be important in competitive situations (Caldwell, 2002).

The arista and a3 together are the fly's sound receivers that oscillate sympathetically when stimulated acoustically. Laser vibrometry has been performed to analyze the biomechanics of the Drosophila antenna. Measurements of oscillations taken from different locations along the arista and a3 indicate that the arista and a3 vibrate together as a stiff unit and rotate about the longitudinal axis of a3; however, a2 and the head capsule remain stationary. Thus, the major articulation is at the a2/3 joint. This articulation causes stretching of the sound transducer, the Johnston's Organ (JO). The JO is a sense organ that mediates hearing in a2 and is a cluster of about 200 scolopidia, which are the functional units of chordotonal organs (CHOs) (Caldwell, 2002).

A useful feature of the antenna, as an asymmetric sound receiver that represents a moderately damped simple harmonic oscillator when presented with sound, is that the resonance frequency of the arista and a3 increases with the sound intensity, permitting the fly to tune a large dynamic range of sound: acute hearing at low intensities and damped sensitivity at high intensities (Göpfert, 2002). This broad tuning ensures that antennal vibrations are detectable both at frequencies below antennal resonance (when elicited at close range) and when the distance of the courting male from the female's receiver increases (Göpfert, 2002). The arista and a3 rotate relative to a2 and vibrate visibly when presented with acoustic stimulation. This vibration, in turn, stimulates the mechanosensitive scolopidia of JO in a2. Indeed, the JO scolopidia are arrayed in such a way as to easily detect acoustic vibrations in the more distal antennal segments and immobilization or loss of antennal segments drastically reduces sound-evoked potentials (Caldwell, 2002).

The nonlinearity represented by the intensity-dependent frequency response of Drosophila antennal vibrations has minimal effects on tuning sharpness and sensitivity (Göpfert, 2002). Therefore, this nonlinearity is in the stiffness of the resonator. This is in contrast to that found in the vertebrate ear (which underlies the cochlear amplifier) and the mosquito antenna where the nonlinearity is introduced by negative damping. Whether the Drosophila nonlinearity in the stiffness is mediated by active processes, such as power generated by the sensory organs, or by passive processes remains to be tested (Caldwell, 2002).

The mechanoreceptive auditory organ of the Drosophila antenna is a large CHO in a2, comprising 150-200 sensory units. The homologous CHO in the mosquito had been proposed as the auditory organ by Christopher Johnston (1855); hence, this CHO is referred to as Johnston's organ (JO). Chordotonal sensory units, called scolopidia, are classified as type I sense organs: they have monodendritic, ciliated neurons associated with accessory cells. By comparison, type II sense organs are multidendritic, nonciliated neurons with no accessory cells. CHOs are not associated with external structures, in contrast to other type I sense organs, such as bristle organs and campaniform sensilla that together are called external sensory (es) organs. Rather, CHOs act as stretch receptors with attachments at two points of cuticle, often at the joints of appendages. In adult flies, each scolopidium has two or three bipolar neurons and several accessory cells. The precise composition of accessory cells in JO has not yet been well defined, but likely includes cap cells, scolopale cells, and ligament cells similar to those in larval CHOs. Cap cells are responsible for apical attachments of scolopidia, although there may also be accessory epidermal cells that secrete specialized cuticular elements for attachment. Larval CHO ligament cells are responsible for basal attachments; the JO equivalents must perform a similar function. The scolopale cell wraps several times around the neuronal outer dendritic segment (the cilium) in myelin sheath-like layers that are joined by extensive septate junctions. The scolopale cell also seals around the inner dendritic segment of the neurons with desmosomal junctions. All these junctions allow for a sealed extracellular receptor lymph space, the scolopale space, which serves as an endolymph-like ionic compartment that can drive receptor potentials. Finally, the scolopale cell also elaborates a prominent series of scolopale rods composed of thick bundles of filamentous actin. It is not yet clear whether these rods are fixed structural components or contribute to adaptation by adjusting their length (Caldwell, 2002).

In general, CHOs develop in the same way as other PNS elements. They arise from epithelia, cells of which are equipotent for CHO fates. A prepattern defined by genes that establish dorso-ventral, anterior-posterior and proximo-distal axes influences the positions at which clusters of cells acquire competence for CHO development. Competence of these proneural clusters is achieved by proneural gene expression; in the case of CHO development, atonal (ato) is the primary proneural gene. From each cluster, a single chordotonal organ precursor (COP) is selected by upregulation of the transmembrane ligand, Delta (Dl). The Delta ligand binds to the Notch (N) receptor on the other cells in the cluster. N activation blocks proneural gene expression and the non-COPs lose their competence for chordotonal fate, via lateral inhibition. In the case of CHO clusters such as the leg CHO, COPs first recruit additional COPs in a reiterative fashion. The first-specified COPs delaminate from the epithelium and activate the rhomboid (rho) gene, which results in signaling through the epidermal growth factor receptor [Egfr] on the N-activated non-COP cells of the cluster. Egfr activation mitigates N signaling and activates Dl and ato, generating additional COPs. Once COPs are specified, they undergo several asymmetric divisions to produce differentiated neuron(s) and the supporting cells of a mature scolopidium (Caldwell, 2002).

In recent years, large strides have been made towards identifying the molecular machinery that underlies JO mechanotransduction as well as the effects mutations of these components have on hearing and other mechanosenses in the fly. Genetic screens for hearing, proprioceptive, and mechanosensory mutants are well suited for isolating the molecular machinery involved in these processes regardless of the abundance or nature of the component. The sole prerequisite is that loss of function mutants will exhibit an easily recognizable phenotype (Caldwell, 2002).

Kernan (1994) isolated numerous mutants in an EMS mutagenesis screen for genes encoding mechanosensory transduction components because they exhibited reduced larval behavioral response to gentle touch. X-linked mutations in uncoordinated (unc), uncoordinated-like (uncl), and touch insensitive larva B (tilB) were isolated in the primary screen. An additional screen for mutants on the second chromosome that exhibited uncoordination resembling that of unc mutants resulted in several no mechanoreceptor potential (nomp) mutants and one reduced mechanoreceptor potential (remp) mutant. Mutations in these nomp genes produce flies with aberrant touch-sensitivity, coordination, and acoustic reception. Bristle function was measured by attaching a voltage recording pipette to the K+-rich receptor lymph of an exposed mechanosensory bristle and using a piezoelectric stage to deliver a precise movement of the bristle shaft. In wild-type flies, a bristle in the resting position exhibits a positive transepithelial potential (TEP: the voltage difference between the apical and basal sides of the sensory epithelium), reflecting the K+-richness of the receptor lymph. Deflections of the bristle towards the body elicit a stereotypic strongly depolarizing current, carried by the flow of K+ions from the receptor lymph into the neurons, presumably through the mechanotransduction channel. This transduction current results in a mechanoreceptor potential (MRP: the voltage change upon deflection). The unc, uncl, nomp, and remp mutations all eithe reduce or abolish the MRP, while the TEP is relatively unaffected (Caldwell, 2002 and references therein).

These mutants were later shown to also disrupt sound-evoked electrophysiologic responses measured from the antennal nerve (Eberl, 2000). A number of mutants were isolated in an EMS mutagenesis screen for mutations on chromosome 2 that disrupt an auditory response in Drosophila adults. Mutant strains were identified based on defects in the vigorous group courting behavior normally seen when males are presented with the pulse song. The previous assay for auditory function was female receptivity as measured statistically by a difference in latency of copulation, an assay prohibitive to screening efficiency. From this screen, 15 mutant lines exhibiting a loss or reduction of male chaining behavior were further characterized. Of these, the 5P1 mutant [later named beethoven (btv)] was the only one to affect JO sensory electrophysiology severely (Eberl, 2000). It has since been found that the 5D10 mutant also has a moderate effect on JO physiology. Finally, Drosophila homologs to vertebrate deafness genes have begun to show promise. Perhaps the best example of this is the crinkled gene, which encodes a myosin VIIa homolog. In addition, one mutant, smetana, was discovered as an unrelated mutation on a chromosome carrying another mechanosensory mutation (Caldwell, 2002 and references therein).

Mutations analyzed from these various sources could potentially disrupt several different structures or processes involved in mechanosensation in JO. (1) The acoustically induced mechanical vibrations must be propagated to the mechanosensory neurons. This is likely achieved by a tensioned system that relies on counteracting forces, namely, elasticity of the cuticle opposing tension from the JO scolopidia. Propagation of the signal requires intact structural linkages of the vibrating distal elements. (2) Given that mechanical vibrations are delivered to the neurons, the mechano-transduction machinery within the neuron must be intact to allow activation of a physiologic sensory response. This must occur somewhere along the outer dendritic segment, which is bathed in the receptor lymph, although the precise location is not yet known. Transduction likely occurs by direct activation of an ion channel, which may be part of a multiprotein complex. Specialized cytoskeletal architecture is likely required not only for localization of transduction components by motor proteins, but also for anchoring the transduction complex to allow direct activation (Caldwell, 2002).

The crinkled (ck) gene of Drosophila encodes myosin VIIa. Vertebrate myosin VIIa is an unconventional myosin expressed primarily in sensory hair cells. Orthologs of crinkled have been studied in humans (MYO7A), mouse (shaker-1) and zebrafish (mariner). In humans, defects in MYO7A primarily underlie Usher syndrome type 1B, characterized by sensorineural deafness, vestibular dysfunction, and retinitis pigmentosa, and specific mutations are responsible for two forms of nonsyndromic deafness, DFNB2 and DFNA11. In Drosophila, strong ck mutants are completely deaf, as determined electrophysiologically by the absence of sound-evoked potentials in the antennal nerve. In preliminary histologic examination of ck mutant antennae, the JO scolopidia appear detached from the a2/a3 joint. This suggests that the mechanical vibrations of the arista and a3 cannot be propagated to the scolopidia. Although this defect would adequately explain the deafness phenotype, it is quite possible that the myosin motor encoded by ck is required not only for apical scolopidial attachments or their maintenance, but also for physiologic function of the scolopidia. Such a dualism appears to be true for vertebrate myosin VIIa as well because mutants not only show gross structural defects in the stereocilia, suggesting a morphologic maintenance role, but also show defects in gating properties of the transduction channel, suggesting an additional more intimate role in transduction (Caldwell, 2002 and references therein).

Bristle shafts in ck adults are also shorter and appear bent when compared with the wild type. Bristle MRP amplitudes are severely reduced in ck mutants. This is consistent with broad sharing of mechano-transduction components between bristle organs and CHOs (Eberl, 2000). The structural or physiological basis of ck bristle dysfunction is not yet clear (Caldwell, 2002).

