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

Saiki T, Ogata G, Sawamura S, Asai K, Razvina O, Watanabe K, Kato R, Zhang Q, Akiyama K, Madhurantakam S, Ahmad NB, Ino D, Nashimoto H, Matsumoto Y, Moriyama M, Horii A, Kondo C, Ochiai R, Kusuhara H, Saijo Y, Einaga Y, Hibino H

Heliyon （Elsevier {BV}） 9 （ 5 ） e15963 - e15963 2023年05月

査読有り, ISSN 2405-8440

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.

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Calcium/calmodulin-dependent protein kinase II associates with the K<sup>+</sup> channel isoform Kv4.3 in adult rat optic nerve

Ogata G., Partida G.J., Fasoli A., Ishida A.T.

Frontiers in Neuroanatomy （Frontiers in Neuroanatomy） 16 2022年09月

筆頭著者, 査読有り, ISSN 16625129

Spikes are said to exhibit “memory” in that they can be altered by spikes that precede them. In retinal ganglion cell axons, for example, rapid spiking can slow the propagation of subsequent spikes. This increases inter-spike interval and, thus, low-pass filters instantaneous spike frequency. Similarly, a K+ ion channel blocker (4-aminopyridine, 4AP) increases the time-to-peak of compound action potentials recorded from optic nerve, and we recently found that reducing autophosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII) does too. These results would be expected if CaMKII modulates spike propagation by regulating 4AP-sensitive K+ channels. As steps toward identifying a possible substrate, we test whether (i) 4AP alters optic nerve spike shape in ways consistent with reducing K+ current, (ii) 4AP alters spike propagation consistent with effects of reducing CaMKII activation, (iii) antibodies directed against 4AP-sensitive and CaMKII-regulated K+ channels bind to optic nerve axons, and (iv) optic nerve CaMKII co-immunoprecipitates with 4AP-sensitive K+ channels. We find that, in adult rat optic nerve, (i) 4AP selectively slows spike repolarization, (ii) 4AP slows spike propagation, (iii) immunogen-blockable staining is achieved with anti-Kv4.3 antibodies but not with antibodies directed against Kv1.4 or Kv4.2, and (iv) CaMKII associates with Kv4.3. Kv4.3 may thus be a substrate that underlies activity-dependent spike regulation in adult visual system pathways.

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

Moriyama H, Ogata G, Nashimoto H, Sawamura S, Furukawa Y, Hibino H, Kusuhara H, Einaga Y

The Analyst （Analyst） 147 （ 20 ） 4442 - 4449 2022年09月

筆頭著者, 査読有り, ISSN 0003-2654

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.

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Electrochemical detection of triamterene in human urine using boron-doped diamond electrodes.

Ishii K, Ogata G, Einaga Y

Biosensors & bioelectronics （Biosensors and Bioelectronics） 217 114666 - 114666 2022年09月

査読有り, ISSN 0956-5663

Urine is one of the most used biological fluids for screening drug delivery and the resultant metabolites. In sports, the use of diuretics such as triamterene is considered a violation of anti-doping rules and is stipulated to be present at less than 79 nM in urine by the World Anti-Doping Agency (WADA). It is therefore important to develop effective rapid and low-cost tests for this diuretic. Here we apply electrochemical analysis using boron-doped diamond (BDD) electrodes, which have superior properties such as low background current, a wide potential window, and high resistance to deactivation. Since real urine samples show clear oxidation current peaks in the potential range more positive than 0.5 V (vs. Ag/AgCl) due to the presence of bio-components such as protein, uric acid, and ascorbic acid, to detect triamterene effectively, the electrochemical protocol was optimized towards a potential range where the other components have limited effect. Our results show that reduced triamterene exhibits an oxidation peak at 0.1 V (vs. Ag/AgCl) in 0.1 M phosphate buffer (PB) and at 0.2 V (vs. Ag/AgCl) in pooled human urine. The peak current value increased according to the triamterene concentration. The limit of detection (LOD) was 3.15 nM in the PB and 7.80 nM in pooled human urine. Finally, triamterene detection was attempted in individual urine samples. Triamterene was electrochemically detectable in individual urine samples, excluding urine samples containing an excess amount of ascorbic acid. The limit of detection (LOD) in individual urine samples was determined to be 20.8 nM.

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Application of boron doped diamond electrodes to electrochemical gas sensor

Triana Y., Ogata G., Einaga Y.

