Disturbance in the protein landscape of cochlear perilymph in an Alzheimer's disease mouse model.

Hearing loss is a pivotal risk factor for dementia. It has recently emerged that a disruption in the intercommunication between the cochlea and brain is a key process in the initiation and progression of this disease. However, whether the cochlear properties can be influenced by pathological signals associated with dementia remains unclear. In this study, using a mouse model of Alzheimer's disease (AD), we investigated the impacts of the AD-like amyloid ß (Aß) pathology in the brain on the cochlea. Despite little detectable change in the age-related shift of the hearing threshold, we observed quantitative and qualitative alterations in the protein profile in perilymph, an extracellular fluid that fills the path of sound waves in the cochlea. Our findings highlight the potential contribution of Aß pathology in the brain to the disturbance of cochlear homeostasis.

Classification of multiple emotional states from facial expressions in head-fixed mice using a deep learning-based image analysis.

Facial expressions are widely recognized as universal indicators of underlying internal states in most species of animals, thereby presenting as a non-invasive measure for assessing physical and mental conditions. Despite the advancement of artificial intelligence-assisted tools for automated analysis of voluminous facial expression data in human subjects, the corresponding tools for mice still remain limited so far. Considering that mice are the most prevalent model animals for studying human health and diseases, a comprehensive characterization of emotion-dependent patterns of facial expressions in mice could extend our knowledge on the basis of emotions and the related disorders. Here, we present a framework for the development of a deep learning-powered tool for classifying facial expressions in head-fixed mouse. We demonstrate that our machine vision was capable of accurately classifying three different emotional states from lateral facial images in head-fixed mouse. Moreover, we objectively determined how our classifier characterized the differences among the facial images through the use of an interpretation technique called Gradient-weighted Class Activation Mapping. Importantly, our machine vision presumably discerned the data by leveraging multiple facial features. Our approach is likely to facilitate the non-invasive decoding of a variety of emotions from facial images in head-fixed mice.

A strategy for low-cost portable monitoring of plasma drug concentrations using a sustainable boron-doped-diamond chip.

On-site monitoring of plasma drug concentrations is required for effective therapies. Recently developed handy biosensors are not yet popular owing to insufficient evaluation of accuracy on clinical samples and the necessity of complicated costly fabrication processes. Here, we approached these bottlenecks via a strategy involving engineeringly unmodified boron-doped diamond (BDD), a sustainable electrochemical material. A sensing system based on a ∼1 cm2 BDD chip, when analysing rat plasma spiked with a molecular-targeting anticancer drug, pazopanib, detected clinically relevant concentrations. The response was stable in 60 sequential measurements on the same chip. In a clinical study, data obtained with a BDD chip were consistent with liquid chromatography-mass spectrometry results. Finally, the portable system with a palm-sized sensor containing the chip analysed ∼40 µL of whole blood from dosed rats within ∼10 min. This approach with the 'reusable' sensor may improve point-of-monitoring systems and personalised medicine while reducing medical costs.

Two Loci Contribute to Age-Related Hearing Loss Resistance in the Japanese Wild-Derived Inbred MSM/Ms Mice.

An MSM/Ms strain was established using Japanese wild mice, which exhibit resistance to several phenotypes associated with aging, such as obesity, inflammation, and tumorigenesis, compared to common inbred mouse strains. MSM/Ms strain is resistant to age-related hearing loss, and their auditory abilities are sustained for long durations. The age-related hearing loss 3 (ahl3) locus contributes to age-related hearing in MSM/Ms strain. We generated ahl3 congenic strains by transferring a genomic region on chromosome 17 from MSM/Ms mice into C57BL/6J mice. Although C57BL/6J mice develop age-related hearing loss because of the ahl allele of the cadherin 23 gene, the development of middle- to high-frequency hearing loss was significantly delayed in an ahl3 congenic strain. Moreover, the novel age-related hearing loss 10 (ahl10) locus associated with age-related hearing resistance in MSM/Ms strain was mapped to chromosome 12. Although the resistance effects in ahl10 congenic strain were slightly weaker than those in ahl3 congenic strain, slow progression of age-related hearing loss was confirmed in ahl10 congenic strain despite harboring the ahl allele of cadherin 23. These results suggest that causative genes and polymorphisms of the ahl3 and ahl10 loci are important targets for the prevention and treatment of age-related hearing loss.

