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provided by Elsevier - Publisher Connector Journal of Otology 2008 Vol. 3 No. 1 · 9 · Review Supporting Cells-a New Area in Cochlear Physiology Study

ZHAO Li-dong, LIU Jun, HU Yin-yan, SUN Jian-he, YANG Shi-ming Institute of Otolaryngology, PLA General Hospital, Beijing 100853, China

Abstract For many years, studies about the cochlea have been mainly focused on sensory cells, i.e. the in⁃ ner hair cell(IHC) and outer hair cell (OHC), and the neuron system. Supporting cells, such as Hensen’s cells and Deiters’cells are less studied. Their physiological functions and other characteristics are not well documented. Nowadays，supporting cells are a new world attracting to scientists' interests. The scope of this review is to detail the biological properties of the supporting cells, mainly Hensen’s cells and Deiters’cells in the cochlea. Studies on this subject will be helpful in understanding physiology of the cochlea, and hopefully provide new approaches in treating diseases of inner ear.

Key words Supporting cells; Cochlea gy properties, relation to the NO/cGMP pathway, Introduction contribution to the cochlear function and their re⁃ Supporting cells in the cochlea include Hensen’s lationship with hair cells. cells, Deiters’cells, inner and outer pillar cells, Structure of Hensen’s cells and Deiters’cells Claudius cells and inner supporting cells (mainly the inner phalangeal cells). The inner and outer pil⁃ General anatomy lar cells lean toward each other at the head, form⁃ Deiters’cells consist of three parts: 1) the cell ing a triangular tunnel, which is filled with corti⁃ body, which forms a cup at the topic face attached lymph. The structure of the organ of Corti is based to the OHC on the basilar membrane, 2) the on this structurally sound geometric object of the straight phalangeal process, extending obliquely triangle 1. The inner hair cells and outer hair cells and passing two or three OHCs, free of mechanical lie on the two side of the Corti’s tunnel respective⁃ contact, and 3) the head of the phalangeal process, ly. The inner phalangeal cells and Deiters’cells forming tight junctions with the apical support the inner and outer hair cells respectively. circumference of the OHC at the level of reticular Hensen’s cells form a Hensen’s bar on the outer laminar 2. There are relatively few organelles such edge of the organ of Corti. Hensen’s cells, the as rough endoplasm reticule, lysosomes and third row OHCs and Deiters’cells together consti⁃ mitochondria in the Deiters cell. The round tute the outer tunnel. Studies on Hensen’s cells nuclear appears to be of low electronic density3. and Deiters’cells have been mainly focused on Consisted of microtubes and microfilaments, the their structure, ion metabolism, electric physiolo⁃ phalangeal process is a prominent structure characteristic of the Deiters’cell. The complex and yet precisely arranged microfilaments play an Correspondence to: ZHAO Li-dong M.D., Institute of Otolaryngology, PLA General Hospital, Beijing, 100853. important role in the micromechanical aspect of E-mail: [email protected] cochlear function. They can transmit mechanical ·10· Journal of Otology 2008 Vol. 3 No. 1 force between cells and coordinate shift of play an important role in recycling and regulating adjacent cells 4. intracellular K + , as well as pH homeostatic mecha⁃ Hensen’s cells are somewhat column like, upper nisms 8. Electrical coupling and dye coupling be⁃ part wide and lower part narrow. Under electronic tween supporting cells are very strong, but cou⁃ microscope, their cytoplasm is thin and endoplas⁃ pling between supporting cells and hair cells are mic reticulum undeveloped, with relatively few mi⁃ relatively weak 9. When injected into Hensen’s tochondria dispersed in the cytoplasm. The copu⁃ cells, Lucifer yellow always diffuses into adjacent lar part is toward the scala media, with numerous Hensen’s cells and often into Deiters’cells. Dyes microvilli, which are 0.4-0.6 micrometer long. A injected into outer hair cells can also diffuse into characteristic of Hensen’s cell is its phagosomes Hensen’s cells and Deiters’cells, as well as neigh⁃ in the cytoplasm, indicating a function of phagocy⁃ boring outer hair cells. Gentamycin can suppress tosis. Lipid droplets are another morphologic fea⁃ conduction between supporting cells by 90% , but ture of Hensen’s cell and most noticeable at the catalase can attenuate this effect. It is assumed third and fourth turns of the cochlear. Hensen’s that gentamycin induces production of oxygen cells at the basal and second turns contain few lip⁃ free radical, which restrain cell coupling between id droplets. The lipid content increase progressive⁃ supporting cells and ruin tuning function of the or⁃ ly at the second and third turns compared to the gan of Corti10.H + and Ca2 + , as well as Calmodulin basal turn. The distribution of lipid droplets is par⁃ antagonists W7 and trifluoperazine（TFP）can also allel to the innervation, suggesting their relation to induce gap junction uncoupling at unchanged glob⁃ the auditory process. The lipid was observed to be al［Ca2+］(i) in Hensen’s cells11, 12. Gap junctions be⁃ expulsed into the endolymphatic space, which tween supporting cells may have another role, i.e. may cause the height of Hensen’s cell to change. to provide a pathway for rapid removal of ions This change may modulate the interaction be⁃ from the region of the sensory cells during sound tween the tectorial membrane and hair cells to fa⁃ conduction in order to maintain sensitivity13. As cilitate conduction of mechanical energy 5. Recent⁃ gap junctions between supporting cells conduct in⁃ ly, observation of fresh human organ of Corti un⁃ formation rapidly while linking adjacent cells to⁃ der scanning electron microscopic revealed that gether, Hensen’s cells may work as a syncytium there was extensive folding of the lateral cell mem⁃ and may be involved in adjusting cochlear func⁃ branes of Hensen’s cells. In addition, there are lat⁃ tion. eral membrane interdigitation between Hensen’s Nerve innervation Cells（HC）and Claudius cells（CC）6. In recent years, some new interesting results have Gap junctions been discovered with synaptophysin and neurofila⁃ There are gap junctions not only among support⁃ ments immunostaining, acetycholine sterase histo⁃ ing cells but also between supporting cells and chemistry and transmission electron microscopy hair cells. Gap junctions between supporting cells technique-there are nerve fibers innervating sup⁃ consist of two identical hemi channels. They are porting cells in many species including the guinea made of connexins that are encoded by connexins pig 14, 15. Most Hensen’s cells in the apical turn re⁃ genes（e.g., Cx26, Cx30, etc.）. Expression of Cx26 ceive innervation at their basal parts, where mito⁃ and Cx30 is not the same throughout in the co⁃ chondria and other organelles such as Golgi com⁃ chlea. In the organ of Corti, its distribution shows plex and dense core vesicles are rich 16. The nerve a threefold longitudinal gradient of reduction from terminals attached to Deiters’and Hensen’s cells the apex to the base. The reduction is more pro⁃ seem to be chemical synapses, which are more nounced in the Deiters cells and pillar cells than in common in the apical turn than in the basal turn of the Hensen’s cells7. These channels of connexins the cochlea. As mentioned above, this is parallel Journal of Otology 2008 Vol. 3 No. 1 ·11· with the lipid droplet volume and distribution in that was mainly carried by Cl-. The Cl- current ap⁃ Hensen’s cells. This structural characteristic may peared to be activated by Ca2+ secondary to Ca2+ in⁃ contribute to the physical properties of the Organ flux（Ca2 + influx-induced Ca2 + release）, as it re⁃ of Corti such as resonance vibration as well as the quired the presence of extracellular Ca2+ and could hearing sensitivity distributed along the basal be suppressed by adding 10 mM 1, 2-bis（2-amino⁃ membrane. These nerve fibers are neither collater⁃ phenoxya）ethane-N, N, N’,N’-tetra acetic acid. al of cholinergic olivo-cochlear fibers nor GA⁃ At the same time, ATP caused Ca2+ release from in⁃ BA-energic olivo-cochlear fibers because few of tracellular Ca2 + stores when Ca2 + was absent in the them are acetyl cholinesterase positive and the extracellular medium. These data suggest that 1) number of these fibers is far greater than GA⁃ there are inotropic and metatropic channels in BA-energic fibers 14, 15. Furthermore, most of these Hensen's cell 29; 2) Hensen’s cell can act as an ad⁃ fibers are not olivo-cochlear originated17. Some justing conditioner of ion concentration in inner scientists suggested that these fibers might be ear liquids (endolymph and perilymph) and of wa⁃ branches of type Ⅱ afferent fibers to outer hair ter homeostasis in the organ of Corti; and 3) in De⁃ cells and form a local axon reflex in the apical iters’cells, Ca2 + may be a intracellular messenger turns of the guinea pig cochlea, so information and contributes to their active mechanic move⁃ could be transmitted among outer hair cells, Deit⁃ ment and therefore affects the micromechanic ers’cells and Hensen’s cells through this loop 18. property of the organ of Corti.

