A novel copy number variant in the murine Cdh23 gene gives rise to profound deafness and vestibular dysfunction.

Hearing loss is the most common congenital sensory deficit worldwide and exhibits high genetic heterogeneity, making molecular diagnoses elusive for most individuals. Detecting novel mutations that contribute to hearing loss is crucial to providing accurate personalized diagnoses, tailored interventions, and improving prognosis. Copy number variants (CNVs) are structural mutations that are understudied, potential contributors to hearing loss. Here, we present the Abnormal Wobbly Gait (AWG) mouse, the first documented mutant exhibiting waltzer-like locomotor dysfunction, hyperactivity, circling behaviour, and profound deafness caused by a spontaneous CNV deletion in cadherin 23 (Cdh23). We were unable to identify the causative mutation through a conventional whole-genome sequencing (WGS) and variant detection pipeline, but instead found a linked variant in hexokinase 1 (Hk1) that was insufficient to recapitulate the AWG phenotype when introduced into C57BL/6J mice using CRISPR-Cas9. Investigating nearby deafness-associated genes revealed a pronounced downregulation of Cdh23 mRNA and a complete absence of full-length CDH23 protein, which is critical for the development and maintenance of inner ear hair cells, in whole head extracts from AWG neonates. Manual inspection of WGS read depth plots of the Cdh23 locus revealed a putative 10.4 kb genomic deletion of exons 11 and 12 that was validated by PCR and Sanger sequencing. This study underscores the imperative to refine variant detection strategies to permit identification of pathogenic CNVs easily missed by conventional variant calling to enhance diagnostic precision and ultimately improve clinical outcomes for individuals with genetically heterogenous disorders such as hearing loss.

Doppler detection triggers instantaneous escape behavior in scanning bats.

Animals must instantaneously escape from predators for survival, which requires quick detection of approaching threats. Although the neural mechanisms underlying the perception of looming objects have been extensively studied in the visual system, little is known about their auditory counterparts. Echolocating bats use their auditory senses to perceive not only the soundscape, but also the physical environment through active sensing. Although object movement induces both echo delay changes and Doppler shifts, the actual information required to perceive movement has been unclear. Herein, we addressed this question by playing back phantom echoes mimicking an approaching target to horseshoe bats and found that they relied only on Doppler shifts. This suggests that the bats do not perceive object motion in the spatiotemporal dimension (i.e., positional variation), as in vision, but rather take advantage of acoustic sensing by directly detecting velocity, thereby enabling them to respond instantaneously to approaching threats.

Echo reception in group flight by Japanese horseshoe bats, Rhinolophus ferrumequinum nippon.

The ability to detect behaviourally relevant sensory information is crucial for survival. Especially when active-sensing animals behave in proximity, mutual interferences may occur. The aim of this study was to examine how active-sensing animals deal with mutual interferences. Echolocation pulses and returning echoes were compared in spaces of various sizes (wide and narrow) in Rhinolophus ferrumequinum nippon flying alone or in a group of three bats. We found that in the narrow space, the group-flying bats increased the duration and bandwidth of the terminal frequency-modulated component of their vocalizations. By contrast, the frequency of the returning echoes did not differ in the presence of conspecifics. We found that their own echo frequencies were compensated within the narrow frequency ranges by Doppler shift compensation. By contrast, the estimated frequencies of the received pulses emitted by the other bats were much more broadly distributed than their echoes. Our results suggest that the bat auditory systems are sharply tuned to a narrow frequency to filter spectral interference from other bats.

Three forebrain structures directly inform the auditory midbrain of echolocating bats.