Current Opinion in Electrochemistry （Current Opinion in Electrochemistry） 36 101113 - 101113 2022年08月

査読有り, ISSN 2451-9103

Boron-doped diamond (BDD) electrodes have been attracting attention as superior electrode materials for electrochemical sensing applications. Here, we focused on applications of the BDD electrodes to electrochemical gas sensing especially dissolved gas in aqueous solution. This article discusses arsine, hydrogen sulfide, nitrogen dioxide gas for environmental sensing, and oxygen in the blood for biomedical sensing. The analytical performances suggest that BDD electrodes are more than adequate for determining the concentration of these gas species.

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Blood Oxygen Sensor Using a Boron-Doped Diamond Electrode.

Triana Y, Ogata G, Tomisaki M, Irkham, Einaga Y

Analytical chemistry （Analytical Chemistry） 94 （ 9 ） 3948 - 3955 2022年02月

査読有り, ISSN 0003-2700

The electrochemical behavior of oxygen (O2) in blood was studied using boron-doped diamond (BDD) electrodes. Cyclic voltammogram of O2 in a 0.1 M phosphate buffer solution solution containing 1 × 10-6 M of bovine hemoglobin exhibits a reduction peak at -1.4 V (vs Ag/AgCl). Moreover, the scan rate dependence was investigated to study the reduction reaction mechanism, which was attributable to the reduction of O2 to H2O2 via two electrons. A linear calibration curve was observed in the concentration range of 86.88-314.63 mg L-1 (R2 = 0.99) with a detection limit of 1.0 mg L-1 (S/B = 3). The analytical performance was better than those with glassy carbon or platinum electrodes as the working electrode. In addition, an application to bovine blood was performed. The O2 concentration in the blood measured on the BDD electrodes was compared to that measured using an OxyLite Pro fiber-optic oxygen sensor device. Both methods gave similar values of the O2 concentration in the range of ∼40 to 150 mmHg. This result confirms that BDD electrodes could potentially be used to detect the O2 concentration in blood.

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Author Correction: Computer modeling defines the system driving a constant current crucial for homeostasis in the mammalian cochlea by integrating unique ion transports.

Nin F, Yoshida T, Murakami S, Ogata G, Uetsuka S, Choi S, Doi K, Sawamura S, Inohara H, Komune S, Kurachi Y, Hibino H

NPJ systems biology and applications （NPJ systems biology and applications） 7 （ 1 ） 39 - 39 2021年10月

ISSN 20567189

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Analysis of Pharmacokinetics in the Cochlea of the Inner Ear

Seishiro Sawamura, Genki Ogata, Kai Asai, Olga Razvina, Takeru Ota, Qi Zhang, Sasya Madhurantakam, Koei Akiyama, Daisuke Ino, Sho Kanzaki, Takuro Saiki, Yoshifumi Matsumoto, Masato Moriyama, Yasuo Saijo, Arata Horii, Yasuaki Einaga, Hiroshi Hibino

Frontiers in Pharmacology （Frontiers Media SA） 12 633505 2021年05月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1663-9812

Hearing loss affects &gt;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.

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

Ai Hanawa, Genki Ogata, Seishiro Sawamura, Kai Asai, Sho Kanzaki, Hiroshi Hibino, Yasuaki Einaga

Analytical chemistry （Analytical Chemistry） 92 （ 20 ） 13742 - 13749 2020年08月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0003-2700

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.

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

Takeru Ota, Fumiaki Nin, Samuel Choi, Shogo Muramatsu, Seishiro Sawamura, Genki Ogata, Mitsuo P. Sato, Katsumi Doi, Kentaro Doi, Tetsuro Tsuji, Satoyuki Kawano, Tobias Reichenbach, Hiroshi Hibino

PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY （SPRINGER HEIDELBERG） 472 （ 5 ） 625 - 635 2020年04月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0031-6768

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.

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Extra-Retinal Spike Normalization in Retinal Ganglion Cell Axons.

Alex Fogli Iseppe, Genki Ogata, Jeffrey S Johnson, Gloria J Partida, Nicholas Johnson, Christopher L Passaglia, Andrew T Ishida