Like ck, the no mechanoreceptor potential A (nompA) mutants disrupt the physical propagation of the mechanical signal to the sensory neurons. However, in nompA the disruption appears as a detachment of the sensory dendrites from the dendritic cap elaborated by the support cells. Thus, the disruption is farther along the mechanical stimulus chain. Nevertheless, some apical detachment of scolopidia from the a2/a3 joint is also seen. nompA was recovered as a complementation group of two mutant alleles associated with severe adult uncoordination and a third allele was identified later. Mutations in nompA affect both es and CHOs as evidenced by the lack of bristle MRPs and lack of sound-evoked potentials from JO, both of which are rescued with a nompA+transgene. nompA encodes a type I sensilla-specific, single-pass transmembrane protein with extracellular domains that include a Zona Pellucida (ZP) domain and five plasminogen N-terminal (PAN) modules. In CHO, the NompA protein is a component of the dendritic cap produced by the scolopale cell, but is not expressed in the neuron. The ZP domain is necessary and sufficient for incorporation into the cap matrix, while the divergent PAN domains may permit the cap to bind to a diverse array of attachment sites. In es organs, the shaft (trichogen) and socket (tormogen) cells secrete K+-rich endolymph that generates the TEP while the sheath cell (thecogen) ensheathes the sensory cilium and produces the dendritic cap. nompA is expressed primarily in the sheath cell of es organs; in mutants the dendrite is usually detached from the dendritic cap, consistent with uncoupling of the mechanical stimulus from the mechano-transduction machinery. Clearly, nompA encodes a protein essential for organization of the cap and its proper attachment to mechanosensory cilia and to apical cuticular structures (Caldwell, 2002).

no mechanoreceptor potential C (nompC) mutants were of interest because of severe uncoordination, loss of larval touch-sensitivity, and loss of MRPs in bristle organs. The nompC gene has been cloned; it encodes a six-transmembrane domain protein, distantly related to the TRP (transient receptor potential) family of ion channels, and at the cytoplasmic amino terminus it has a long array of 29 ankyrin repeats, which could mediate associations with a variety of cellular components, including the cytoskeleton. Under voltage clamp conditions, three nonsense alleles of nompC, all causing strong uncoordination, exhibit an almost complete loss of mechanoreceptor current (MRC: receptor current evoked by mechanical stimulation) when the bristle is deflected toward the body and a severe reduction in the number of action potentials fired. A fourth missense allele with less severe uncoordination shows retention of the robust mechanically evoked response, but exhibits adaptation to mechanical perturbations much more quickly than in wild-type controls and a twofold reduction of action potentials. Therefore, these flies produce fewer action potentials due to the rapid adaptation that limits the time of receptor depolarization. These features argue strongly that nompC represents the major mechano-transduction channel in bristle organs and mimics very closely the biophysical properties of vertebrate hair cell transduction channels. Surprisingly, nompC mutants show only a modest reduction of sound-evoked potentials recorded from the JO when presented with the pulse song. In fact, of all bristle MRP mutants tested for JO response, nompC was the only one that retained much of the sound-evoked response. Thus, although nompC is absolutely required for meaningful bristle organ function, it appears to play only a minor role in transducing auditory stimuli in JO. Therefore, an additional transduction channel is inferred to operate in CHOs. This additional inferred channel may be CHO-specific, or it may be responsible for the small MRC remaining in bristle organs in nompC mutants (Caldwell, 2002 and references therein).

nompB flies, like nompA, exhibit no bristle MRP and no sound-evoked potentials in response to the pulse song. As with nomp A, nompB mutants show gaps between the tips of sensory dendrites and the external sensory structures of campaniform sensilla. Unlike nompA, it is the sensory ending of the neuron rather than the dendritic cap that is abnormal. This is clear in the long outer segments of CHOs, which are missing or malformed in nompB mutants. Molecular analysis of the nompB locus suggests that the basis of the dendritic detachment phenotype is different from that of nompA. nompB encodes a protein containing ten tetratricopeptide repeats that compose two potential protein interacting domains. It is homologous to the mouse and human Tg737/orpk gene, to osm-5 in C. elgans and to IFT88 in Chlamydomonas. These proteins are all associated with ciliary elongation defects, which, in some of these systems, have been shown to result from defects in intraflagellar transport (IFT). IFT is a mechanism by which proteins required for assembly and maintenance of the axoneme are trafficked along the axonemal microtubules of cilia and flagella microtubules. Thus, the mutant phenotype of nompB is consistent with the role of the putative IFT component it encodes (Caldwell, 2002 and references therein).

The 5P1 mutant, recovered as an auditory behavior mutant, was later named beethoven (btv) because it disrupts JO function but leaves bristle MRPs intact. These flies are slightly uncoordinated and sedentary, consistent with defects in all CHOs. Preliminary electron microscopy reveals ciliary defects in the outer dendritic segment of JO neurons, as well as other abnormalities in the appearance of the scolopidia. Homozygous and hemizygous btv males have motile sperm and are fertile, whereas males deficient for the btv region in overlapping chromosomal deficiency combinations are sterile. It is not yet known whether the male sterility results from complete deletion of btv or from a separate genetic function, although the latter interpretation is favored. The btv gene maps to the 36DE region of chromosome 2. The sequence in the candidate region encodes two compelling candidates for the btv gene. One is a new cadherin gene (CadN2) which is adjacent to CadN, and which likely arose from a tandem duplication-divergence of a single ancestral gene. The other major candidate is a dynein heavy chain, Dhc36C, specifically the 1b isoform implicated in IFT in other organisms (Caldwell, 2002).

The two touch insensitive larva B (tilB) mutations, recovered in a larval touch screen, disrupt CHOs but have no effect on bristle physiology or MRP, mirroring the defect seen in btv. Unlike the nomp genes, tilB mutants only show slight motor uncoordination. tilB sound-evoked antennal nerve potentials are completely absent. In addition to auditory dysfunction, these mutants are also male sterile because of sperm amotility. Ultrastructurally, tilB spermatid axonemes have defects in the outer microtubule doublet arrangement. The axonemes of wild-type spermatids are 9 X 2 + 2. Each microtubule doublet has inner and outer dynein arms extending from the A microtubule and a nexin linkage between AB microtubules. The dynein arms, required for microtubule sliding to effect sperm motility, are absent in tilB mutants (as perhaps is the nexin bridge). tilB maps genetically to an unsequenced gap in the genome on the X chromosome (Caldwell, 2002). Similar to tilB, smetana (smet) flies are defective for hearing and male fertility. This combination suggests that smet is also required for axonemal integrity of sperm flagella and CHOs. smet was discovered fortuitously as an additional unrelated mutation on a nompC mutant chromosome that resulted in complete loss of sound-evoked potentials. smet has been mapped to an unsequenced gap near the histone gene cluster on chromosome 2 (Caldwell, 2002).

The function of unc in Drosophila is also currently under investigation. In addition to their uncoordination and bristle MRP defects, unc mutants are also deaf and male sterile. Males do not produce mature sperm, and the flagella of primary spermatids show gross defects in their axonemal structure. unc encodes a coiled-coil protein that is expressed solely in neurons of type I ciliated mechanoreceptors and in the male germline. It is localized to the centrioles of primary spermatocytes and the junction of the nucleus and flagellum in differentiating spermatids, but not in mature sperm. The inner dendritic segments of leg and JO scolopidia are normal, but the ciliated outer dendritic segments fail to connect to the dendritic cap. Clearly, unc has an important role in axonemal formation in sensory cilia and flagella, because it appears to be involved in the conversion of mitotic centrioles into ciliogenic basal bodies (Caldwell, 2002).

Recently, the role in hearing has been investigated of technical knockout (tko), a nuclear gene encoding mitochondrial ribosomal protein S12. In humans, mitochondrial DNA (mtDNA) mutations are responsible for a large number of pathologic syndromes with which sensorineural deafness is often associated. In some cases, the only physiologic defect in patients is hearing loss. tko flies are bang-sensitive, exhibiting a temporary paralysis from the mechanical perturbation. The paralysis could be explained in part by altered sensory feedback from the mechanosensitive bristles. This sensitivity to mechanical vibration is similarly found in humans as a result of aberrant sensory signaling from mechanoreceptors in the inner ear. In addition to lengthened developmental times, tko males also show a reduced male courtship response upon presentation of the pulse song, suggesting a hearing deficiency. These phenotypes are associated with a single missense mutation (L85H) of tko, and mutant larvae have reduced activity of mitochondrial redox enzymes and mitochondrial small subunit rRNA. Raising wild-type flies on doxycyclin generates a phenocopy of the tko phenotypes. It is likely that hearing is an energetically costly process and, consistent with this, reduced levels of ATP were found in tko flies. Electrophysiologic recordings will be necessary in the future to determine the anatomical location of tko behavioral deafness. Nevertheless, this mutant provides a compelling model for mitochondrial deafness that could be used to study not only the precise role of mitochondria themselves, but perhaps also their intracellular transport to relevant parts of the cell (Caldwell, 2002).

It is concluded that construction of a functional auditory receptor requires proper integration of developmental and mechanical processes. Specification, asymmetric divisions and differentiation of the cells producing sense organs must proceed unperturbed. Then, the sense organs must express the diverse assortment of cellular components that establish the intercellular and intracellular environment for the sense organ to be poised for mechanosensation. Furthermore, the mechanoreceptive cells must be mechanically linked to the acoustically sensitive vibrating structures. The development of JO, the Drosophila auditory organ in the antenna, is understood in many fundamental ways, at least vicariously. Although few studies have focused specifically on JO, it is believed that the basic steps of PNS development, which are generally well understood, will also hold for JO. Thus, further studies on JO development must focus on determining the extent of the variations on a well-known theme. In some cases it has already been seen that the mechanisms can differ in JO, such as in the role of sal/salr. Specifically flies in which sal/salr expression is absent in the antenna are completely deaf to the courtship song because of defects in the JO. In these mutants, the specialized cuticle at the a2/a3 joint where JO is attached is missing. a2 and a3 in these flies are effectively fused, restricting rotation of the a3 relative to a2. This fusion prevents the propagation of the mechanical signal to JO; thus, sal/salr mutants have conductive hearing loss. Furthermore, most or all scolopidia of JO are absent in sal/salr mutants. Mutant scolopidia appear to be specified, but are not maintained (Caldwell, 2002).

Many future prospects remain for research in Drosophila hearing: (1) it is important to understand the developmental issues that make JO like other sense organs, and those that set it apart as a specialized organ for hearing; (2) cloning more transduction components will illuminate understanding of the nature of the mechanosensory machinery. In particular, the transduction channel that operates in JO and acts in parallel with nompC, must be identified. (3) To understand the function of each component, determining the relationship of components to each other will be crucial. Epistasis experiments, through interaction screens or localization of components in genetic backgrounds mutant for various other auditory components, would be useful experimental paradigms. (4) The fundamental role of ciliary action in transduction of CHOs is still not understood. However, analysis of auditory mutants with laser vibrometry may add greatly to this endeavor. (5) For Drosophila to achieve its greatest usefulness in understanding of the relationship between insect and vertebrate auditory mechanisms, continual comparison, and testing of homology with vertebrate auditory genes and mechanisms must be carried out. The Drosophila auditory system is poised to become an important test system for dissecting the function of human homologs, even human-specific auditory components (Caldwell, 2002).