Echolocating bats emit various types of vocalizations for navigation and communication, and need to pay attention to vocal sounds. Projections from forebrain centers to auditory centers are involved in the attention to vocalizations, with the inferior colliculus (IC) being the main target of the projections. Here, using a retrograde tracer, we demonstrate that three forebrain structures, namely, the medial prefrontal cortex (mPFC), amygdala, and auditory cortex (AC), send direct descending projections to the central nucleus of IC. We found that all three structures projected to the bilateral IC. A comparison of the patterns of retrogradely labeled cells across animals suggests that the ipsilateral AC-IC projection is topographically organized, whereas mPFC-IC or amygdala-IC projections did not show clear topographic organization. Together with evidence from previous studies, these results suggest that three descending projections to the IC form loops between the forebrain and IC to make attention to various vocal sounds.

Hearing sensitivity evaluated by the auditory brainstem response in Miniopterus fuliginosus.

This study evaluated the hearing sensitivity of Miniopterus fuliginosus, a frequency-modulating (FM) bat species, by measuring the auditory brainstem responses in the inferior colliculus. The average audiogram was U-shaped. The mean threshold decreased gradually as the frequency increased from 16 to 40 kHz and then decreased rapidly as the frequency reached 46 kHz, with the peak sensitivity occurring at the terminal portion of the echolocation pulse between frequencies of 44 and 56 kHz. The shape of audiogram of M. fuliginosus is consistent with other FM bats, and is compared with its vocalization behavior.

Adaptive frequency shifts of echolocation sounds in Miniopterus fuliginosus according to the frequency-modulated pattern of jamming sounds.

When flying in a group, echolocating bats have to separate their own echoes from pulses and echoes belonging to other individuals to extract only the information necessary for their own navigation. Previous studies have demonstrated that frequency-modulated (FM) bats change the terminal frequencies (TFs) of downward FM pulses under acoustic interference. However, it is not yet clear which acoustic characteristics of the jamming signals induce the TF shift according to the degree of acoustic interference. In this study, we examined changes in the acoustic characteristics of pulses emitted by Miniopterus fuliginosus while presenting jamming stimuli with different FM patterns to the bat flying alone. Bats significantly altered their TFs when responding to downward (dExp) and upward (uExp) exponential FM sounds as well as to a constant-frequency (CF) stimulus, by approximately 1-2 kHz (dExp: 2.1±0.9 kHz; uExp: 1.7±0.3 kHz; CF: 1.3±0.4 kHz) but not for linear FM sounds. The feature common to the spectra of these three jamming stimuli is a spectrum peak near the TF frequency, demonstrating that the bats shift the TF to avoid masking of jamming sounds on the TF frequency range. These results suggest that direct frequency masking near the TF frequency range induces the TF shift, which simultaneously decreases the similarity between their own echolocation sounds and jamming signals.

Bats enhance their call identities to solve the cocktail party problem.

Echolocating bats need to solve the problem of signal jamming by conspecifics when they are in a group. However, while several mechanisms have been suggested, it remains unclear how bats avoid confusion between their own echoes and interfering sounds in a complex acoustic environment. Here, we fixed on-board microphones onto individual frequency-modulating bats flying in groups. We found that group members broaden the inter-individual differences in the terminal frequencies of pulses, thereby decreasing the similarity of pulses among individuals. To understand what features most affect similarity between pulses, we calculated the similarity of signals mimicking pulses. We found that the similarity between those artificial signals was decreased most by manipulation of terminal frequency. These results demonstrate that the signal jamming problem is solved by this simple strategy, which may be universally used by animals that use active sensing, such as echolocating bats and electric fish, thereby transcending species and sensory modalities.

Organization of subcortical auditory nuclei of Japanese house bat (Pipistrellus abramus) identified with cytoarchitecture and molecular expression.