eNeuro （eNeuro） 7 （ 2 ） 2020年02月

共著, 査読有り, ISSN 23732822

Spike conduction velocity characteristically differs between myelinated and unmyelinated axons. Here we test whether spikes of myelinated and unmyelinated paths differ in other respects by measuring rat retinal ganglion cell (RGC) spike duration in the intraretinal, unmyelinated nerve fiber layer and the extraretinal, myelinated optic nerve and optic chiasm. We find that rapid spike firing and illumination broaden spikes in intraretinal axons but not in extraretinal axons. RGC axons thus initiate spikes intraretinally and normalize spike duration extraretinally. Additionally, we analyze spikes that were recorded in a previous study of rhesus macaque retinogeniculate transmission and find that rapid spike firing does not broaden spikes in optic tract. The spike normalization we find reduces the number of spike properties that can change during RGC light responses. However, this is not because identical spikes fire in all axons. Instead, our recordings show that different subtypes of RGC generate axonal spikes of different durations and that the differences resemble spike duration increases that alter neurotransmitter release from other neurons. Moreover, previous studies have shown that RGC spikes of shorter duration can fire at higher maximum frequencies. These properties should facilitate signal transfer by different mechanisms at RGC synapses onto subcortical target neurons.SIGNIFICANCE Myelination increases spike conduction velocity in axons. To test whether unmyelinated and myelinated segments of axons differ only in propagation speed, we compared spikes in unmyelinated intraretinal and myelinated extraretinal portions of retinal ganglion cell (RGC) axons. Short interspike intervals and illumination increased spike duration intraretinally but not extraretinally. Consequently, light can change only whether and how often extraretinal spikes fire. Notably, this does not eliminate axonal spike duration differences between subtypes of RGC. This would allow previously described properties of narrower and broader spikes to facilitate signal transfer by different mechanisms at RGC synapses onto subcortical target neurons.

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

Asai K, Yamamoto T, Nagashima S, Ogata G, Hibino H, Einaga Y

The Analyst （Royal Society of Chemistry (RSC)） 145 （ 2 ） 544 - 549 2019年11月

共著, 査読有り, ISSN 0003-2654

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Autophosphorylated CaMKII Facilitates Spike Propagation in Rat Optic Nerve.

Partida GJ, Fasoli A, Fogli Iseppe A, Ogata G, Johnson JS, Thambiaiyah V, Passaglia CL, Ishida AT

The Journal of neuroscience : the official journal of the Society for Neuroscience 38 （ 37 ） 8087 - 8105 2018年09月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0270-6474

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Electrochemical measurement of lamotrigine using boron-doped diamond electrodes

Ai Hanawa, Kai Asai, Genki Ogata, Hiroshi Hibino, Yasuaki Einaga

Electrochimica Acta （Elsevier Ltd） 271 35 - 40 2018年05月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0013-4686

In this paper we report on a study of the electrochemical behavior of lamotrigine (LTG) using boron-doped diamond (BDD) electrodes. Cyclic voltammetry of LTG in a phosphate buffer solution exhibited an oxidation signal at ca. +1.4 V (vs. Ag/AgCl). A reduction signal at −0.5 to −1.0 V (vs. Ag/AgCl) was observed only after oxidation of LTG. The oxidation signal was due to polymerization of LTG adsorbed on the BDD electrode, while the reduction signal was a consequence of a process involving the reduction of the azo group in the LTG-oxidized product. Continuous measurements of LTG aiming at in vivo monitoring were successfully investigated and an optimized chronoamperometry technique, in which the BDD microelectrode was clamped to the LTG oxidation potential (+1.4 V) for 2 s, then to the LTG reduction potential (−0.6 V) for 2 s, was implemented. The limits of detection (S/N = 3) were estimated to be 29 nM and 130 nM for the oxidation and reduction of LTG, respectively. The use of this chronoamperometry technique enables a high temporal resolution of 4 s for LTG measurements. BDD microelectrodes, therefore, are the ideal electrodes for performing continuous in vivo measurements of LTG.

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A microsensing system for the in vivo real-time detection of local drug kinetics.

Ogata G, Ishii Y, Asai K, Sano Y, Nin F, Yoshida T, Higuchi T, Sawamura S, Ota T, Hori K, Maeda K, Komune S, Doi K, Takai M, Findlay I, Kusuhara H, Einaga Y, Hibino H

Nature biomedical engineering （NATURE PUBLISHING GROUP） 1 （ 8 ） 654 - 666 2017年08月

査読有り, ISSN 2157-846X

Real-time recording of the kinetics of systemically administered drugs in in vivo microenvironments may accelerate the development of effective medical therapies. However, conventional methods require considerable analyte quantities, have low sampling rates and do not address how drug kinetics correlate with target function over time. Here, we describe the development and application of a drug-sensing system consisting of a glass microelectrode and a microsensor composed of boron-doped diamond with a tip of around 40 mu m in diameter. We show that, in the guinea pig cochlea, the system can measure-simultaneously and in real time-changes in the concentration of bumetanide (a diuretic that is ototoxic but applicable to epilepsy treatment) and the endocochlear potential underlying hearing. In the rat brain, we tracked the kinetics of the drug and the local field potentials representing neuronal activity. We also show that the actions of the antiepileptic drug lamotrigine and the anticancer reagent doxorubicin can be monitored in vivo. Our microsensing system offers the potential to detect pharmacological and physiological responses that might otherwise remain undetected.