Distinct sensory representations of wind and near-field sound in the Drosophila brain

Behavioural responses to wind are thought to have a critical role in controlling the dispersal and population genetics of wild Drosophila species, as well as their navigation in flight, but their underlying neurobiological basis is unknown. This study shows that Drosophila, like wild-caught Drosophila strains, exhibits robust wind-induced suppression of locomotion in response to air currents delivered at speeds normally encountered in nature. Wind-sensitive neurons were identified in Johnston's organ, an antennal mechanosensory structure implicated in near-field sound detection (Caldwell, 2002; Kernan, 2007). Using enhancer trap lines targeted to different subsets of Johnston's organ neurons, and a genetically encoded calcium indicator, it was shown that wind and near-field sound (courtship song) activate distinct populations of Johnston's organ neurons, which project to different regions of the antennal and mechanosensory motor centre in the central brain. Selective genetic ablation of wind-sensitive Johnston's organ neurons in the antenna abolishes wind-induced suppression of locomotion behaviour, without impairing hearing. Moreover, different neuronal subsets within the wind-sensitive population respond to different directions of arista deflection caused by air flow and project to different regions of the antennal and mechanosensory motor centre, providing a rudimentary map of wind direction in the brain. Importantly, sound- and wind-sensitive Johnston's organ neurons exhibit different intrinsic response properties: the former are phasically activated by small, bi-directional, displacements of the aristae, whereas the latter are tonically activated by unidirectional, static deflections of larger magnitude. These different intrinsic properties are well suited to the detection of oscillatory pulses of near-field sound and laminar air flow, respectively. These data identify wind-sensitive neurons in Johnston's organ, a structure that has been primarily associated with hearing, and reveal how the brain can distinguish different types of air particle movements using a common sensory organ (Yorozu, 2010).

Drosophila exhibit a rapid and reversible arrest of walking activity under gentle air currents (0.7-1.6 m/s). This behavior is also exhibited by wild-caught Drosophila species, at wind speeds (1.7 m/s - 2.8 m/s) within the range measured in their natural habitats. This behavior, called wind-induced suppression of locomotion (WISL), could be observed whether or not locomotor activity was enhanced by mechanical startle prior to the introduction of airflow. Importantly, WISL was not observed in response to near-field sound stimuli such as courtship song (280 Hz pulse song: 75-100 dB (Yorozu, 2010).

Recent antennal-gluing experiments have implicated the antenna, and by extension JO, in wind-sensation in Drosophila. Surgical removal of the third antennal segment (a3), or gluing of a3 to the second antennal segment (a2), both of which cause a functional impairment of JO, eliminated WISL. Genetic ablation of mechanosensory chordotonal neurons using nanchung-Gal4 and UAS-hid, a Drosophila cell death gene, also eliminated WISL. Taken together, these results support the idea that JO is required for WISL, a conclusion confirmed by genetic ablation of specific JO subpopulations (Yorozu, 2010).

To investigate how wind and sound are discriminated by the brain, extracellular recordings were performed from the antennal nerve. In some electrode placements, spike trains were evoked by both wind (0.3-0.9m/s) and courtship song (pulse-song). The short duration of the wind-evoked action potentials (<1 msec) is consistent with neuronal, rather than muscle, action potentials. In other cases, responses were evoked by sound but not wind; a few spikes were detected at the onset and offset of the wind stimulus), or by wind but not sound. These results suggested that different axons within the antennal nerve might respond differentially to wind vs. sound (Yorozu, 2010).

To determine whether distinct subsets of JO neurons are activated by wind vs. near-field sound, functional imaging experiments were performed, using a genetically encoded calcium sensor (GCaMP-1.3), controlled by different Gal4 enhancer trap lines expressed in JO. These lines identify 5 major groups of JO axonal projections in the AMMC, called zones A, B, C, D, and E. Each Gal4 driver labels a subset of zones, but mosaic analysis has revealed that individual JO neurons innervate only one zone. Since it is difficult to distinguish the cell bodies of these 5 groups of neurons in JO itself, activity was imaged in JO axon terminals in the AMMC, where the 5 zones are easily discriminated. To do this, live flies were mounted in an inverted orientation under a 2-photon microscope, while airflow and/or near-field sound were delivered from tubing and a speaker, respectively (Yorozu, 2010).

Using an enhancer trap line (JO-AB) that selectively labels neurons in zones A and B, strong GCaMP activation was observed by courtship song (pulse song; 400 Hz, 90 dB SPL, but not by wind (0.9 m/s). Conversely, using a different line (JO-CE) that selectively labels zones C and E, responses to airflow were observed, but not to courtship song. To directly compare responses to wind and sound in the same preparation, a third line, which labels neurons in zones A, C and E, was observed. These experiments confirmed that zone A was activated by sound but not by airflow, while zone E was activated by airflow but not by sound. The same selective responses were observed when the two stimuli were presented sequentially or simultaneously. Together, these data indicated that JO contains distinct populations of sound- and wind-responsive neurons that project to different regions of the AMMC (Yorozu, 2010).

To determine whether the wind-sensitive JO neurons are also required for WISL behavior, these neurons were genetically ablated using a toxin, Ricin A chain. Because the JO-CE Gal4 driver is expressed not only in JO neurons but also in the central brain, an intersectional strategy was employed to restrict ablation to the antenna using eyeless-FLP recombinase. The specificity of this manipulation was confirmed using a FLP-dependent mCD8GFP reporter (Yorozu, 2010).

Following ablation of JO-C and -E neurons, WISL behavior was eliminated, while basal locomotor activity (prior to wind exposure) and phototaxis behavior were unaffected. Importantly, female flies lacking JO-CE neurons had normal hearing, as evidenced by their unperturbed receptivity to courtship by wild-type males, a behavior that depends on the females'; ability to hear male courtship song. In contrast, females lacking nanchung, a gene required for hearing, or whose aristae were glued to their head, exhibited a greatly increased latency to copulation. These data indicate that JO-CE neurons are necessary for WISL behavior, but dispensable for a hearing-dependent behavior (Yorozu, 2010).

Next, the functional significance of the two wind-sensitive JO subpopulations was investigated. Axons innervating zones C and E terminate in lateral vs. medial domains of the AMMC, respectively. When airflow was applied to the front of the head (0°), or at 45°, there was strong activation in zone E, and little activation in zone C. Conversely, airflow applied from the rear (180°) activated zone C, and slightly inhibited zone E. Airflow applied to the side of the head (90°) activated zone C ipsilaterally, and zone E contralaterally. Thus zone C and E neurons are differentially sensitive to airflow directionality (Yorozu, 2010).

High magnification video analysis suggested a simple hypothesis to account for these observations: airflow from different directions moves the aristae either anteriorly or posteriorly, and the direction of arista deflection determines whether zone C or E neurons are activated. Arista ablation experiments indicated that the activation of wind-sensitive JO neurons, like that of sound-sensitive JO neurons, is dependent upon this structure. To test the hypothesis directly, the aristae were moved in different directions using a probe controlled by a DC motor. Displacing the arista posteriorly with a probe activated the E zone almost as strongly as wind delivered from the front, and weakly inhibited the C zone, while displacing it anteriorly activated the C zone and inhibited the E zone. These data demonstrate that zones C and E are sensitive to different directions of arista deflection. This model can explain the asymmetric activation of zones C and E in ipsi- and contral-lateral hemi-brains during wind stimulation from 90°, because this stimulus produces opposite deflection of the aristae on the ipsi- and contra-lateral sides of the head. An internal comparison of activity between zones C and E, both within and between each hemi-brain, could provide a basis for computing wind direction (Yorozu, 2010).

What stimulus features are responsible for the selective activation of sound vs. wind-sensitive neurons in JO? It was first asked whether these two classes of mechanoreceptors are sensitive to different stimulus amplitudes, i.e., air particle velocities (vair). A pressure gradient microphone positioned at the antenna yielded a vair = 0.011 m/s for the 400 Hz sound stimulus played at 90dB, which maximally activated JO-AB neurons. Yet this sound stimulus did not activate zone E neurons, even though these neurons are activated by airflow at a vair as low as 0.005 m/s. Thus, the selectivity of JO-CE and -AB neurons for wind vs. sound is not simply due to differences in stimulus magnitude (Yorozu, 2010).

It was next asked whether JO-AB and -CE neurons might have different intrinsic sensitivities to different types of arista movements, by moving the aristae in steps of different magnitudes and patterns using a probe controlled by a DC motor. Sound-sensitive neurons in zone A, were activated by displacements as small as 0.01 mm, while wind-sensitive neurons in zone E were only weakly activated at displacements below 0.04 mm. Thus, zone A neurons have a lower displacement threshold than zone E neurons (Yorozu, 2010).

Strikingly, it was observed that zone E neurons remained active for as long as the aristae were displaced, while zone A neurons were only transiently activated at the onset and offset of probe displacement. This suggested that zone E neurons might adapt slowly, and therefore respond tonically, while zone A neurons might adapt rapidly, and therefore respond phasically. To confirm this, the aristae were moved in three successive steps of 0.033 mm each (total displacement of 0.099 mm). Zone A neurons exhibited transient (phasic) responses after each displacement, while zone E neurons were tonically activated for the duration of each displacement, and were maximally activated after the second step. These data indicate that JO-AB and JO-CE neurons respond phasically and tonically to arista displacement, with low vs. high activation thresholds, respectively. Furthermore, zone A neurons were activated by bidirectional movements, while zone E neurons were activated only unidirectionally. These different intrinsic response properties are well matched to the oscillatory arista movements caused by pulses of near-field sound vs. uni-directional arista deflections caused by wind. The fly';s ability to discriminate wind vs. sound using a common sensory organ is thus explained by different population of JO neurons with different intrinsic response properties, which project to distinct area of the AMMC (Yorozu, 2010).

The identification of different subpopulations of JO neurons with tonic vs. phasic response properties illustrates a general and conserved feature of mechanosensation. In mammalian skin, slowly adapting, tonically activated Merkel cells, and rapidly adapting, phasically activated Meissner';s corpuscles are used for different types of light touch sensation. In Drosophila, these two properties have been adapted to detect different types of bulk air particle movements by different subsets of JO neurons. Zone AB neurons are activated by sound and required for hearing. Zone CE neurons are required for the behavioral response to gravity (negative gravitaxis), a force that could also produce static deflections of the arista, albeit of a smaller magnitude than those produced by wind (Yorozu, 2010).

