Optogenetics Reveals Roles for Supporting Cells in Force Transmission to and From Outer Hair Cells in the Mouse Cochlea.

Outer hair cells (OHCs) of the organ of Corti (OoC), acting as bidirectional cellular mechanoelectrical transducers, generate, receive, and exchange forces with other major elements of the cochlear partition, including the sensory inner hair cells (IHCs). Force exchange is mediated via a supporting cell scaffold, including Deiters' (DC) and outer pillar cells (OPC), to enable the sensitivity and exquisite frequency selectivity of the mammalian cochlea and to transmit its responses to the auditory nerve. To selectively activate DCs and OPCs in male and female mice, we conditionally expressed in them a hyperpolarizing halorhodopsin (HOP), a light-gated inward chloride ion pump, and measured extracellular receptor potentials (ERPs) and their DC component (ERPDCs) from the cortilymph, which fills the OoC fluid spaces, and compared the responses with similar potentials from HOP-/- littermates. The compound action potentials (CAP) of the auditory nerve were measured as an indication of IHC activity and transmission of cochlear responses to the CNS. HOP light-activated hyperpolarization of DCs and OPCs suppressed cochlear amplification through changing the timing of its feedback, altered basilar membrane (BM) responses to tones at all measured levels and frequencies, and reduced IHC excitation. HOP activation findings reported here complement recent studies that revealed channelrhodopsin activation depolarized DCs and OPCs and effectively bypassed, rather than blocked, the control of OHC mechanical and electrical responses to sound and their contribution to timed and directed electromechanical feedback to the mammalian cochlea. Moreover, our findings identify DCs and OPCs as potential targets for the treatment of noise-induced hearing loss.

A Gap-Junction Mutation Reveals That Outer Hair Cell Extracellular Receptor Potentials Drive High-Frequency Cochlear Amplification.

Cochlear amplification enables the enormous dynamic range of hearing through amplifying cochlear responses to low- to moderate-level sounds and compressing them to loud sounds. Amplification is attributed to voltage-dependent electromotility of mechanosensory outer hair cells (OHCs) driven by changing voltages developed across their cell membranes. At low frequencies, these voltage changes are dominated by intracellular receptor potentials (RPs). However, OHC membranes have electrical low-pass filter properties that attenuate high-frequency RPs, which should potentially attenuate amplification of high-frequency cochlear responses and impede high-frequency hearing. We made in vivo intracellular and extracellular electrophysiological measurements from the organ of Corti of male and female mice of the CBA/J strain, with excellent high-frequency hearing, and from the CD-1 mouse strain, which has sensitive hearing below 12 kHz but loses high-frequency hearing within a few weeks postpartum. The CD-1 mouse strain was transfected with an A88V mutation of the connexin 30 gap-junction protein. By blocking the action of the GJ protein to reduce input resistance, the mutation increased the OHC extracellular RP (ERP) magnitude and rescued high-frequency hearing. However, by increasing the organ of Corti resistance, the mutation rescued high-frequency hearing through preserving the OHC extracellular RP (ERP) magnitude. We measured the voltage developed across the basolateral membranes of OHCs, which controls their electromotility, for low- to high-frequency sounds in male and female mice of the CD-1 strain that expressed the A88V mutation. We demonstrate that ERPs, not RPs, drive OHC motility and cochlear amplification at high frequencies because at high frequencies, ERPs are not frequency attenuated, exceed RPs in magnitude, and are appropriately timed to provide cochlear amplification.SIGNIFICANCE STATEMENT Cochlear amplification, which enables the enormous dynamic range of hearing, is attributed to voltage-dependent electromotility of the mechanosensory outer hair cells (OHCs) driven by sound-induced voltage changes across their membranes. OHC intracellular receptor potentials are electrically low-pass filtered, which should hinder high-frequency hearing. We measured the intracellular and extracellular voltages that control OHC electromotility in vivo in a mouse strain with impaired high-frequency hearing. A gap-junction mutation of the strain rescued high-frequency hearing, increased organ of Corti resistance, and preserved large OHC extracellular receptor potentials but reduced OHC intracellular receptor potentials and impaired low-frequency hearing. We concluded intracellular potentials drive OHC motility at low frequencies and extracellular receptor potentials drive OHC motility and cochlear amplification at high frequencies.