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Computer modeling defines the system driving a constant current crucial for homeostasis in the mammalian cochlea by integrating unique ion transports.

Nin F, Yoshida T, Murakami S, Ogata G, Uetsuka S, Choi S, Doi K, Sawamura S, Inohara H, Komune S, Kurachi Y, Hibino H

NPJ systems biology and applications 3 （ 1 ） 24 - 24 2017年

査読有り, ISSN 20567189

The cochlear lateral wall-an epithelial-like tissue comprising inner and outer layers-maintains +80 mV in endolymph. This endocochlear potential supports hearing and represents the sum of all membrane potentials across apical and basolateral surfaces of both layers. The apical surfaces are governed by K+ equilibrium potentials. Underlying extracellular and intracellular [K+] is likely controlled by the "circulation current," which crosses the two layers and unidirectionally flows throughout the cochlea. This idea was conceptually reinforced by our computational model integrating ion channels and transporters; however, contribution of the outer layer's basolateral surface remains unclear. Recent experiments showed that this basolateral surface transports K+ using Na+, K+-ATPases and an unusual characteristic of greater permeability to Na+ than to other ions. To determine whether and how these machineries are involved in the circulation current, we used an in silico approach. In our updated model, the outer layer's basolateral surface was provided with only Na+, K+-ATPases, Na+ conductance, and leak conductance. Under normal conditions, the circulation current was assumed to consist of K+ and be driven predominantly by Na+, K+-ATPases. The model replicated the experimentally measured electrochemical properties in all compartments of the lateral wall, and endocochlear potential, under normal conditions and during blocking of Na+, K+-ATPases. Therefore, the circulation current across the outer layer's basolateral surface depends primarily on the three ion transport mechanisms. During the blockage, the reduced circulation current partially consisted of transiently evoked Na+ flow via the two conductances. This work defines the comprehensive system driving the circulation current.

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

Sato MP, Higuchi T, Nin F, Ogata G, Sawamura S, Yoshida T, Ota T, Hori K, Komune S, Uetsuka S, Choi S, Masuda M, Watabe T, Kanzaki S, Ogawa K, Inohara H, Sakamoto S, Takebayashi H, Doi K, Tanaka KF, Hibino H

Frontiers in molecular neuroscience （FRONTIERS MEDIA SA） 10 300 2017年

査読有り, ISSN 1662-5099

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 b -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 C -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 acuteonset, 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.

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Fibrocytes in the cochlea of the mammalian inner ear: their molecular architecture, physiological properties, and pathological relevance

T. Yoshida, S. Sawamura, T. Ota, T. Higuchi, G. Ogata, K. Hori, T. Nakagawa, K. Doi, M.P. Sato, Y. Nonomura, A. Horii, S. Takahashi, S. Komune, F. Nin, H. Hibino

Medical Research Archives 5 1 - 16 2017年

査読有り, ISSN 2375-1916

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The unique electrical properties in an extracellular fluid of the mammalian cochlea; their functional roles, homeostatic processes, and pathological significance.

Nin F, Yoshida T, Sawamura S, Ogata G, Ota T, Higuchi T, Murakami S, Doi K, Kurachi Y, Hibino H

Pflugers Archiv : European journal of physiology （SPRINGER） 468 （ 10 ） 1637 - 49 2016年10月

査読有り, ISSN 0031-6768

The cochlea of the mammalian inner ear contains an endolymph that exhibits an endocochlear potential (EP) of +80 mV with a [K+] of 150 mM. This unusual extracellular solution is maintained by the cochlear lateral wall, a double-layered epithelial-like tissue. Acoustic stimuli allow endolymphatic K+ to enter sensory hair cells and excite them. The positive EP accelerates this K+ influx, thereby sensitizing hearing. K+ exits from hair cells and circulates back to the lateral wall, which unidirectionally transports K+ to the endolymph. In vivo electrophysiological assays demonstrated that the EP stems primarily from two K+ diffusion potentials yielded by [K+] gradients between intracellular and extracellular compartments in the lateral wall. Such gradients seem to be controlled by ion channels and transporters expressed in particular membrane domains of the two layers. Analyses of human deafness genes and genetically modified mice suggested the contribution of these channels and transporters to EP and hearing. A computational model, which reconstitutes unidirectional K+ transport by incorporating channels and transporters in the lateral wall and connects this transport to hair cell transcellular K+ fluxes, simulates the circulation current flowing between the endolymph and the perilymph. In this model, modulation of the circulation current profile accounts for the processes leading to EP loss under pathological conditions. This article not only summarizes the unique physiological and molecular mechanisms underlying homeostasis of the EP and their pathological relevance but also describes the interplay between EP and circulation current.