The data presented here indicate that JO is not simply a hearing organ, but also mediates wind detection, in a direction sensitive manner. Wind-activated neurons in JO are, moreover, required for an innate behavioral response to wind. The function of WISL in nature is not clear. Field studies have suggested that wind is a major environmental factor affecting the dispersal of wild Drosophila populations. WISL may have evolved to control population dispersal, and thereby maintain genetic homogeneity. Alternatively, WISL may represent a defense mechanism that serves to protect individual flies from injury, or to prevent dispersal from food resources. Identification of the sensory neurons that mediate WISL opens the way to a systematic analysis of the genes and neural circuitry that underlie this robust, innate behavioral response to wind (Yorozu, 2010).

Functional maps of mechanosensory features in the Drosophila brain

Johnston's organ is the largest mechanosensory organ in Drosophila. It contributes to hearing, touch, vestibular sensing, proprioception, and wind sensing. This study used in vivo 2-photon calcium imaging and unsupervised image segmentation to map the tuning properties of Johnston's organ neurons (JONs) at the site where their axons enter the brain. The same methodology was then applied to study two key brain regions that process signals from JONs: the antennal mechanosensory and motor center (AMMC) and the wedge, which is downstream of the AMMC. First, a diversity of JON response types was identified that tile frequency space and form a rough tonotopic map. Some JON response types are direction selective; others are specialized to encode amplitude modulations over a specific range (dynamic range fractionation). Next, it was discovered that both the AMMC and the wedge contain a tonotopic map, with a significant increase in tonotopy-and a narrowing of frequency tuning-at the level of the wedge. Whereas the AMMC tonotopic map is unilateral, the wedge tonotopic map is bilateral. Finally, a subregion of the AMMC/wedge was identified that responds preferentially to the coherent rotation of the two mechanical organs in the same angular direction, indicative of oriented steady air flow (directional wind). Together, these maps reveal the broad organization of the primary and secondary mechanosensory regions of the brain. They provide a framework for future efforts to identify the specific cell types and mechanisms that underlie the hierarchical re-mapping of mechanosensory information in this system (Patella, 2018).

This study imaged activity in specific neuropil regions in the Drosophila brain. That focus was placed on the neuropil, rather than neural somata, may require explanation for readers unfamiliar with Drosophila neuroanatomy. Briefly, most of the Drosophila brain volume is exclusively neuropil, i.e., axons and dendrites. Axons and dendrites with similar tuning properties are often co-localized, and so pan-neuronal GCaMP imaging can reveal orderly maps of sensory stimulus features in Drosophila brain neuropil. By contrast, neural somata are excluded from the neuropil and are instead confined to a thin rind around the neuropil core; there is no simple rule that relates the location of a neuron's soma to the location of its axon and dendrites. Thus, similarly tuned cells often have dissimilar soma locations, and a sensory stimulus typically evokes activity in widely dispersed somata on the surface of the brain. The goal of this study was to visualize maps of mechanosensory stimulus features in the brain, and so neuropil imaging was an appropriate choice (Patella, 2018).

One limitation of neuropil imaging (as compared to somatic imaging) is that signals are being measured from groups of neurons, not single neurons. Cell type diversity may disappear due to optical mixing. Alternatively, diversity might actually be overestimated. Consider a hypothetical scenario with only two cell types, occurring in 10 different ratios in 10 different neuropil subregions; the algorithm would identify 10 response types because of optical mixing. It is reasonably certain that diversity is not being overestimated diversity because of optical mixing, because sparse Gal4 lines (where only scattered cells are labeled) collectively revealed the same sort of diversity seen with a broad Gal4 line. It seems more likely that diversity is being underestimated rather than being overestimated. Calcium imaging also has limited temporal resolution. This is especially relevant to mechanosensation, which is the fastest known sensory modality. Both JONs and AMMC neurons can phase-lock to vibrations as fast as 500 Hz. GCaMP6f signals will reveal only the amplitude modulation envelope of these responses, not their fine structure (Patella, 2018).

All the stimuli transduced by Johnston's organ can be reduced to a single variable, namely, the rotational angle of the distal antennal segment, and its evolution over time. This single variable fully describes the rich content of the natural stimuli that impinge on Johnston's organ. These include stimuli as diverse as courtship song, wind, self-generated wingbeat patterns, vestibular cues, and tactile stimuli. In much the same way, the rich content of human speech and music is also described by a single variable, i.e., the position of the tympanal membrane (Patella, 2018).

Any one-dimensional time-varying signal can be described in terms of three fundamental features: frequency, amplitude, and phase. The results show that mechanosensory neurons in this system show specializations for encoding all three of these fundamental features. Collectively, they divide up frequency space, amplitude space, and phase space. Below each of these features is discussed in turn (Patella, 2018).

Almost all of the neural response types that were identified were tuned to frequency. Moreover, frequency tuning was consistently related to spatial position. In other words, tonotopy was found at every level of this system, from JONs to AMMC to WED (Patella, 2018).

Tonotopy may be useful because it allows co-tuned neurons to interact with each other using a minimal expenditure of 'wire'. This arrangement should maximize speed and minimize metabolic costs. Tonotopic maps are a prominent feature of vertebrate auditory systems in peripheral cells and CNS neurons. In insects, tonotopic maps have been described in peripheral cells, but there is less evidence for tonotopy in CNS neurons. Coarse tonotopy has been reported in some CNS neurons, but in other cases there is a lack of tonotopy. Indeed, it has been proposed that the insect CNS generally discards the tonotopic organization of the periphery. Surprisingly, this study found that tonotopy is a prominent feature of the AMMC and WED in the Drosophila brain, suggesting there may be more functional similarity than previously suspected in the auditory systems of insects and vertebrates. The prominence of tonotopy in the AMMC and WED (and the narrowness of frequency tuning in the WED) is also surprising for yet another reason: spectral cues are reportedly irrelevant for determining behavioral responses to courtship song in Drosophila. However, courtship behaviors are not the only behaviors that depend on Johnston's organ. Spectral cues may be important for other Drosophila behaviors that are much less well studied, e.g., suppression of locomotion by turbulent wind, flight-steering maneuvers, or defensive reactions to predator sounds (Patella, 2018).

Different neural channels were found that were specialized to encode stimulus amplitude over different ranges. At one extreme, some channels responded to vibrations as small as 225 nm. This is close to the smallest vibration amplitude that elicits a detectable electrophysiological or behavioral response (Patella, 2018).

Interestingly, the most sensitive coding channels were already approaching saturation at low-stimulus amplitudes. These channels should be relatively insensitive to amplitude modulations at high amplitudes. Indeed, when the stimulus is the sound of the fly's own flight (which is a loud buzz, from the fly's perspective), high-sensitivity JONs are saturated, and so they cannot follow the sound amplitude modulation envelope. This illustrates the need for low-sensitivity channels as well. Accordingly, this study found low-sensitivity coding channels that did not saturate at high-stimulus amplitudes. Together, these findings illustrate the principle of dynamic range fractionation: different stimulus intensity ranges are allocated to different coding channels. This principle applies to many peripheral sensory cells, including insect auditory receptors and proprioceptors. In other sensory systems, distinct cell types comprise low- and high-threshold subtypes. For example, in the vertebrate somatosensory system, each patch of skin contains low- and high-threshold afferents. In the vertebrate auditory system, each frequency band contains low- and high-threshold auditory fibers. Similarly, in the Drosophila brain, each frequency band is found to contain both high- and low-sensitivity channels. In other words, these neural channels tile both frequency space and amplitude space (Patella, 2018).

Phase is the third fundamental feature of time-varying signals. Like frequency and amplitude, phase is also represented systematically in JONs and downstream CNS neurons. The push/pull channels are a case in point. When the antenna is pushed and pulled, the push/pull channels respond ~180 degrees out of phase. This is equivalent to saying that these two channels have opposing preferred directions (Patella, 2018).

Direction sensitivity has obvious utility in wind sensing. Walking flies use wind direction as a guidance cue. Johnston's organ is a wind-sensing organ, and so it is interesting to compare it with the best-studied insect wind-sensing organ, the cricket cercus. The mechanisms of direction sensitivity are quite different in the cricket cercus and the fly antenna. The cercus is covered by tiny hairs. Due to the asymmetric structure of the hair socket, each hair has a preferred direction of movement, and wind from different directions will maximally deflect different hairs. Each hair is innervated by one mechanoreceptor neuron, which only spikes when the hair is pushed in its preferred direction. By contrast, in the wind-sensing system of Drosophila, there is a single mechanical receiver (i.e., the arista, which is rigidly coupled to the distal antennal segment); this stands in contrast to the many mechanical receivers on the cricket cercus (i.e., the many hairs that move independently). Whereas each receiver in the cricket cercus is innervated by a single neuron, the single receiver in Johnston's organ is innervated by many neurons (JONs), with some cells having opposing responses to the same receiver movement. Thus, in the cercus peripheral complexity is mechanical, whereas in Johnston's organ peripheral complexity is neural (Patella, 2018).

Direction sensitivity is also potentially useful in sound sensing, because the relative phase (direction) of right and left antennal movements can carry information about the sound source location in the azimuth. Thus, direction sensitivity may exist in vibration-preferring JONs. Indeed, direction sensitivity does exist in certain vibration-preferring neurons in the AMMC that project to the WED. However, in this study, it was not possible resolve direction sensitivity in vibration-preferring JONs or AMMC/WED subregions, because GCaMP signals cannot fluctuate rapidly enough to capture any direction sensitivity (phase preferences) in vibration responses (Patella, 2018).

This study provides the first physiological evidence of bilateral integration downstream of Johnston's organ. Notably, it was found that each strip of the WED tonotopic map receives convergent input from both antennae. Within each strip, ipsi- and contralateral frequency preferences are matched. One potential function of bilateral integration is sound localization. In vertebrates and large insects, lateralized sound produces a detectable difference in the amplitude and/or timing of sound pressure cues at the two auditory organs. However, as body size decreases, sound pressure differences become difficult to resolve. For example, in the fly Ormia ochracea, the two auditory organs are only ~500 μm apart; this species has evolved mechanisms for amplifying left/right differences in sound pressure. Drosophila melanogaster is even smaller than Ormia ochracea, meaning left/right differences in sound pressure are correspondingly smaller as well. Accordingly, Drosophila has evolved an auditory organ that does not sense sound pressure: instead, the distal antennal segment blows back and forth with air particle velocity fluctuations (like a flag), rather than expanding and compressing with air pressure fluctuations (like a balloon). Thus, each antenna has intrinsic direction sensitivity. Because the two aristae are positioned at different angles, they have different preferred directions. Bilateral comparisons would still be needed for true directional hearing, because one organ alone could not tell the difference between a quiet sound coming from a preferred direction and a loud sound coming from a nonpreferred direction. The key point is that each organ is inherently directional, so there is no need for them to be separated by a large distance (Patella, 2018).