Auditory sensory range of male mosquitoes for the detection of female flight sound.

Male mosquitoes detect and localize conspecific females by their flight-tones using the Johnston's organ (JO), which detects antennal deflections under the influence of local particle motion. Acoustic behaviours of mosquitoes and their JO physiology have been investigated extensively within the frequency domain, yet the auditory sensory range and the behaviour of males at the initiation of phonotactic flights are not well known. In this study, we predict a maximum spatial sensory envelope for flying Culex quinquefasciatus by integrating the physiological tuning response of the male JO with female aeroacoustic signatures derived from numerical simulations. Our sensory envelope predictions were tested with a behavioural assay of free-flying males responding to a female-like artificial pure tone. The minimum detectable particle velocity observed during flight tests was in good agreement with our theoretical prediction formed by the peak JO sensitivity measured in previous studies. The iso-surface describing the minimal detectable particle velocity represents the quantitative auditory sensory range of males and is directional with respect to the female body orientation. Our results illuminate the intricacy of the mating behaviour and point to the importance of observing the body orientation of flying mosquitoes to understand fully the sensory ecology of conspecific communication.

In vivo optogenetics reveals control of cochlear electromechanical responses by supporting cells.

Cochlear sensitivity, essential for communication and exploiting the acoustic environment, results from sensory-motor outer hair cells (OHCs) operating in a structural scaffold of supporting cells and extracellular cortilymph (CL) within the organ of Corti (OoC). Cochlear sensitivity control is hypothesized to involve interaction between the OHCs and OoC supporting cells (e.g., Deiters' cells (DCs) and outer pillar cells (OPCs)), but this has never been established in vivo Here, we conditionally expressed channelrhodopsins (ChR2) specifically in male and female mouse DCs and OPCs. illumination of the OoC activated the nonselective ChR2 cation conductance and depolarized DCs when measured in vivo and in isolated OoC. Measurements of sound-induced cochlear mechanical and electrical responses revealed OoC illumination suppressed the normal functions of OoC supporting cells transiently and reversibly. OoC illumination blocked normally occurring continuous minor adjustments of tone-evoked basilar membrane (BM) displacements over their entire dynamic range and OHC voltage responses to tones at levels and frequencies subject to cochlear amplification. OoC illumination altered the OHC MET conductance operating point, which reversed the asymmetry of OHC voltage responses to high level tones. OoC illumination accelerated recovery from temporary loud sound-induced acoustic desensitization. We concluded that DCs and OPCs are involved in both the control of cochlear responses that are essential for normal hearing, and the recovery from temporary acoustic desensitization. This is the first direct in vivo evidence for the interdependency of the structural, mechanical, and electrochemical arrangements of OHCs and OoC supporting cells that together provide fine control of cochlear responses.Significance statement:A striking feature of the mammalian cochlear sensory epithelium, the organ of Corti, is the cellular architecture and supporting cell arrangement that provides a structural scaffold for the sensory-motor outer hair cells. The role of the supporting cell scaffold, however, has never been elucidated in vivo, although in vitro and modelling studies indicate the scaffold is involved in exchange of forces between the outer hair cells and the organ of Corti. We used in vivo techniques, including optogenetics, that do not disrupt arrangements between the outer hair cells and supporting cells, but selectively, transiently, and reversibly interfere with supporting cell normal function. We revealed the supporting cells provide continuous adjustment of cochlear sensitivity, which is instrumental in normal hearing.

Aerodynamic imaging by mosquitoes inspires a surface detector for autonomous flying vehicles.

Some flying animals use active sensing to perceive and avoid obstacles. Nocturnal mosquitoes exhibit a behavioral response to divert away from surfaces when vision is unavailable, indicating a short-range, mechanosensory collision-avoidance mechanism. We suggest that this behavior is mediated by perceiving modulations of their self-induced airflow patterns as they enter a ground or wall effect. We used computational fluid dynamics simulations of low-altitude and near-wall flights based on in vivo high-speed kinematic measurements to quantify changes in the self-generated pressure and velocity cues at the sensitive mechanosensory antennae. We validated the principle that encoding aerodynamic information can enable collision avoidance by developing a quadcopter with a sensory system inspired by the mosquito. Such low-power sensing systems have major potential for future use in safer rotorcraft control systems.