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The unique ion permeability profile of cochlear fibrocytes and its contribution to establishing their positive resting membrane potential.

Yoshida T, Nin F, Murakami S, Ogata G, Uetsuka S, Choi S, Nakagawa T, Inohara H, Komune S, Kurachi Y, Hibino H

Pflugers Archiv : European journal of physiology （SPRINGER） 468 （ 9 ） 1609 - 19 2016年09月

査読有り, ISSN 0031-6768

Eukaryotic cells exhibit negative resting membrane potential (RMP) owing to the high K+ permeability of the plasma membrane and the asymmetric [K+] between the extracellular and intracellular compartments. However, cochlear fibrocytes, which comprise the basolateral surface of a multi-layer epithelial-like tissue, exhibit a RMP of +5 to +12 mV in vivo. This positive RMP is critical for the formation of an endocochlear potential (EP) of +80 mV in a K+-rich extracellular fluid, endolymph. The epithelial-like tissue bathes fibrocytes in a regular extracellular fluid, perilymph, and apically faces the endolymph. The EP, which is essential for hearing, represents the potential difference across the tissue. Using in vivo electrophysiological approaches, we describe a potential mechanism underlying the unusual RMP of guinea pig fibrocytes. The RMP was + 9.0 +/- 3.7 mV when fibrocytes were exposed to an artificial control perilymph (n = 28 cochleae). Perilymphatic perfusion of a solution containing low [Na+] (1 mM) markedly hyperpolarized the RMP to -31.1 +/- 11.2 mV (n = 10; p < 0.0001 versus the control, TukeyKramer test after one-way ANOVA). Accordingly, the EP decreased. Little change in RMP was observed when the cells were treated with a high [K+] of 30 mM (+ 10.4 +/- 2.3 mV; n= 7; p = 0.942 versus the control). During the infusion of a low [Cl-] solution (2.4 mM), the RMP moderately hyperpolarized to -0.9 +/- 3.4 mV (n = 5; p < 0.01 versus the control), although the membranes, if governed by Cl-permeability, should be depolarized. These observations imply that the fibrocyte membranes are more permeable to Na+ than K+ and Cl-, and this unique profile and [Na+] gradient across the membranes contribute to the positive RMP.

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Theoretical and experimental analysis of ototoxic mechanism in the spiral ligament fibrocytes by multi-level simulation with ion transports in the cochlea.

Nin F, Yoshida T, Murakami S, Uetsuka S, Ogata G, Kurachi Y, Hibino H

Nihon yakurigaku zasshi. Folia pharmacologica Japonica 147 （ 2 ） 80 - 3 2016年02月

ISSN 0015-5691

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Molecular architecture of the stria vascularis membrane transport system, which is essential for physiological functions of the mammalian cochlea.

Uetsuka S, Ogata G, Nagamori S, Isozumi N, Nin F, Yoshida T, Komune S, Kitahara T, Kikkawa Y, Inohara H, Kanai Y, Hibino H

The European journal of neuroscience （WILEY-BLACKWELL） 42 （ 3 ） 1984 - 2002 2015年08月

査読有り, ISSN 0953-816X

Stria vascularis of the mammalian cochlea transports K+ to establish the electrochemical property in the endolymph crucial for hearing. This epithelial tissue also transports various small molecules. To clarify the profile of proteins participating in the transport system in the stria vascularis, membrane components purified from the stria of adult rats were analysed by liquid chromatography tandem mass spectrometry. Of the 3236 proteins detected in the analysis, 1807 were membrane proteins. Ingenuity Knowledge Base and literature data identified 513 proteins as being expressed on the plasma membrane', these included 25 ion channels and 79 transporters. Sixteen of the former and 62 of the latter had not yet been identified in the stria. Unexpectedly, many Cl- and Ca2+ transport systems were found, suggesting that the dynamics of these ions play multiple roles. Several transporters for organic substances were also detected. Network analysis demonstrated that a few kinases, including protein kinase A, and Ca2+ were key regulators for the strial transports. In the library of channels and transporters, 19 new candidates for uncloned deafness-related genes were identified. These resources provide a platform for understanding the molecular mechanisms underlying the epithelial transport essential for cochlear function and the pathophysiological processes involved in hearing disorders.