In crickets, bilateral integration for sound localization occurs in cells directly postsynaptic to peripheral auditory afferents. These cells receive antagonistic input from ipsi- and contralateral auditory organs. By contrast, in Drosophila, bilaterality does not seem to emerge in cells directly postsynaptic to JONs (AMMC neurons). Instead, this study found the first evidence for bilaterality in the WED. In addition to finding bilaterality in vibration-preferring subregions, bilaterality was also found in one subregion of the CNS that preferred steady antennal displacements. This subregion spans the border between the AMMC and WED. This subregion is particularly interesting because it has antagonistic directional preferences for ipsi- and contralateral displacements: it responds best when the ipsilateral antenna is pulled while the contralateral antenna is pushed. This pattern of bilateral antagonism confers selectivity for wind directed at the ipsilateral side of the head (Patella, 2018).

Of course, bilateral integration does not necessarily involve left/right antagonism. Instead, excitatory signals from the two auditory organs may simply be added together. This sort of bilateral pooling could improve the accuracy of behavioral decisions based on the temporal or spectral features of sound stimuli (Patella, 2018).

Drosophila neurobiologists refer to the little-studied regions of the fly brain as terra incognita. New tools have recently opened these brain regions to functional characterization. Like explorers in an unknown land, Drosophila neurobiologists are now facing the task of mapmaking (Patella, 2018).

This study illustrates a general approach to mapmaking that makes no assumptions about the scale or shape of functional compartments or the functional properties that distinguish them. This approach yielded fine-grained maps of mechanosensory feature representations. Maps like these will complement new bioinformatic tools that allow researchers to search genetic driver lines using fine-grained anatomical criteria. Together, these tools will enable detailed investigations of specific cell types and the neural computations they implement (Patella, 2018).

NompC TRP channel is essential for Drosophila sound receptor function

The idea that the NompC TRPN1 channel is the Drosophila transducer for hearing has been challenged by remnant sound-evoked nerve potentials in nompC nulls. This study reports that NompC is essential for the function of Drosophila sound receptors and that the remnant nerve potentials of nompC mutants are contributed by gravity/wind receptor cells. Ablating the sound receptors reduces the amplitude and sensitivity of sound-evoked nerve responses, and the same effects ensued from mutations in nompC. Ablating the sound receptors also suffices to abolish mechanical amplification, which arises from active receptor motility, is linked to transduction, and also requires NompC. Calcium imaging shows that the remnant nerve potentials in nompC mutants are associated with the activity of gravity/wind receptors and that the sound receptors of the mutants fail to respond to sound. Hence, Drosophila sound receptors require NompC for mechanical signal detection and amplification, demonstrating the importance of this transient receptor potential channel for hearing and reviving the idea that the fly's auditory transducer might be NompC (Effertz, 2011).

Ever since NompC (also known as TRPN1) was implicated in Drosophila touch sensation, it has been speculated that this transient receptor potential (TRP) channel could be one of the elusive transduction channels for hearing. Bearing a predicted pore region and an N-terminal ankyrin spring, NompC seems structurally qualified for being a gating spring-operated ion channel as implicated in auditory transduction. Though displaying a rather spotty phylogenetic appearance, NompC is required for the function of certain Drosophila and nematode mechanoreceptors and zebrafish hair cells. NompC is also expressed in hair cells of frogs and in mechanoreceptors of the Drosophila ear, but even though NompC demonstrably can serve as mechanotransduction channel, its importance for auditory transduction and hearing remains uncertain: in frog hair cells, NompC localizes to kinocilia that are dispensable for transduction. And in the Drosophila ear, loss of nompC function reduces the amplitude of sound-evoked afferent nerve responses by only approximately one-half (Effertz, 2011).

A possible explanation for the mild latter effect has emerged with the recent discovery that the antennal hearing organ of Drosophila, Johnston's organ (JO), houses sound and gravity/wind receptors: about half of the fly's approximately 480 JO receptor cells preferentially respond to dynamic antennal vibrations and serve sound detection, whereas the other half preferentially respond to static antennal deflections and mediate the detection of gravity and wind. Driving reporter genes via a nompC-Gal4 promoter fusion construct only labeled the sound receptors, suggesting that the sound-evoked nerve potentials that persist in nompC mutants may be contributed by nompC-independent JO gravity/wind receptor cells. nompC-Gal4, however, reproduces endogenous nompC expression only partially, and an antibody detected NompC protein in virtually all receptors of JO. To explore whether the two JO receptor types nonetheless differ in their nompC dependence, JO function was analyzed in nompC mutants and in flies with ablated sound or gravity/wind receptor cells (Effertz, 2011).

To selectively ablate JO sound or gravity/wind receptors, UAS-ricin toxin A was expressed in these cells using receptor type-specific GAL4 drivers in conjunction with the ey-FLP/FRT system to restrict toxin expression to GAL4-expressing cells in the antenna and eye. To assess JO function, the flies were exposed to pure tones of different intensities and the resulting mechanical input and electrical output of JO were simultaneously monitored. The mechanical input was measured as sound-induced displacement of the antenna's arista, whereas the electrical output was recorded in the form of sound-evoked compound action potentials (CAPs) from the receptor axons in the antennal nerve. The frequency of the tones was adjusted to the mechanical best frequency of the antenna, which was deduced from the power spectrum of the antenna's free fluctuations. The intensity of the tones was measured as the sound particle velocity at the position of the fly (Effertz, 2011).

In accord with previous observations, it was found that remnant sound-evoked nerve potentials persist in nompC nulls: varying the sound particle velocity between approximately 0.001 and 10 mm/s evoked CAPs in nompC2 and nompC3 null mutants whose maximum amplitudes were ~6 times smaller than those of the wild-type and controls. Mutant flies carrying the weaker allele nompC4 displayed equally reduced CAP amplitudes, but the amplitudes were normal when a UAS-nompC-L rescue construct was expressed in all JO receptors of nompC3 nulls. Reduced CAP amplitudes as observed in nompC mutants also ensued from the targeted ablation of JO sound receptors. When JO gravity/wind receptors were ablated, however, CAP amplitudes remained normal, resembling those of wild-type flies and controls. Hence, sound-evoked potentials in the fly's antennal nerve are not only contributed by JO sound receptors: if these receptors are ablated, residual CAPs persist whose amplitudes resemble those of nompC nulls (Effertz, 2011).

Mutations in nompC, in addition to reducing sound-evoked nerve potentials, impair sensitive hearing. This reduction in auditory sensitivity became apparent when the relative CAP amplitudes were plotted against the corresponding sound-induced antennal displacement. In wild-type and control flies, antennal displacements equal to or greater than ~50 nm were sufficient to elicit CAPs, and the CAP amplitude increased monotonously for displacements between approximately 50 and 600 nm. In nompC mutants, this dynamic range of the CAP response consistently shifted up to antennal displacements between approximately 160 and 2000nm, corresponding to an ~3-fold sensitivity drop. This sensitivity drop, which was rescued by expressing UAS-nompC-L in the JO receptors of nompC3 mutants, was also observed in flies with ablated JO sound receptor cells. When the gravity/wind receptors were ablated, however, auditory sensitivity remained unchanged (Effertz, 2011).

When the relative CAP amplitudes were plotted against the sound particle velocity instead of the antennal displacement, the sensitivity drop observed in nompC mutants and flies with ablated sound receptors was even more pronounced. Accordingly, loss of nompC function and loss of sound receptor function reduce both the sensitivity of JO to antennal displacements and, in addition, the mechanical sensitivity of the antenna to sound (Effertz, 2011).

To assess the mechanical sensitivity of the antenna, its displacement varies with sound intensity was determined. In wild-type and control flies, the antenna's displacement nonlinearly increased with sound particle velocity, displaying a compressive nonlinearity that, arising from mechanical activity of JO receptors, enhanced the mechanical sensitivity ~8-fold when sound was faint. Consistent with previous observations, it was found that this nonlinear mechanical amplification was lost in nompC mutants, rendering their antennae mechanically less sensitive to acoustic stimuli so that louder sounds were required to displace their antennae by a given distance, in addition to the larger antennal displacements that were required to elicit CAPs in their antennal nerves. It was also found that this nonlinear amplification could be rescued by expressing UAS-nompC-L in JO receptors and that it specifically required JO sound receptor cells: ablating only the sound receptors abolished mechanical amplification, and the same effect was caused by mutations in nompC. In nompC mutants, this loss of amplification was associated with alterations of the antenna's tuning and fluctuation power that were quantitatively mimicked in flies with ablated sound receptor cells. If the gravity/wind receptors were ablated, however, mechanical amplification remained normal, with the antenna's compressive nonlinearity, its tuning, and its fluctuation power resembling those of wild-type, nompC-L rescue, and control flies. Hence, nonlinear mechanical amplification in the Drosophila ear requires both the NompC channel and JO sound receptors but is independent of JO gravity/wind receptor cells (Effertz, 2011).

Ablating JO sound receptors phenocopies the auditory defects of nompC mutants, suggesting that NompC is essential for the mechanosensory function of these cells. To test this hypothesis, mechanically evoked calcium signals were monitored in the somata of JO receptors of nompC3 null mutants and controls while simultaneously recording the displacement of the antenna and the ensuing CAPs from the antennal nerve. Calcium signals were measured through the cuticle of the antenna using the genetically encoded ratiometric calcium sensor Cameleon2.1 (Cam2.1). To evoke calcium signals, the antenna was sinusoidally actuated at its mechanical best frequency with electrostatic force (Effertz, 2011).

When Cam2.1 was expressed in either the sound receptors alone or all JO receptors, antennal vibrations evoked robust calcium signals in controls. The calcium signals of the sound receptors were entirely abolished in nompC3 mutants, but when Cam2.1 was expressed in all of their JO receptors, small calcium signals were detected that closely resembled those of the gravity/wind receptors of controls. To assess the relation between JO calcium signals and antennal nerve potentials, their respective amplitudes were plotted against the antennal displacement. The large calcium signals of the sound receptors of controls superimposed with the relative amplitudes of the simultaneously recorded CAPs and the CAPs of flies with ablated gravity/wind receptor cells. The small calcium signals of the gravity/wind receptors were shifted to larger antennal displacements and superimposed with the CAPs of flies with ablated sound receptor cells. Calcium signals obtained from all JO receptors of controls had intermediate amplitudes, identifying them as mixed signals contributed by sound and gravity/wind receptor cells. The residual CAPs of nompC3 mutants did not associate with calcium signals in their sound receptors, yet they superimposed with the small calcium signals obtained from all JO receptors of the mutants and from JO gravity/wind receptors of the controls. Although unsuccessful recombination prevented selectively expressing Cam2.1 in the gravity/wind receptors of the mutants, the above findings show that calcium signals that can be ascribed to these receptors are associated with the residual CAPs in nompC nulls. Additional evidence that the calcium signals in the mutants arise from gravity/wind receptors was obtained when the time course of these signals was inspected: in controls, the onset of the calcium signals of all JO receptors followed two exponentials. The exponential with the larger time constant well fitted the calcium signals of their sound receptors. The exponential with the smaller time constant well fitted the calcium signals of their gravity/wind receptors and also those of nompC3 nulls. Hence, instead of being contributed by JO sound receptors, the residual CAPs of nompC mutants are deemed to reflect the activity of JO gravity/wind receptor cells (Effertz, 2011).