A fluorescent sensor for real-time measurement of extracellular oxytocin dynamics in the brain.

Oxytocin (OT), a hypothalamic neuropeptide that acts as a neuromodulator in the brain, orchestrates a variety of animal behaviors. However, the relationship between brain OT dynamics and complex animal behaviors remains largely elusive, partly because of the lack of a suitable technique for its real-time recording in vivo. Here, we describe MTRIAOT, a G-protein-coupled receptor-based green fluorescent OT sensor that has a large dynamic range, suitable affinity, ligand specificity for OT orthologs, minimal effects on downstream signaling and long-term fluorescence stability. By combining viral gene delivery and fiber photometry-mediated fluorescence measurements, we demonstrate the utility of MTRIAOT for real-time detection of brain OT dynamics in living mice. MTRIAOT-mediated measurements indicate variability of OT dynamics depending on the behavioral context and physical condition of an animal. MTRIAOT will likely enable the analysis of OT dynamics in a variety of physiological and pathological processes.

A rapid and simple electrochemical detection of the free drug concentration in human serum using boron-doped diamond electrodes.

Monitoring drug concentration in blood and reflecting this in the dosage are crucial for safe and effective drug treatment. Most drug assays are based on total concentrations of bound and unbound proteins in the serum, although only the unbound concentration causes beneficial and adverse events. Monitoring the unbound concentration alone is expected to provide a means for further optimisation of drug treatment. However, unbound concentration monitoring has not been routinely used for drug treatment due to the long analysis time and the high cost of conventional methods. Here, we have developed a rapid electrochemical method to determine the unbound concentration in ultrafiltered human serum using boron-doped diamond (BDD) electrodes. When the anticancer drug doxorubicin was used as the test drug, the catalytic doxorubicin-mediated reduction of dissolved oxygen provided a sensitive electrochemical signal, with a detection limit of 0.14 nM. In contrast, the sensitivity of glassy carbon (GC) was inferior under the same conditions due to interference from the dissolved oxygen reduction current. The signal background ratio (S/B) of BDD and GC was 11.5 (10 nM doxorubicin) and 1.1 (50 nM), respectively. The results show that a fast measurement time within ten seconds is possible in the clinical concentration range. Additionally, in the ultrafiltered human serum, the obtained values of unbound doxorubicin concentration showed good agreement with those quantified by conventional liquid chromatography-mass spectrometry. This approach has the potential for application in clinical settings where rapid and simple analysis methods would be beneficial.

Electrochemical properties of the non-excitable tissue stria vascularis of the mammalian cochlea are sensitive to sounds.