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NKCCs in the fibrocytes of the spiral ligament are silent on the unidirectional K⁺ transport that controls the electrochemical properties in the mammalian cochlea.

Yoshida T, Nin F, Ogata G, Uetsuka S, Kitahara T, Inohara H, Akazawa K, Komune S, Kurachi Y, Hibino H

Pflugers Archiv : European journal of physiology （SPRINGER） 467 （ 7 ） 1577 - 1589 2015年07月

査読有り, ISSN 0031-6768

Unidirectional K+ transport across the lateral cochlear wall contributes to the endocochlear potential (EP) of +80 mV in the endolymph, a property essential for hearing. The wall comprises two epithelial layers, the syncytium and the marginal cells. The basolateral surface of the former and the apical membranes of the latter face the perilymph and the endolymph, respectively. Intrastrial space (IS), an extracellular compartment between the two layers, exhibits low [K+] and a potential similar to the EP. This IS potential (ISP) dominates the EP and represents a K+ diffusion potential elicited by a large K+ gradient across the syncytial apical surface. The K+ gradient depends on the unidirectional K+ transport driven by Na+,K+-ATPases on the basolateral surface of each layer and the concomitant Na+,K+,2Cl(-)-cotransporters (NKCCs) in the marginal cell layer. The NKCCs coexpressed with the Na+,K+-ATPases in the syncytial layer also seem to participate in the K+ transport. To test this hypothesis, we examined the electrochemical properties of the lateral wall with electrodes measuring [K+] and potential. Blocking NKCCs by perilymphatic perfusion of bumetanide suppressed the ISP. Unexpectedly and unlike the inhibition of the syncytial Na+,K+-ATPases, the perfusion barely altered the electrochemical properties of the syncytium but markedly augmented [K+] of the IS. Consequently, the K+ gradient decreased and the ISP declined. These observations resembled those when the marginal cells' Na+,K+-ATPases or NKCCs were blocked with vascularly applied inhibitors. It is plausible that NKCCs in the marginal cells are affected by the perilymphatically perfused bumetanide, and these transporters, but not those in the syncytium, mediate the unidirectional K+ transport.

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内耳の特殊体液の恒常性が立脚する上皮イオン輸送の分子構築の解析

上塚学, 緒方元気, 永森收志, 五十棲規嘉, 吉田崇正, 任書晃, 北原糺, 吉川欣亮, 猪原秀典, 金井好克, 日比野浩

日本プロテオーム学会大会要旨集（日本プロテオーム学会（日本ヒトプロテオーム機構）） 2015 191 - 191 2015年

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Functional analysis of Na+,K+,2Cl(-)-cotransporters contributing to the endocochlear potential

Yoshida, Takamasa and Nin, Fumiaki and Ogata, Genki and Uetsuka, Satoru and Komune, Shizuo and Kurachi, Yoshihisa and Hibino, Hiroshi

Journal of Pharmacological Sciences 124 138P - 138P 2014年

ISSN 1347-8613

The mechanism underlying maintenance of the endocochlear potential by the K+ transport system in fibrocytes of the inner ear.

Adachi N, Yoshida T, Nin F, Ogata G, Yamaguchi S, Suzuki T, Komune S, Hisa Y, Hibino H, Kurachi Y

The Journal of physiology （WILEY-BLACKWELL） 591 （ 18 ） 4459 - 72 2013年09月

査読有り, ISSN 0022-3751

The endocochlear potential (EP) of +80 mV in the scala media, which is indispensable for audition, is controlled by K+ transport across the lateral cochlear wall. This wall includes two epithelial barriers, the syncytium and the marginal cells. The former contains multiple cell types, such as fibrocytes, which are exposed to perilymph on their basolateral surfaces. The apical surfaces of the marginal cells face endolymph. Between the two barriers lies the intrastrial space (IS), an extracellular space with a low K+ concentration ([K+]) and a potential similar to the EP. This intrastrial potential (ISP) dominates the EP and represents the sum of the diffusion potential elicited by a large K+ gradient across the apical surface of the syncytium and the syncytium's potential, which is slightly positive relative to perilymph. Although a K+ transport system in fibrocytes seems to contribute to the EP, the mechanism remains uncertain. We examined the electrochemical properties of the lateral wall of guinea pigs with electrodes sensitive to potential and K+ while perfusing into the perilymph of the scala tympani blockers of Na+,K+-ATPase, the K+ pump thought to be essential to the system. Inhibiting Na+,K+-ATPase barely affected [K+] in the IS but greatly decreased [K+] within the syncytium, reducing the K+ gradient across its apical surface. The treatment hyperpolarized the syncytium only moderately. Consequently, both the ISP and the EP declined. Fibrocytes evidently use the Na+,K+-ATPase to achieve local K+ transport, maintaining the syncytium's high [K+] that is crucial for the K+ diffusion underlying the positive ISP.