Judged from the intracellular calcium signals, the responses of JO gravity/wind receptors to sinusoidal forcing are independent of NompC. Because these receptors preferentially respond to static forcing, the flies' antennae were statically deflected, and the ensuing calcium signals were measured. In accord with previous observations, JO sound receptors hardly responded to antennal deflections, and the calcium signals obtained from all of the JO receptors of nompC3 mutants were indistinguishable from those of controls. Hence, whereas NompC is essential for the mechanosensory function of JO sound receptors, the mechanosensory function of JO gravity/wind receptors seems independent of NompC. Because NompC is detectable in the dendritic tips of virtually all JO receptors other proteins may compensate for the loss of NompC in JO gravity/wind receptors. Possibly, both JO receptor types also use different NompC isoforms, which could also explain why certain nompC promoter fusion constructs are selectively expressed in JO sound receptor cells. The isoform NompC-L rescues the auditory defects of nompC mutants and accordingly seems crucial for JO sound receptor function. Determining NompC isoform patterns in JO may help understanding why gravity/wind receptors express, but apparently do not need, this TRP (Effertz, 2011).

This study has shown that NompC is essential for the mechanosensory function of Drosophila sound receptors, making this TRP channel a strong candidate for the fly's auditory mechanotransducer. Precedence that NompC can serve as a mechanotransduction channel comes from work on C. elegans, and the importance of NompC for Drosophila auditory transduction is supported by its requirement for nonlinear mechanical amplification: in the Drosophila ear, the source of this amplification has been traced down to mechanotransducers that, judged from the present study, reside in the sound receptors. Loss of amplification in flies with ablated sound receptors and in nompC mutants indicates that these auditory transducers require NompC. Clearly, more work is needed to dissect the specific roles of NompC in auditory transduction, and such dissection now seems most worthwhile given the auditory importance of this TRP (Effertz, 2011).

Prestin is an anion transporter dispensable for mechanical feedback amplification in Drosophila hearing

In mammals, the membrane-based protein Prestin confers unique electromotile properties to cochlear outer hair cells, which contribute to the cochlear amplifier. Like mammals, the ears of insects, such as those of Drosophila melanogaster, mechanically amplify sound stimuli and have also been reported to express Prestin homologs. To determine whether the D. melanogaster Prestin homolog (dpres) is required for auditory amplification, dpres mutant flies were generated and analyzed. dpres is robustly expressed in the fly's antennal ear. However, dpres mutant flies show normal auditory nerve responses, and intact non-linear amplification. Thus it is concluded that, in D. melanogaster, auditory amplification is independent of Prestin. This finding resonates with prior phylogenetic analyses, which suggest that the derived motor function of mammalian Prestin replaced, or amended, an ancestral transport function. Indeed, this study shows that dpres encodes a functional anion transporter. Interestingly, the acquired new motor function in the phylogenetic lineage leading to birds and mammals coincides with loss of the mechanotransducer channel NompC (=TRPN1), which has been shown to be required for auditory amplification in flies. The advent of Prestin (or loss of NompC, respectively) may thus mark an evolutionary transition from a transducer-based to a Prestin-based mechanism of auditory amplification (Kavlie, 2014).

Flying Drosophila stabilize their vision-based velocity controller by sensing wind with their antennae

Flies and other insects use vision to regulate their groundspeed in flight, enabling them to fly in varying wind conditions. Compared with mechanosensory modalities, however, vision requires a long processing delay (~100 ms) that might introduce instability if operated at high gain. Flies also sense air motion with their antennae, but how this is used in flight control is unknown. This study manipulated the antennal function of fruit flies by ablating their aristae, forcing them to rely on vision alone to regulate groundspeed. Arista-ablated flies in flight exhibited significantly greater groundspeed variability than intact flies. They were then subjected to a series of controlled impulsive wind gusts delivered by an air piston and experimentally manipulated antennae and visual feedback. The results show that an antenna-mediated response alters wing motion to cause flies to accelerate in the same direction as the gust. This response opposes flying into a headwind, but flies regularly fly upwind. To resolve this discrepancy, a dynamic model of the fly's velocity regulator was obtained by fitting parameters of candidate models to the experimental data. The model suggests that the groundspeed variability of arista-ablated flies is the result of unstable feedback oscillations caused by the delay and high gain of visual feedback. The antenna response drives active damping with a shorter delay (~20 ms) to stabilize this regulator, in exchange for increasing the effect of rapid wind disturbances. This provides insight into flies' multimodal sensory feedback architecture and constitutes a previously unknown role for the antennae (Fuller, 2014).

Engrailed alters the specificity of synaptic connections of Drosophila auditory neurons with the giant fiber

A subset of sound-detecting Johnston's Organ neurons (JONs) in Drosophila melanogaster that express the transcription factors Engrailed (En) and Invected (Inv) form mixed electrical and chemical synaptic inputs onto the giant fiber (GF) dendrite. These synaptic connections are detected by trans-synaptic Neurobiotin (NB) transfer and by colocalization of Bruchpilot-short puncta. Misexpressing En postmitotically in a second subset of sound-responsive JONs causes them to form ectopic electrical and chemical synapses with the GF, in turn causing that postsynaptic neuron to redistribute its dendritic branches into the vicinity of these afferents. A simple electrophysiological recording paradigm was introduced for quantifying the presynaptic and postsynaptic electrical activity at this synapse, by measuring the extracellular sound-evoked potentials (SEPs) from the antennal nerve while monitoring the likelihood of the GF firing an action potential in response to simultaneous subthreshold sound and voltage stimuli. Ectopic presynaptic expression of En strengthens the synaptic connection, consistent with there being more synaptic contacts formed. Finally, RNAi-mediated knockdown of En and Inv in postmitotic neurons reduces SEP amplitude but also reduces synaptic strength at the JON-GF synapse. Overall, these results suggest that En and Inv in JONs regulate both neuronal excitability and synaptic connectivity (Pezier, 2014).

Shaking B mediates synaptic coupling between auditory sensory neurons and the giant fiber of Drosophila melanogaster

The Johnston's Organ neurons (JONs) form chemical and electrical synapses onto the giant fiber neuron (GF), as part of the neuronal circuit that mediates the GF escape response in Drosophila. This study examined which of the 8 Drosophila innexins (invertebrate gap junction proteins) mediates the electrical connection at this synapse. The GF is known to express Shaking B (ShakB), specifically the ShakBN+16 isoform only, at its output synapses in the thorax. The shakB2 mutation disrupts these GF outputs and also abolishes JON-GF synaptic transmission. The amplitude of the compound action potential recorded in response to sound from the base of the antenna (sound-evoked potential, or SEP) was reduced by RNAi of the innexins Ogre, Inx3, Inx6 and, to a lesser extent Inx2, suggesting that they could be required in JONs for proper development, excitability, or synchronization of action potentials. The strength of the JON-GF connection itself was reduced to background levels only by RNAi of shakB, not of the other seven innexins. ShakB knockdown prevented Neurobiotin coupling between GF and JONs and removed the plaques of ShakB protein immunoreactivity that are present at the region of contact. Specific shakB RNAi lines that are predicted to target the ShakBL or ShakBN isoforms alone did not reduce the synaptic strength, implying that it is ShakBN+16 that is required in the presynaptic neurons. It was also suggested that gap junction proteins may have an instructive role in synaptic target choice (Pezier, 2016).

Cell-type-specific roles of Na+/K+ ATPase subunits in Drosophila auditory mechanosensation

Ion homeostasis is a fundamental cellular process particularly important in excitable cell activities such as hearing. It relies on the Na(+)/K(+) ATPase (also referred to as the Na pump), which is composed of a catalytic α subunit and a β subunit required for its transport to the plasma membrane and for regulating its activity. This study shows that α and β subunits are expressed in Johnston's organ (JO), the Drosophila auditory organ. Expression of α subunits (ATPα and α-like) and β subunits (nrv1, nrv2, and nrv3) were knocked down individually in JO with UAS/Gal4-mediated RNAi. ATPα shows elevated expression in the ablumenal membrane of scolopale cells, which enwrap JO neuronal dendrites in endolymph-like compartments. Knocking down ATPα, but not α-like, in the entire JO or only in scolopale cells using specific drivers, resulted in complete deafness. Among β subunits, nrv2 is expressed in scolopale cells and nrv3 in JO neurons. Knocking down nrv2 in scolopale cells blocked Nrv2 expression, reduced ATPα expression in the scolopale cells, and caused almost complete deafness. Furthermore, knockdown of either nrv2 or ATPα specifically in scolopale cells causes abnormal, electron-dense material accumulation in the scolopale space. Similarly, nrv3 functions in JO but not in scolopale cells, suggesting neuron specificity that parallels nrv2 scolopale cell-specific support of the catalytic ATPα. These studies provide an amenable model to investigate generation of endolymph-like extracellular compartments (Roy, 2013).

Using the energy of ATP hydrolysis, the Na pump extrudes cytoplasmic Na+ (out) and extracellular K+ (in) in a 3:2 ratio and maintains the gradient of these cations across the membrane, thus controlling the electrolytic and fluid balance in the cells and organs throughout the body. Among its other functions, the Na pump helps maintain the resting potential of cells, regulates cellular volume, and facilitates transport of solutes in and out of cells. Ion homeostasis of most biological systems depends on the Na pump. In the auditory system, this pump has been linked to the maintenance of the inner ear osmotic balance (Bartolami, 2011). The scala media of the inner ear is filled with a K+-rich extracellular fluid known as endolymph, which is essential for preserving the sensory structures and supporting transduction. Maintaining the endolymph homeostasis is critical to sustain auditory functions. Loss of endolymphatic balance causes collapse of the endolymphatic compartment, leading to hearing loss in mammals. K+ channels and pumps, including the Na pump, ensure proper cycling and secretion of K+ ions in the stria vascularis cells of the cochlea. The Na pump has also been linked to age-related hearing loss and Ménière disease. A detailed functional analysis of this pump is therefore necessary to gain insight into the molecular physiology of hearing loss resulting from loss of auditory ionic homeostasis (Roy, 2013).