Pre-copula acoustic behaviour of males in the malarial mosquitoes Anopheles coluzzii and Anopheles gambiae s.s. does not contribute to reproductive isolation.

We reveal that males of two members of the Anopheles gambiae s.l. species complex, Anopheles coluzzii and Anopheles gambiae s.s. (hereafter A. gambiae), which are both malaria vectors, perform a stereotypical acoustic behaviour in response to pure tones at frequencies that encompass the frequency range of the female's flight-tones. This behaviour resembles that described for Culex quinquefasciatus and consists of phonotactic flight initiated by a steep increase in wing-beat frequency (WBF) followed by rapid frequency modulation (RFM) of WBF when in close proximity to the sound source. RFM was elicited without acoustic feedback or the presence of a live female, but it appears to be a stereotypic behaviour in the immediate lead up to copula formation. RFM is an independent and different behavioural process from harmonic convergence interactions used by male-female pairs for mate recognition at earlier stages of mating. Acoustic threshold for RFM was used to plot behavioural audiograms from free-flying A coluzzii and A gambiae males. These audiograms were almost identical (minima ∼400 Hz) and encompassed the WBF ranges of A coluzzii (378-601 Hz) and A gambiae (373-590 Hz) females, indicating that males of the two species share similar frequency tuning and range. Furthermore, no differences were found between the two species in their WBFs, RFM behaviour or harmonic convergence ratios. These results indicate that assortative mating between A coluzzii and A gambiae is unlikely to be based on male-specific acoustic behaviours during RFM. The significance of these findings in relation to possible mechanisms for assortative mating is discussed.

Environmental Adaptation, Phenotypic Plasticity, and Associative Learning in Insects: The Desert Locust as a Case Study.

The ability to learn and store information should be adapted to the environment in which animals operate to confer a selective advantage. Yet the relationship between learning, memory, and the environment is poorly understood, and further complicated by phenotypic plasticity caused by the very environment in which learning and memory need to operate. Many insect species show polyphenism, an extreme form of phenotypic plasticity, allowing them to occupy distinct environments by producing two or more alternative phenotypes. Yet how the learning and memories capabilities of these alternative phenotypes are adapted to their specific environments remains unknown for most polyphenic insect species. The desert locust can exist as one of two extreme phenotypes or phases, solitarious and gregarious. Recent studies of associative food-odor learning in this locust have shown that aversive but not appetitive learning differs between phases. Furthermore, switching from the solitarious to the gregarious phase (gregarization) prevents locusts acquiring new learned aversions, enabling them to convert an aversive memory formed in the solitarious phase to an appetitive one in the gregarious phase. This conversion provides a neuroecological mechanism that matches key changes in the behavioral environments of the two phases. These findings emphasize the importance of understanding the neural mechanisms that generate ecologically relevant behaviors and the interactions between different forms of behavioral plasticity.

A role for acoustic distortion in novel rapid frequency modulation behaviour in free-flying male mosquitoes.

We describe a new stereotypical acoustic behaviour by male mosquitoes in response to the fundamental frequency of female flight tones during mating sequences. This male-specific free-flight behaviour consists of phonotactic flight beginning with a steep increase in wing-beat frequency (WBF) followed by rapid frequency modulation (RFM) of WBF in the lead up to copula formation. Male RFM behaviour involves remarkably fast changes in WBF and can be elicited without acoustic feedback or physical presence of the female. RFM features are highly consistent, even in response to artificial tones that do not carry the multi-harmonic components of natural female flight tones. Comparison between audiograms of the robust RFM behaviour and the electrical responses of the auditory Johnston's organ (JO) reveals that the male JO is tuned not to the female WBF per se but, remarkably, to the difference between the male and female WBFs. This difference is generated in the JO responses as a result of intermodulation distortion products (DPs) caused by non-linear interaction between male-female flight tones in the vibrations of the antenna. We propose that male mosquitoes rely on their own flight tones in making use of DPs to acoustically detect, locate and orientate towards flying females. We argue that the previously documented flight-tone harmonic convergence of flying male and female mosquitoes could be a consequence of WBF adjustments so that DPs generated through flight-tone interaction fall within the optimal frequency ranges for JO detection.