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Dopamine and full-field illumination activate D1 and D2-D5-type receptors in adult rat retinal ganglion cells

Ogata, G. and Stradleigh, T.W. and Partida, G.J. and Ishida, A.T.

Journal of Comparative Neurology （WILEY-BLACKWELL） 520 （ 17 ） 4032 - 4049 2012年12月

査読有り, ISSN 0021-9967

Dopamine can regulate signal generation and transmission by activating multiple receptors and signaling cascades, especially in striatum, hippocampus, and cerebral cortex. Dopamine modulates an even larger variety of cellular properties in retina, yet has been reported to do so by only D1 receptor-driven cyclic adenosine monophosphate (cAMP) increases or D2 receptor-driven cAMP decreases. Here, we test the possibility that dopamine operates differently on retinal ganglion cells, because the ganglion cell layer binds D1 and D2 receptor ligands, and displays changes in signaling components other than cAMP under illumination that should release dopamine. In adult rat retinal ganglion cells, based on patch-clamp recordings, Ca2+ imaging, and immunohistochemistry, we find that 1) spike firing is inhibited by dopamine and SKF 83959 (an agonist that does not activate homomeric D1 receptors or alter cAMP levels in other systems); 2) D1 and D2 receptor antagonists (SCH 23390, eticlopride, raclopride) counteract these effects; 3) these antagonists also block light-induced rises in cAMP, light-induced activation of Ca2+/calmodulin-dependent protein kinase II, and dopamine-induced Ca2+ influx; and 4) the Ca2+ rise is markedly reduced by removing extracellular Ca2+ and by an IP3 receptor antagonist (2-APB). These results provide the first evidence that dopamine activates a receptor in adult mammalian retinal neurons that is distinct from classical D1 and D2 receptors, and that dopamine can activate mechanisms in addition to cAMP and cAMP-dependent protein kinase to modulate retinal ganglion cell excitability. J. Comp. Neurol. 520: 4032-4049, 2012. (C) 2012 Wiley Periodicals, Inc.

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Thy1 associates with the cation channel subunit HCN4 in adult rat retina.

Partida GJ, Stradleigh TW, Ogata G, Godzdanker I, Ishida AT

Investigative ophthalmology & visual science （ASSOC RESEARCH VISION OPHTHALMOLOGY INC） 53 （ 3 ） 1696 - 703 2012年03月

査読有り, ISSN 0146-0404

PURPOSE. The membrane expression and gene promoter of the glycosylphosphatidylinositol (GPI)-anchored protein Thy1 have been widely used to examine the morphology and distribution of retinal ganglion cells in normal eyes and disease models. However, it is not known how adult mammalian retinal neurons use Thy1. Because Thy1 is not a membrane-spanning protein and, instead, complexes with structural and signaling proteins in other tissues, the aim of this study was to find protein partners of retinal Thy1.
METHODS. Coimmunoprecipitation, immunohistochemistry, confocal imaging, and patch-clamp recording were used to test for association of Thy1 and HCN4, a cation channel subunit, in adult rat retina.
RESULTS. Hyperpolarization of cells immunopanned by an anti-Thy1 antibody activated HCN channels. Confocal imaging showed that individual somata in the ganglion cell layer bound antibodies against Thy1 and HCN4, that the majority of these bindings colocalized, and that some of the immunopositive cells also bound antibody against a ganglion cell marker (Brn3a). Consistent with these results, Thy1 and HCN4 were coimmunoprecipitated by magnetic beads coated with either anti-Thy1 antibody or anti-HCN4 antibody. In control experiments, beads coated with these antibodies did not immunoprecipitate a photoreceptor rim protein (ABCR) and uncoated beads did not immunoprecipitate either Thy1 or HCN4.
CONCLUSIONS. This is the first report that Thy1 colocalizes and coimmunoprecipitates with a membrane-spanning protein in retina, that Thy1 complexes with an ion channel protein in any tissue, and that a GPI-anchored protein associates with an HCN channel subunit protein. (Invest Ophthalmol Vis Sci. 2012; 53: 1696-1703) DOI:10.1167/iovs.11-9307

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Colocalization of hyperpolarization-activated, cyclic nucleotide-gated channel subunits in rat retinal ganglion cells.