Although vertebrate and invertebrate auditory systems differ structurally, they evolved from the same primitive mechanosensors, and there are striking developmental genetic similarities between the two lineages. The fly auditory organ, Johnston's organ (JO), is a chordotonal organ (cho) housed in the second antennal segment (9-9292369817">9). The JO comprises an array of ~250 auditory units or scolopidia. Each scolopidium comprises two to three ciliated sensory neurons associated with several support cells. These bipolar neurons are monodendritic with a single distal cilium and a proximal axon. The scolopale cell, a principal support cell, encloses the neuronal dendrites in a fluid-filled lumen, the scolopale space. This fluid, the receptor lymph, resembles cochlear endolymph and, like the endolymph, is believed to be rich in K+ ions. The scolopale cells are structurally enforced with actin-based scolopale rods. Auditory mechanosensation involves the transduction of the mechanical sound stimulus through the rotation of distal antennal segments into a neuronal response in JO. Using electrophysiological techniques it is possible to record sound-evoked potentials (SEPs) from the auditory nerve. The JO also mediates gravity and wind detection, in addition to auditory mechanosensation (Roy, 2013).

This study used the auditory mechanosensory system of Drosophila melanogaster, with its molecular genetic and electrophysiological techniques, to understand the role of the Na pump in maintaining auditory ion homeostasis. It was hypothesized that the Na pump is important in maintaining the ion homeostasis of the auditory receptor lymph. This study shows that ATPα is the sole Na pump α subunit in JO and that it has elevated expression in scolopale cells. The β subunits show cell-type specific expression and functions in JO, with nrv2 being specific to scolopale cells and nrv3 specific to neurons. It was also shown that ATPα preferentially localizes to the scolopale cell ablumenal membrane. Such functional pump localization is consistent with a role in pumping K+ ions into the scolopale cell en route to the receptor lymph, a role that resembles its contribution to generating vertebrate inner ear endolymph (Roy, 2013).

This study has shown that the Na pump is localized preferentially to the scolopale cell ablumenal plasma membrane, from where it likely pumps K+ ions into the scolopale cell cytoplasm en route to the scolopale space. Several lines of evidence support this conclusion. First, it was observed that ATPα, the principal JO α subunit, has a strikingly high expression in the scolopale cell. This suggested that the ATPα gene has a scolopale cell-specific specialized role. Second, most ATPα protein localizes outside the scolopale rods, supporting an ablumenal plasma membrane localization. Third, ATPα knockdown in the scolopale cell resulted in deafness, loss of scolopale cell integrity, and morphological defects such as presence of distended cilia, implying ionic imbalance in the scolopale space. Taken together, the Na pump is likely to be involved in maintaining JO receptor lymph ion homeostasis. Other molecular players must work in conjunction with the Na pump to maintain the ion homeostasis of the system. However, their identification must await further study (Roy, 2013).

The Na pump may also have alternative functions that are not dependent on pump activity. The current results show subcellular localization of the ATPα-GFP fusion protein accumulating near the scolopale cell-cap cell junction and the scolopale cell-neuron inner dendritic segment junction. Septate junctions are known to be present between these cell types. Insect septate junctions form a transepithelial diffusion barrier that limits solute passage through the spaces between adjacent cells in an epithelium. The Na pump has a pump-independent cell junctional activity responsible for maintaining the epithelial barrier function in the Drosophila tracheal system, mediated by the Nrv2 β subunit (Paul, 2003; Paul, 2007). In the fly auditory system, failure to preserve junctional integrity of the scolopale cells because of a lack of functional pumps may also cause fluid retention inside the scolopale space, which could manifest as the morphological abnormalities seen when either ATPα or nrv2 were knocked down in the scolopale cell. However, a Lucifer Yellow dye exclusion assay, in animals in which ATPα has been knocked down with nompA-Gal4, argues against such a junctional role of the Na pump in scolopale cells, although one cannot absolutely rule out a junctional role of the pump because the Lucifer Yellow molecules may be too large to detect a mild junctional compromise, or RNAi may not completely inhibit the pump. Furthermore, septate junctions require numerous other components, so loss of only one component may not completely dismantle the septate junctions in these cells. Future genetic rescue experiments using constructs with inactivated pump function in an ATPα knockdown background would indicate whether the observed knockdown phenotype is a pump-independent function of the Na pump. However, because of the early requirement of the Na pump during development and cross-reactivity of the RNAi to both the endogenous and rescue construct gene copies, such experiments are currently not feasible (Roy, 2013).

The finding of organelles such as mitochondria in the scolopale space, often devoid of plasma membrane enclosure, raises the question of their origin. The most likely source of these is the scolopale cell itself. One possibility is that concomitantly compromised ion homeostasis and osmotic balance results in scolopale cell membrane rupture, releasing cellular contents. Torn membranes can rapidly reseal themselves through a Ca2+-dependent process. In Drosophila embryos, cells undergoing such a cell membrane tear form a membrane plug to reseal the gap in the lipid bilayer through a coordinated activity of the cell membrane and the cytoskeleton. A second possibility is that the extraneous material in the scolopale space results primarily from a developmental requirement of the Na pump. The cho cell lineage for each scolopidium comes from a single sense organ-precursor cell, specified in the imaginal disk epithelium. Lineage cell division occurs early, before massive changes in cell shape, with enormous subsequent elongation. Loss of ion homeostasis during this process may prevent the high developmental fidelity required for these cell shape changes. A third possible explanation may be partial apoptosis of the scolopale cell. Ultrastructurally, it is clear that the scolopale cell is still alive in the knockdown animals because the nuclei are not heteropyknotic and no cell shrinkage is seen; both of which are hallmarks of apoptotic cells. Nevertheless, ionic or osmotic imbalances in the cell may trigger a subset of apoptotic features such as blebbing of the plasma membrane. Such blebs into the scolopale space would initially be membrane-bound, but this membrane may be unstable and break down in the context of the scolopale space. The mitochondria found in the scolopale space also frequently display swelling or disrupted cristae, observations that are also consistent with apoptotic features in Drosophila (Roy, 2013).

Neuronal receptor currents resulting from auditory transduction likely cause depletion of ions in the receptor lymph; therefore, a mechanism must exist by which the receptor lymph is replenished with a constant supply of K+ ions. The receptor lymph filling the scolopale space in JO is likely to be highly enriched in K+ ions in analogy to the bristle receptors and campaniform sensilla in insects that have been shown to be rich in K+ ions. In addition, vertebrate endolymph is K+ enriched. The model of the Na pump in the fly auditory system is that it is present in the ablumenal plasma membrane of the scolopale cell where it actively transports K+ ions into the scolopale cell en route to the scolopale space to help maintain its K+-rich ionic composition. RNAi-mediated knockdown of Na pump subunits results in deafness and loss of morphological integrity. All of these findings are consistent with the model. However, to absolutely confirm the relevance of this model, additional experiments are required. First, it would be informative to measure the receptor lymph ionic concentration and directly demonstrate the high K+ concentration within the scolopale space. However, such experiments may prove to be technically challenging because of the small size of the scolopidium. It is also necessary to to identify other molecules that work in combination with the Na pump to maintain the receptor lymph for efficient auditory transduction (Roy, 2013).

In the vertebrate inner ear, the endocochlear potential is achieved by maintaining the ionic composition of the endolymph in the fluid compartment into which the stereocilia project. Deregulation of the ion concentration or fluid volume in the endolymph, mediated by cells in the stria vascularis and the lateral walls of the organ of Corti, is thought to underlie hearing disorders such as Alzheimer's disease, and may contribute to age-related hearing loss. Although the Na pump is thought to participate in enriching K+ in the endolymph and to contribute to fluid volume homeostasis in the endolymph, the precise mechanisms of Alzheimer disease and related diseases are not well understood. This article has taken advantage of rapidly advancing understanding of the Drosophila auditory system to systematically investigate the expression and functional roles of each Na pump subunit in the auditory organ. The cell-type specificity of Na pump subunits was defined as well as the functional and morphological consequences of cell type-specific loss of function of these subunits. It also sets the stage for future studies to elucidate the detailed pathways and mediators of ion transport and fluid regulation. The model of the Na pump subcellular localization in the ablumenal membrane of the scolopale cell provides a useful system to investigate endocochlear potential generation in endolymph-like extracellular compartments and its malfunctions in connection to inner ear disorders such as age-related hearing loss and Ménière disease (Roy, 2013).

Diverse roles of axonemal dyneins in Drosophila auditory neuron function and mechanical amplification in hearing

Much like vertebrate hair cells, the chordotonal sensory neurons that mediate hearing in Drosophila are motile and amplify the mechanical input of the ear. Because the neurons bear mechanosensory primary cilia whose microtubule axonemes display dynein arms, it was hypothesized that their motility is powered by dyneins. This study describes two axonemal dynein proteins that are required for Drosophila auditory neuron function, localize to their primary cilia, and differently contribute to mechanical amplification in hearing. Promoter fusions revealed that the two axonemal dynein genes Dmdnah3 (=CG17150) and Dmdnai2 (=CG6053) are expressed in chordotonal neurons, including the auditory ones in the fly's ear. Null alleles of both dyneins equally abolished electrical auditory neuron responses, yet whereas mutations in Dmdnah3 facilitated mechanical amplification, amplification was abolished by mutations in Dmdnai2. Epistasis analysis revealed that Dmdnah3 acts downstream of Nan-Iav channels in controlling the amplificatory gain. Dmdnai2, in addition to being required for amplification, is essential for outer dynein arms in auditory neuron cilia. Mutant defects in sperm competition suggest that both dyneins also function in sperm motility (Karak, 2015).

Ciliary phosphoinositide regulates ciliary protein trafficking in Drosophila

Cilia are highly specialized antennae-like cellular organelles. Inositol polyphosphate 5-phosphatase E (INPP5E) converts PI(4,5)P2 into PI4P and is required for proper ciliary function. Although Inpp5e mutations are associated with ciliopathies in humans and mice, the precise molecular role INPP5E plays in cilia remains unclear. This study reports that Drosophila INPP5E (dINPP5E) regulates ciliary protein trafficking by controlling the phosphoinositide composition of ciliary membranes. Mutations in dInpp5e lead to hearing deficits due to the mislocalization of dTULP and mechanotransduction channels, Inactive and NOMPC, in chordotonal cilia. Both loss of dINPP5E and ectopic expression of the phosphatidylinositol-4-phosphate 5-kinase Skittles increase PI(4,5)P2 levels in the ciliary base. The fact that Skittles expression phenocopies the dInpp5e mutants confirms a central role for PI(4,5)P2 in the regulation of dTULP, Inactive, and NOMPC localization. These data suggest that the spatial localization and levels of PI(4,5)P2 in ciliary membranes are important regulators of ciliary trafficking and function (Park, 2015).