Phenotypic transformation affects associative learning in the desert locust.

In desert locusts, increased population densities drive phenotypic transformation from the solitarious to the gregarious phase within a generation [1-4]. Here we show that when presented with odor-food associations, the two extreme phases differ in aversive but not appetitive associative learning, with solitarious locusts showing a conditioned aversion more quickly than gregarious locusts. The acquisition of new learned aversions was blocked entirely in acutely crowded solitarious (transiens) locusts, whereas appetitive learning and prior learned associations were unaffected. These differences in aversive learning support phase-specific feeding strategies. Associative training with hyoscyamine, a plant alkaloid found in the locusts' habitat [5, 6], elicits a phase-dependent odor preference: solitarious locusts avoid an odor associated with hyoscyamine, whereas gregarious locusts do not. Remarkably, when solitarious locusts are crowded and then reconditioned with the odor-hyoscyamine pairing as transiens, the specific blockade of aversive acquisition enables them to override their prior aversive memory with an appetitive one. Under fierce food competition, as occurs during crowding in the field, this provides a neuroecological mechanism enabling locusts to reassign an appetitive value to an odor that they learned previously to avoid.

A long-latency aversive learning mechanism enables locusts to avoid odours associated with the consequences of ingesting toxic food.

Avoiding food that contains toxins is crucial for the survival of many animals, particularly herbivores, because many plants defend themselves with toxins. Some animals can learn to avoid food containing toxins not through its taste but by the toxins' effects following ingestion, though how they do so remains unclear. We studied how desert locusts (Schistocerca gregaria), which are generalist herbivores, form post-ingestive aversive memories and use them to make appropriate olfactory-based decisions in a Y-maze. Locusts form an aversion gradually to an odour paired with food containing the toxin nicotine hydrogen tartrate (NHT), suggesting the involvement of a long-latency associative mechanism. Pairing of odour and toxin-free food accompanied by NHT injections at different latencies showed that locusts could form an association between an odour and toxic malaise, which could be separated by up to 30 min. Tasting but not swallowing the food, or the temporal separation of odour and food, prevents the formation of these long-latency associations, showing that they are post-ingestive. A second associative mechanism not contingent upon feeding operates only when odour presentation is simultaneous with NHT injection. Post-ingestive memory formation is not disrupted by exposure to a novel odour alone but can be if the odour is accompanied by simultaneous NHT injection. Thus, the timing with which food, odour and toxin are encountered whilst foraging is likely to influence memory formation and subsequent foraging decisions. Therefore, locusts can form specific long-lasting aversive olfactory associations that they can use to avoid toxin-containing foods whilst foraging.

Associative olfactory learning in the desert locust, Schistocerca gregaria.

Locusts can learn associations between olfactory stimuli and food rewards, and use the acquired memories to choose between foods according to their nutrient requirements. They are a model system for both the study of olfactory coding and insect nutritional regulation. Previous studies have used operant paradigms for conditioning freely moving locusts, restricting the study of the neural mechanisms underlying the acquisition of olfactory memories, which requires restrained preparations for electrophysiological recordings. Here we present two complementary paradigms for the classical conditioning of olfactory memories in restrained desert locusts (Schistocerca gregaria). These paradigms allow precise experimental control over the parameters influencing learning. The first paradigm is based on classical (Pavlovian) appetitive conditioning. We show that opening of the maxillary palps can be used as a measure of memory acquisition. Maxillary palp opening in response to odour presentation is significantly higher in locusts trained with paired presentation of an odour and a food reward than in locusts trained either with unpaired presentation of food and odour or the odour alone. The memory formed by this conditioning paradigm lasts for at least 24 h. In the second paradigm, we show that classical conditioning of an odour memory in restrained locusts influences their decisions in a subsequent operant task. When locusts that have been trained to associate an odour with a food reward are placed in a Y-maze, they choose the arm containing that odour significantly more often than naïve locusts. A single conditioning trial is sufficient to induce a significant bias for that odour for up to 4 h. Multiple- and block-trial training induce a significant bias that lasts at least 24 h. Thus, locusts are capable of forming appetitive olfactory memories in classical conditioning paradigms and can use these memories to modify their decisions.