Stradleigh TW, Ogata G, Partida GJ, Oi H, Greenberg KP, Krempely KS, Ishida AT

The Journal of comparative neurology （WILEY-BLACKWELL） 519 （ 13 ） 2546 - 73 2011年09月

査読有り, ISSN 0021-9967

The current-passing pore of mammalian hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels is formed by subunit isoforms denoted HCN1-4. In various brain areas, antibodies directed against multiple isoforms bind to single neurons, and the current (I-h) passed during hyperpolarizations differs from that of heterologously expressed homomeric channels. By contrast, retinal rod, cone, and bipolar cells appear to use homomeric HCN channels. Here, we assess the generality of this pattern by examining HCN1 and HCN4 immunoreactivity in rat retinal ganglion cells, measuring I-h in dissociated cells, and testing whether HCN1 and HCN4 proteins coimmunoprecipitate. Nearly half of the ganglion cells in whole-mounted retinae bound antibodies against both isoforms. Consistent with colocalization and physical association, 8-bromo-cAMP shifted the voltage sensitivity of I-h less than that of HCN4 channels and more than that of HCN1 channels, and HCN1 coimmunoprecipitated with HCN4 from membrane fraction proteins. Finally, the immunopositive somata ranged in diameter from the smallest to the largest in rat retina, the dendrites of immunopositive cells arborized at various levels of the inner plexiform layer and over fields of different diameters, and I-h activated with similar kinetics and proportions of fast and slow components in small, medium, and large somata. These results show that different HCN subunits colocalize in single retinal ganglion cells, identify a subunit that can reconcile native I-h properties with the previously reported presence of HCN4 in these cells, and indicate that I-h is biophysically similar in morphologically diverse retinal ganglion cells and differs from I-h in rods, cones, and bipolar cells. J. Comp. Neurol. 519:2546-2573, 2011. (C) 2011 Wiley-Liss, Inc.

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Inhibition of adult rat retinal ganglion cells by D1-type dopamine receptor activation.

Hayashida Y, Rodríguez CV, Ogata G, Partida GJ, Oi H, Stradleigh TW, Lee SC, Colado AF, Ishida AT

The Journal of neuroscience : the official journal of the Society for Neuroscience （SOC NEUROSCIENCE） 29 （ 47 ） 15001 - 16 2009年11月

査読有り, ISSN 0270-6474

The spike output of neural pathways can be regulated by modulating output neuron excitability and/or their synaptic inputs. Dopaminergic interneurons synapse onto cells that route signals to mammalian retinal ganglion cells, but it is unknown whether dopamine can activate receptors in these ganglion cells and, if it does, how this affects their excitability. Here, we show D-1a receptor-like immunoreactivity in ganglion cells identified in adult rats by retrogradely transported dextran, and that dopamine, D-1-type receptor agonists, and cAMP analogs inhibit spiking in ganglion cells dissociated from adult rats. These ligands curtailed repetitive spiking during constant current injections and reduced the number and rate of rise of spikes elicited by fluctuating current injections without significantly altering the timing of the remaining spikes. Consistent with mediation by D-1-type receptors, SCH-23390 [R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine] reversed the effects of dopamine on spikes. Contrary to a recent report, spike inhibition by dopamine was not precluded by blocking I-h. Consistent with the reduced rate of spike rise, dopamine reduced voltage-gated Na+ current (I-Na) amplitude, and tetrodotoxin, at doses that reduced I-Na as moderately as dopamine, also inhibited spiking. These results provide the first direct evidence that D-1-type dopamine receptor activation can alter mammalian retinal ganglion cell excitability and demonstrate that dopamine can modulate spikes in these cells by a mechanism different from the presynaptic and postsynaptic means proposed by previous studies. To our knowledge, our results also provide the first evidence that dopamine receptor activation can reduce excitability without altering the temporal precision of spike firing.

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The frequency of interictal activity was modulated by the generation of the activity per se

Ogata Genki, Natsume Kiyohisa

NEUROSCIENCE RESEARCH 58 S238 - S238 2007年

ISSN 0168-0102

Synchronization Process of Neurons Depends on Giant EPSP of Hippocampal Pyramidal Cells

OGATA Genki, NATSUME Kiyohisa

J Signal Process （〔信号処理学会〕） 10 （ 6 ） 399 - 406 2006年11月

ISSN 1342-6230

Dependence of Neuronal Synchronization Process on the Giant EPSP Pattern of Hippocampal Pyramidal Cells

OGATA Genki, NATSUME Kiyohisa

J Signal Process 10 （ 4 ） 239 - 242 2006年07月

ISSN 1342-6230

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