The organization of projection neurons and local neurons of the primary auditory center in the fruit fly Drosophila melanogaster

Acoustic communication between insects serves as an excellent model system for analyzing the neuronal mechanisms underlying auditory information processing. To understand the central auditory pathways a large-scale analysis was performed of the interneurons associated with the primary Drosophila auditory center. By screening expression driver strains and performing single-cell labeling of these strains, 44 types of interneurons were identified innervating the primary auditory center - 5 types were local interneurons whereas the other 39 types were projection interneurons connecting the primary auditory center with other brain regions. The projection neurons comprised three frequency-selective pathways and two frequency-embracive pathways. Mapping of their connection targets revealed that five neuropils in the brain - the wedge, anterior ventrolateral protocerebrum, posterior ventrolateral protocerebrum (PVLP), saddle (SAD), and gnathal ganglia (GNG) - were intensively connected with the primary auditory center. In addition, several other neuropils, including visual and olfactory centers in the brain, were directly connected to the primary auditory center. The distribution patterns of the spines and boutons of the identified neurons suggest that auditory information is sent mainly from the primary auditory center to the PVLP, WED, SAD, GNG, and the thoracico-abdominal ganglia. Based on these findings, this study has established the first comprehensive map of secondary auditory interneurons, which indicates the downstream information flow to parallel ascending pathways, multimodal pathways, and descending pathways (Matsuo, 2016).

Shaking B mediates synaptic coupling between auditory sensory neurons and the giant fiber of Drosophila melanogaster

The Johnston's Organ neurons (JONs) form chemical and electrical synapses onto the giant fiber neuron (GF), as part of the neuronal circuit that mediates the GF escape response in Drosophila. This study examined which of the 8 Drosophila innexins (invertebrate gap junction proteins) mediates the electrical connection at this synapse. The GF is known to express Shaking B (ShakB), specifically the ShakBN+16 isoform only, at its output synapses in the thorax. The shakB2 mutation disrupts these GF outputs and also abolishes JON-GF synaptic transmission. The amplitude of the compound action potential recorded in response to sound from the base of the antenna (sound-evoked potential, or SEP) was reduced by RNAi of the innexins Ogre, Inx3, Inx6 and, to a lesser extent Inx2, suggesting that they could be required in JONs for proper development, excitability, or synchronization of action potentials. The strength of the JON-GF connection itself was reduced to background levels only by RNAi of shakB, not of the other seven innexins. ShakB knockdown prevented Neurobiotin coupling between GF and JONs and removed the plaques of ShakB protein immunoreactivity that are present at the region of contact. Specific shakB RNAi lines that are predicted to target the ShakBL or ShakBN isoforms alone did not reduce the synaptic strength, implying that it is ShakBN+16 that is required in the presynaptic neurons. It was also suggested that gap junction proteins may have an instructive role in synaptic target choice (Pezier, 2016).

Auditory efferent system modulates mosquito hearing
The performance of vertebrate ears is controlled by auditory efferents that originate in the brain and innervate the ear, synapsing onto hair cell somata and auditory afferent fibers. Efferent activity can provide protection from noise and facilitate the detection and discrimination of sound by modulating mechanical amplification by hair cells and transmitter release as well as auditory afferent action potential firing. Insect auditory organs are thought to lack efferent control, but when this study inspected mosquito ears, evidence was found for its existence. Antibodies against synaptic proteins recognized rows of bouton-like puncta running along the dendrites and axons of mosquito auditory sensory neurons. Electron microscopy identified synaptic and non-synaptic sites of vesicle release, and some of the innervating fibers co-labeled with somata in the CNS. Octopamine, GABA, and serotonin were identified as efferent neurotransmitters or neuromodulators that affect auditory frequency tuning, mechanical amplification, and sound-evoked potentials. Mosquito brains thus modulate mosquito ears, extending the use of auditory efferent systems from vertebrates to invertebrates and adding new levels of complexity to mosquito sound detection and communication (Andres, 2016).

A mechanosensory circuit that mixes opponent channels to produce selectivity for complex stimulus features

Johnston's organ is the largest mechanosensory organ in Drosophila; it analyzes movements of the antenna due to sound, wind, gravity, and touch. Different Johnston's organ neurons (JONs) encode distinct stimulus features. Certain JONs respond in a sustained manner to steady displacements, and these JONs subdivide into opponent populations that prefer push or pull displacements. This study describes neurons in the brain (aPN3 neurons) that combine excitation and inhibition from push/pull JONs in different ratios. Consequently, different aPN3 neurons are sensitive to movement in different parts of the antenna's range, at different frequencies, or at different amplitude modulation rates. A model was used to show how the tuning of aPN3 neurons can arise from rectification and temporal filtering in JONs, followed by mixing of JON signals in different proportions. These results illustrate how several canonical neural circuit components-rectification, opponency, and filtering-can combine to produce selectivity for complex stimulus features (Chang, 2016).

A set of structural features defines the cis-regulatory modules of antenna-expressed genes in Drosophila melanogaster

Unraveling the biological information within the regulatory region (RR) of genes has become one of the major focuses of current genomic research. It has been hypothesized that RRs of co-expressed genes share similar architecture, but to the best of our knowledge, no studies have simultaneously examined multiple structural features, such as positioning of cis-regulatory elements relative to transcription start sites and to each other, and the order and orientation of regulatory motifs, to accurately describe overall cis-regulatory structure. This work present an improved computational method that builds a feature collection based on all of these structural features. The utility of this approach was demonstrated by modeling the cis-regulatory modules of antenna-expressed genes in Drosophila melanogaster. Six potential antenna-related motifs were predicted initially, including three that appeared to be novel. A feature set was created with the predicted motifs, where a correlation-based filter was used to remove irrelevant features, and a genetic algorithm was designed to optimize the feature set. Finally, a set of eight highly informative structural features was obtained for the RRs of antenna-expressed genes, achieving an area under the curve of 0.841. These features were used to score all D. melanogaster RRs for potentially unknown antenna-expressed genes sharing a similar regulatory structure. Validation of these predictions with an independent RNA sequencing dataset showed that 76.7% of genes with high scoring RRs were expressed in antenna. In addition, it was found that the structural features that were identified are highly conserved in RRs of orthologs in other Drosophila sibling species. This approach to identify tissue-specific regulatory structures showed comparable performance to previous approaches, but also uncovered additional interesting features because it also considered the order and orientation of motifs (Lopez, 2014; PubMed).

Antennal mechanosensory neurons mediate wing motor reflexes in flying Drosophila
Although many behavioral studies have shown the importance of antennal mechanosensation in various aspects of insect flight control, the identities of the mechanosensory neurons responsible for these functions are still unknown. One candidate is the Johnston's organ (JO) neurons that are located in the second antennal segment and detect phasic and tonic rotations of the third antennal segment relative to the second segment. To investigate how different classes of JO neurons respond to different types of antennal movement during flight, 2-photon calcium imaging was combined with a machine vision system to simultaneously record JO neuron activity and the antennal movement from tethered flying fruit flies (Drosophila melanogaster). Most classes of JO neurons were found to respond strongly to antennal oscillation at the wing beat frequency, but not to the tonic deflections of the antennae. To study how flies use input from the JO neurons during flight, specific classes of JO neurons were genetically ablated, and their effect on the wing motion was examined. Tethered flies flying in the dark require JO neurons to generate slow antiphasic oscillation of the left and right wing stroke amplitudes. However, JO neurons are not necessary for this antiphasic oscillation when visual feedback is available, indicating that there are multiple pathways for generating antiphasic movement of the wings. Collectively, these results are consistent with a model in which flying flies use JO neurons to detect increases in the wing-induced airflow and that JO neurons are involved in a response that decreases contralateral wing stoke amplitude (Mamiya, 2015).

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).

Antennae sense heat stress to inhibit mating and promote escaping in Drosophila females

Environmental stress is a major factor that affects courtship behavior and evolutionary fitness. Although mature virgin females of Drosophila melanogaster usually accept a courting male to mate, they may not mate under stressful conditions. Above the temperature optimal for mating (20-25 degrees C), copulation success of D. melanogaster declines with increasing temperature although vigorous courtship attempts were observed by males, and no copulation takes place at temperatures over 36 degrees C. Attempts were made to identify the sensory pathway for detecting heat threat that drives a female to escape rather than to engage in mating that detects hot temperature and suppresses courtship behavior. The artificial activation of warmth-sensitive neurons ('hot cells') in the antennal arista of females completely abrogates female copulation success even at permissive temperatures below 32 degrees C. Moreover, mutational loss of the GR28b.d thermoreceptor protein caused females to copulate even at 36 degrees C. These results indicate that antennal hot cells provide the input channel for detecting the high ambient temperature in the control of virgin female mating under stressful conditions (Miwa, 2018).

Active mechanisms of vibration encoding and frequency filtering in central mechanosensory neurons

To better understand biophysical mechanisms of mechanosensory processing, this study investigated two cell types in the Drosophila brain (A2 and B1 cells) that are postsynaptic to antennal vibration receptors. A2 cells receive excitatory synaptic currents in response to both directions of movement: thus, twice per vibration cycle. The membrane acts as a low-pass filter, so that voltage and spiking mainly track the vibration envelope rather than individual cycles. By contrast, B1 cells are excited by only forward or backward movement, meaning they are sensitive to vibration phase. They receive oscillatory synaptic currents at the stimulus frequency, and they bandpass filter these inputs to favor specific frequencies. Different cells prefer different frequencies, due to differences in their voltage-gated conductances. Both Na+ and K+ conductances suppress low-frequency synaptic inputs, so cells with larger voltage-gated conductances prefer higher frequencies. These results illustrate how membrane properties and voltage-gated conductances can extract distinct stimulus features into parallel channels (Azevedo, 2017).

Fast intensity adaptation enhances the encoding of sound in Drosophila

To faithfully encode complex stimuli, sensory neurons should correct, via adaptation, for stimulus properties that corrupt pattern recognition. This study investigated sound intensity adaptation in the Drosophila auditory system, which is largely devoted to processing courtship song. Mechanosensory neurons (JONs) in the antenna are sensitive not only to sound-induced antennal vibrations, but also to wind or gravity, which affect the antenna's mean position. Song pattern recognition, therefore, requires adaptation to antennal position (stimulus mean) in addition to sound intensity (stimulus variance). Fast variance adaptation was discovered in Drosophila JONs that corrects for background noise over the behaviorally relevant intensity range. This study determined where mean and variance adaptation arises and how they interact. A computational model explains these results using a sequence of subtractive and divisive adaptation modules, interleaved by rectification. These results lay the foundation for identifying the molecular and biophysical implementation of adaptation to the statistics of natural sensory stimuli (Clemens, 2018).


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