Excitable cochlear hair cells convert the mechanical energy of sounds into the electrical signals necessary for neurotransmission. The key process is cellular depolarization via K+ entry from K+ -enriched endolymph through hair cells' mechanosensitive channels. Positive 80 mV potential in endolymph accelerates the K+ entry, thereby sensitizing hearing. This potential represents positive extracellular potential within the epithelial-like stria vascularis; the latter potential stems from K+ equilibrium potential (EK ) across the strial membrane. Extra- and intracellular [K+ ] determining EK are likely maintained by continuous unidirectional circulation of K+ through a putative K+ transport pathway containing hair cells and stria. Whether and how the non-excitable tissue stria vascularis responds to acoustic stimuli remains unclear. Therefore, we analysed a cochlear portion for the best frequency, 1 kHz, by theoretical and experimental approaches. We have previously developed a computational model that integrates ion channels and transporters in the stria and hair cells into a circuit and described a circulation current composed of K+ . Here, in this model, mimicking of hair cells' K+ flow induced by a 1 kHz sound modulated the circulation current and affected the strial ion transport mechanisms; the latter effect resulted in monotonically decreasing potential and increasing [K+ ] in the extracellular strial compartment. Similar results were obtained when the stria in acoustically stimulated animals was examined using microelectrodes detecting the potential and [K+ ]. Measured potential dynamics mirrored the EK change. Collectively, because stria vascularis is electrically coupled to hair cells by the circulation current in vivo too, the strial electrochemical properties respond to sounds. KEY POINTS: A highly positive potential of +80 mV in K+ -enriched endolymph in the mammalian cochlea accelerates sound-induced K+ entry into excitable sensory hair cells, a process that triggers hearing. This unique endolymphatic potential represents an EK -based battery for a non-excitable epithelial-like tissue, the stria vascularis. To examine whether and how the stria vascularis responds to sounds, we used our computational model, in which strial channels and transporters are serially connected to those hair cells in a closed-loop circuit, and found that mimicking hair cell excitation by acoustic stimuli resulted in increased extracellular [K+ ] and decreased the battery's potential within the stria. This observation was overall verified by electrophysiological experiments using live guinea pigs. The sensitivity of electrochemical properties of the stria to sounds indicates that this tissue is electrically coupled to hair cells by a radial ionic flow called a circulation current.

Rapid optical tomographic vibrometry using a swept multi-gigahertz comb.

We propose a rapid tomographic vibrometer technique using an optical comb to measure internal vibrations, transient phenomena, and tomographic distributions in biological tissue and microelectromechanical system devices at high frequencies. This method allows phase-sensitive tomographic measurement in the depth direction at a multi-MHz scan rate using a frequency-modulated broadband electrooptic multi-GHz supercontinuum comb. The frequency spacing was swept instantaneously in time and axisymmetrically about the center wavelength via a dual-drive Mach-Zehnder modulator driven by a variable radio frequency signal. This unique sweeping method permits direct measurement of fringe-free interferometric amplitude and phase with arbitrarily changeable measurement range and scan rate. Therefore, a compressive measurement can be made in only the depth region where the vibration exists, reducing the number of measurement points. In a proof-of-principle experiment, the interferometric amplitude and phase were investigated for in-phase and quadrature phase-shifted interferograms obtained by a polarization demodulator. Tomographic transient displacement measurements were performed using a 0.12 mm thick glass film and piezo-electric transducer oscillating at 10-100 kHz with scan rates in the range 1-20 MHz. The depth resolution and precision of the vibrometer were estimated to be approximately 25 µm and 1.0 nm, respectively.

Analysis of Pharmacokinetics in the Cochlea of the Inner Ear.

Hearing loss affects >5% of the global population and therefore, has a great social and clinical impact. Sensorineural hearing loss, which can be caused by different factors, such as acoustic trauma, aging, and administration of certain classes of drugs, stems primarily from a dysfunction of the cochlea in the inner ear. Few therapeutic strategies against sensorineural hearing loss are available. To develop effective treatments for this disease, it is crucial to precisely determine the behavior of ototoxic and therapeutic agents in the microenvironment of the cochlea in live animals. Since the 1980s, a number of studies have addressed this issue by different methodologies. However, there is much less information on pharmacokinetics in the cochlea than that in other organs; the delay in ontological pharmacology is likely due to technical difficulties with accessing the cochlea, a tiny organ that is encased with a bony wall and has a fine and complicated internal structure. In this review, we not only summarize the observations and insights obtained in classic and recent studies on pharmacokinetics in the cochlea but also describe relevant analytical techniques, with their strengths, limitations, and prospects.

In Vivo Real-Time Simultaneous Examination of Drug Kinetics at Two Separate Locations Using Boron-Doped Diamond Microelectrodes.