The auditory system of echolocating bats shows remarkable specialization likely related to analyzing echoes of sonar pulses. However, significant interspecies differences have been observed in the organization of auditory pathways among echolocating bats, and the homology of auditory nuclei with those of non-echolocating species has not been established. Here, in order to establish the homology and specialization of auditory pathways in echolocating bats, the expression of markers for glutamatergic, GABAergic, and glycinergic phenotypes in the subcortical auditory nuclei of Japanese house bat (Pipistrellus abramus) was evaluated. In the superior olivary complex, we identified the medial superior olive and superior paraolivary nuclei as expressing glutamatergic and GABAergic phenotypes, respectively, suggesting these nuclei are homologous with those of rodents. In the nuclei of the lateral lemniscus (NLL), the dorsal nucleus was found to be purely GABAergic, the intermediate nucleus was a mixture of glutamatergic and inhibitory neurons, the compact part of the ventral nucleus was purely glycinergic, and the multipolar part of the ventral nucleus expressed both GABA and glycine. In the inferior colliculus (IC), the central nucleus was found to be further subdivided into dorsal and ventral parts according to differences in the density of terminals and the morphology of large GABAergic neurons, suggesting specialization to sonar pulse structure. Medial geniculate virtually lacked GABAergic neurons, suggesting that the organization of the tectothalamic pathway is similar with that of rodents. Taken together, our findings revealed that specialization primarily occurs with regard to nuclei size and organization of the NLL and IC.

Organization of projection from brainstem auditory nuclei to the inferior colliculus of Japanese house bat (Pipistrellus abramus).

OBJECTIVES: Echolocating bats show remarkable specialization which is related to analysis of echoes of biosonars in subcortical auditory brainstem pathways. The inferior colliculus (IC) receives inputs from all lower brainstem auditory nuclei, i.e., cochlear nuclei, nuclei of the lateral lemniscus, and superior olivary complex, and create de novo responses to sound, which is considered crucial for echolocation. Inside the central nucleus of the IC (ICC), small domains which receive specific combination of extrinsic inputs are the basis of integration of sound information. In addition to extrinsic inputs, each domain is interconnected by local IC neurons but the cell types related to the interconnection are not well-understood. The primary objective of the current study is to examine whether the ascending inputs are reorganized and terminate in microdomains inside the ICC. METHODS: We made injection of a retrograde tracer into different parts of the ICC, and analyzed distribution of retrogradely labeled cells in the auditory brainstem of Japanese house bat (Pipistrellus abramus). RESULTS: Pattern of ascending projections from brainstem nuclei was similar to other bat species. Percentages of labeled cells in several nuclei were correlated each other. Furthermore, within the IC, we identified that large GABAergic (LG) and glutamatergic neurons made long-range connection. CONCLUSIONS: Synaptic organization of IC of Japanese house bat shows specialization which is likely to relate for echolocation. Input nuclei to the IC make clusters which terminate in specific part of the ICC, implying the presence of microdomains. LG neurons have roles for binding IC microdomains.

Rapid frequency control of sonar sounds by the FM bat, Miniopterus fuliginosus, in response to spectral overlap.

In the presence of multiple flying conspecifics, echolocating bats avoid jamming by adjusting the spectral and/or temporal features of their vocalizations. However, little is known about how bats alter their pulse acoustic characteristics to adapt to an acoustically jamming situation during flight. We investigated echolocation behavior in a bat (Miniopterus fuliginosus) during free flight under acoustic jamming conditions created by downward FM jamming sounds mimicking bat echolocation sounds. In an experimental chamber, the flying bat was exposed to FM jamming sounds with different terminal frequencies (TFs) from loudspeakers. Echolocation pulses emitted by the flying bat were recorded using a telemetry microphone (Telemike) mounted on the back of the bat. The bats immediately (within 150ms) shifted the TFs of emitted pulses upward when FM jamming sounds were presented. Moreover, the amount of upward TF shift differed depending on the TF ranges of the jamming sounds presented. When the TF range was lower than or overlapped the bat's mean TF, the bat TF shifted significantly upward (by 1-2kHz, Student's t-test, P<0.05), corresponding to 3-5% of the total bandwidth of their emitted pulses. These findings indicate that bats actively avoid overlap of the narrow frequency band around the TF.