Methylcobalamin, which is used for the clinical treatment of patients with neuropathy, can have an impact on the sensorineural components associated with the cochlea, and it is possible that the auditory threshold in a certain population of patients with deafness may be recovered. Nonetheless, it remains uncertain whether the action site of methylcobalamin is localized inside or outside the cochlea and which cellular or tissue element is targeted by the drug. In the present work, we developed a method to realize in vivo real-time simultaneous examination of the drug kinetics in two separate locations using boron-doped diamond microelectrodes. First, the analytical performance of methylcobalamin was studied and the measurement protocol was optimized in vitro. Then, the optimized protocol was applied to carry out real-time measurements inside the cochlea and the leg muscle in live guinea pigs while systemically administering methylcobalamin. The results showed that the methylcobalamin concentration in the cochlea was below the limit of detection for the microelectrodes or the drug did not reach the cochlea, whereas the compound clearly reached the leg muscle.

Characterisation of the static offset in the travelling wave in the cochlear basal turn.

In mammals, audition is triggered by travelling waves that are evoked by acoustic stimuli in the cochlear partition, a structure containing sensory hair cells and a basilar membrane. When the cochlea is stimulated by a pure tone of low frequency, a static offset occurs in the vibration in the apical turn. In the high-frequency region at the cochlear base, multi-tone stimuli induce a quadratic distortion product in the vibrations that suggests the presence of an offset. However, vibrations below 100 Hz, including a static offset, have not been directly measured there. We therefore constructed an interferometer for detecting motion at low frequencies including 0 Hz. We applied the interferometer to record vibrations from the cochlear base of guinea pigs in response to pure tones. When the animals were exposed to sound at an intensity of 70 dB or higher, we recorded a static offset of the sinusoidally vibrating cochlear partition by more than 1 nm towards the scala vestibuli. The offset's magnitude grew monotonically as the stimuli intensified. When stimulus frequency was varied, the response peaked around the best frequency, the frequency that maximised the vibration amplitude at threshold sound pressure. These characteristics are consistent with those found in the low-frequency region and are therefore likely common across the cochlea. The offset diminished markedly when the somatic motility of mechanosensitive outer hair cells, the force-generating machinery that amplifies the sinusoidal vibrations, was pharmacologically blocked. Therefore, the partition offset appears to be linked to the electromotile contraction of outer hair cells.

An electrochemical aptamer-based sensor prepared by utilizing the strong interaction between a DNA aptamer and diamond.

Stable and continuous biosensing of electroactive species in vivo has been achieved by using boron-doped diamond (BDD) electrodes owing to their outstanding electrochemical properties. However, the present problem in biosensing using BDD electrodes is how to specifically measure/detect the target molecules, including electrochemically inactive species. A possible solution is to fabricate an electrochemical aptamer-based (E-AB) sensor using a BDD electrode. In a preliminary investigation, we found that DNA aptamers strongly adsorb on the BDD surface and the aptamer-adsorbed BDD apparently worked as an E-AB sensor. The present study reports the performance of the aptamer-adsorbed BDD electrode as an E-AB sensor. Doxorubicin (DOX), a widely used chemotherapeutic, was chosen as a target molecule. The sensor could be prepared by just dipping BDD in an aptamer solution for only 30 min, and the electrochemical signals were dependent on the DOX concentration. The adsorption of DNA was strong enough for continuous measurements and even a sonication treatment. Such behaviors were not observed when using gold and glassy carbon electrodes. In a kinetic measurement, distortion by a sluggish response was observed for both association and dissociation phases, indicating that the interaction between DOX and the aptamer involves several kinetic processes. By fitting to a Langmuir isotherm, a limit of detection of 49 nM and a maximum detectable concentration of 2.3 µM were obtained. Although the sensitivity was lower than those of the well-established E-AB sensors of gold, the values are within a drug's therapeutic range. Overall, the present work demonstrates that a DNA aptamer and a BDD electrode is an effective combination for an E-AB sensor with stable sensitivity, and a wide variety of DNA aptamers can be applied without any special treatment.

In vivo tomographic visualization of intracochlear vibration using a supercontinuum multifrequency-swept optical coherence microscope.

This study combined a previously developed optical system with two additional key elements: a supercontinuum light source characterized by high output power and an analytical technique that effectively extracts interference signals required for improving the detection limit of vibration amplitude. Our system visualized 3D tomographic images and nanometer scale vibrations in the cochlear sensory epithelium of a live guinea pig. The transverse- and axial-depth resolution was 3.6 and 2.7 µm, respectively. After exposure to acoustic stimuli of 21-25 kHz at a sound pressure level of 70-85 dB, spatial amplitude and phase distributions were quantified on a targeted surface, whose area was 522 × 522 µm2.

[Development of in vivo drug sensing system with needle-type diamond microelectrode].

Continuous and real-time measurement of local concentrations of systemically administered drugs in vivo must be crucial for pharmacological studies. Nevertheless, conventional methods require considerable samples quantity and have poor sampling rates. Additionally, they cannot determine how drug kinetics correlates with target function over time. Here, we describe a system with two different sensors. One is a needle-type microsensor composed of boron-doped diamond with a tip of ~40 µm in diameter, and the other is a glass microelectrode. We first tested bumetanide. This diuretic can induce deafness. In the guinea-pig cochlea injected intravenously with bumetanide, the changes of the drug concentration and the extracellular potential underlying hearing were simultaneously measured in real time. We further examined an antiepileptic drug lamotrigine in the rat brain, and tracked its kinetics and at the same time the local field potentials representing neuronal activity. The action of the anticancer reagent doxorubicin was also monitored in the cochlea. This microsensing system may be applied to analyze pharmacokinetics and pharmacodynamics of various drugs at local sites in vivo, and contribute to promoting the pharmacological researches.

Integrative and theoretical research on the architecture of a biological system and its disorder.

An organism stems from assemblies of a variety of cells and proteins. This complex system serves as a unit, and it exhibits highly sophisticated functions in response to exogenous stimuli that change over time. The complete sequencing of the entire human genome has allowed researchers to address the enigmas of life and disease at the gene- or molecular-based level. The consequence of such studies is the rapid accumulation of a multitude of data at multiple levels, ranging from molecules to the whole body, that has necessitated the development of entirely new concepts, tools, and methodologies to analyze and integrate these data. This necessity has given birth to systems biology, an advanced theoretical and practical research framework that has totally changed the directions of not only basic life science but also medicine. During the symposium of the 95th Annual Meeting of The Physiological Society of Japan 2018, five researchers reported on their respective studies on systems biology. The topics included reactions of drugs, ion-transport architecture in an epithelial system, multi-omics in renal disease, cardiac electrophysiological systems, and a software platform for computer simulation. In this review article these authors have summarized recent achievements in the field and discuss next-generation studies on health and disease.

Characterisation of N-glycans in the epithelial-like tissue of the rat cochlea.

Membrane proteins (such as ion channels, transporters, and receptors) and secreted proteins are essential for cellular activities. N-linked glycosylation is involved in stability and function of these proteins and occurs at Asn residues. In several organs, profiles of N-glycans have been determined by comprehensive analyses. Nevertheless, the cochlea of the mammalian inner ear, a tiny organ mediating hearing, has yet to be examined. Here, we focused on the stria vascularis, an epithelial-like tissue in the cochlea, and characterised N-glycans by liquid chromatography with mass spectrometry. This hypervascular tissue not only expresses several ion transporters and channels to control the electrochemical balance in the cochlea but also harbours different transporters and receptors that maintain structure and activity of the organ. Seventy-nine N-linked glycans were identified in the rat stria vascularis. Among these, in 55 glycans, the complete structures were determined; in the other 24 species, partial glycosidic linkage patterns and full profiles of the monosaccharide composition were identified. In the process of characterisation, several sialylated glycans were subjected sequentially to two different alkylamidation reactions; this derivatisation helped to distinguish α2,3-linkage and α2,6-linkage sialyl isomers with mass spectrometry. These data should accelerate elucidation of the molecular architecture of the cochlea.

Erratum to: An approach to the research on ion and water properties in the interphase between the plasma membrane and bulk extracellular solution.

The article An approach to the research on ion and water properties in the interphase between the plasma membrane and bulk extracellular solution, written by Hiroshi Hibino, Madoka Takai, Hidenori Noguchi, Seishiro Sawamura, Yasufumi Takahashi, Hideki Sakai and Hitoshi Shiku, was originally published Online First without open access.

Time-controllable Nkcc1 knockdown replicates reversible hearing loss in postnatal mice.

Identification of the causal effects of specific proteins on recurrent and partially reversible hearing loss has been difficult because of the lack of an animal model that provides reversible gene knockdown. We have developed the transgenic mouse line Actin-tTS::Nkcc1 tetO/tetO for manipulatable expression of the cochlear K+ circulation protein, NKCC1. Nkcc1 transcription was blocked by the binding of a tetracycline-dependent transcriptional silencer to the tetracycline operator sequences inserted upstream of the Nkcc1 translation initiation site. Administration of the tetracycline derivative doxycycline reversibly regulated Nkcc1 knockdown. Progeny from pregnant/lactating mothers fed doxycycline-free chow from embryonic day 0 showed strong suppression of Nkcc1 expression (~90% downregulation) and Nkcc1 null phenotypes at postnatal day 35 (P35). P35 transgenic mice from mothers fed doxycycline-free chow starting at P0 (delivery) showed weaker suppression of Nkcc1 expression (~70% downregulation) and less hearing loss with mild cochlear structural changes. Treatment of these mice at P35 with doxycycline for 2 weeks reactivated Nkcc1 transcription to control levels and improved hearing level at high frequency; i.e., these doxycycline-treated mice exhibited partially reversible hearing loss. Thus, development of the Actin-tTS::Nkcc1 tetO/tetO transgenic mouse line provides a mouse model for the study of variable hearing loss through reversible knockdown of Nkcc1.

Hearing Loss Controlled by Optogenetic Stimulation of Nonexcitable Nonglial Cells in the Cochlea of the Inner Ear.

Light-gated ion channels and transporters have been applied to a broad array of excitable cells including neurons, cardiac myocytes, skeletal muscle cells and pancreatic ß-cells in an organism to clarify their physiological and pathological roles. Nonetheless, among nonexcitable cells, only glial cells have been studied in vivo by this approach. Here, by optogenetic stimulation of a different nonexcitable cell type in the cochlea of the inner ear, we induce and control hearing loss. To our knowledge, deafness animal models using optogenetics have not yet been established. Analysis of transgenic mice expressing channelrhodopsin-2 (ChR2) induced by an oligodendrocyte-specific promoter identified this channel in nonglial cells-melanocytes-of an epithelial-like tissue in the cochlea. The membrane potential of these cells underlies a highly positive potential in a K+-rich extracellular solution, endolymph; this electrical property is essential for hearing. Illumination of the cochlea to activate ChR2 and depolarize the melanocytes significantly impaired hearing within a few minutes, accompanied by a reduction in the endolymphatic potential. After cessation of the illumination, the hearing thresholds and potential returned to baseline during several minutes. These responses were replicable multiple times. ChR2 was also expressed in cochlear glial cells surrounding the neuronal components, but slight neural activation caused by the optical stimulation was unlikely to be involved in the hearing impairment. The acute-onset, reversible and repeatable phenotype, which is inaccessible to conventional gene-targeting and pharmacological approaches, seems to at least partially resemble the symptom in a population of patients with sensorineural hearing loss. Taken together, this mouse line may not only broaden applications of optogenetics but also contribute to the progress of translational research on deafness.