Ion metabolism in supporting cells Electrophysiology of supporting cells

Identified as a neuromodulater or neurotransmit⁃ In the inner ear of guinea pig, the resting mem⁃ ter2, 19, 20, ATP exerts its effects mainly through P2 brane potential of supporting cells is high purinergic receptors. Studies showed that ATP can (range: -50 to -92mV), but their input resistance, induce Potassium current on Hensen’s cells 21, ele⁃ similar to that of glial cells in the neuronal system vate the concentration of free calcium in the inner （typically 0.2 to 48 MΩ 30）, is low（averaged 17.1 and outer hair cells 5, 22-26, and also stimulate Ca2+ re⁃ MΩ）31. Santos-Sacchi reported an input resis⁃ lease from internal stores in Deiters’cells（but not tance of 0.4 MΩ for Hensen’s cells in the guinea in Hensen’s cells27）. ATP mobilized Ca2 + from the pig 32. The discrepancy between different studies phalanges of Deiters’cells, which then propagat⁃ may be attributed to the differences in experimen⁃ ed towards the cell body as a wave, suggestisng tal methods. In alligator lizard, the recorded aver⁃ presence of purinergic receptors in the phalanges. age potential magnitude in the supporting cells There are well-organized cytoskeletons in these is 0.72 mV, which is about 10 fold of the poten⁃ cells, constituted of potentially contractile pro⁃ tial recorded from extracellular space and 1/5 of teins, such as actin microfilaments, microtubules that in sensory cells. This component is non-lin⁃ and tropomyosin28. When the concentration of free early dependent on the intensity of the click calcium in the Deiters’cells was elevated by pho⁃ stimulation 33. When it comes to goldfish, a slow de⁃ to liberation of the caged Ca2+ molecule DM-nitro⁃ polarizing potential was recorded in supporting phen, the head of phalangeal process moved and cells within the saccular macula. It developed and the stiffness of the phalangeal increased2. Being maintained during the sound stimulation and had a held at a negative potential, ATP could activate a latency of less than 5 ms. It rose rapidly upon stim⁃ 2 + biphasic inward current concomitant with［Ca ］i ulation and reached a plateau in 100 to 200 ms. increase in isolated Hensen's cell. The initial cur⁃ Electrical stimuli on the saccular nerve trunk rent could be activated within 50ms and had a re⁃ could also evoke a slow depolarization and sup⁃ versal potential near 0 mV, followed by a current press the excitatory post-synaptic currents while ·12· Journal of Otology 2008 Vol. 3 No. 1 the microphonics were little influenced. The origin supporting cells cannot be ruled out, since the re⁃ of slow depolarization was assumed to be the sup⁃ ceptor current may flow to supporting cells porting cells in the saccular macula and the reason through gap junctions, given the fact that the wave for this phenomenon might be an elevation of ex⁃ phase between outer hair cells and supporting tracellular K + by a few millimoles. This suggests cells is similar. the excitability of supporting cells34. In guinea pig Regulation of supporting cell physiology by the Ni⁃ cochlea, the resting membrane potentials in differ⁃ tro Oxide/cyclic GMP (NO/cGMP) pathway ent supporting cells are as following, Hensen’s Nitric Oxide(NO) is a gaseous product synthesized cell: -68±8mV; Deiters’cells: -70± 12mV; outer by nitric oxide syntheses(NOS). When NO is re⁃ pillar cells: -77 ± 16 mV; inner pillar cells: -79 ± 6 leased from the cells in which it is synthesized, it mV. When a tone burst was given, sinusoidal re⁃ diffuses across the neighboring cell membrane, act sponse could be detected in the third and forth on the enzyme‘soluble guanylate cyclase’（SGC） turn cells and in fluid spaces. The AC potential and result in generation of Cyclic Guanosine Mono⁃ showed non-linear features when the stimulus phosphate（cGMP）, which then mediate cell physi⁃ tone increased. Magnitude responses of support⁃ ological responses by phosphorylating proteins ing cells and hair cells were different, providing in⁃ through specific protein kinase, the cGMP depen⁃ sights into the origin of supporting cell responses. dent Protein kinase-1. This is the NO/cGMP path⁃ Responses of inner and outer hair cells were 33 way 52. NO can cause diverse effects such as mus⁃ and 12 times greater than those of supporting cells cle relaxation, gut peristalsis, neurotransmission respectively at high stimulus sound levels and 10 and hormonal secretion. There are three isoforms and 7 times greater at low stimulus sound level, re⁃ of NOS, namely the neuronal NOS（nNOS）, the in⁃ spectively. The largest peak-to-peak response po⁃ ducible NOS （iNOS） and the endothelial NOS tential magnitude was 41.5mV for inner hair cell, （eNOS）respectively. The nNOS, which was first 34.8mV for outer hair cell and 5.9mV for Hensen’s described in rat brain neurons and also named NOS cell. The AC response of Hensen’s cell was small⁃ Ⅰ（or NOS 1）, is calcium dependent. In the cerebel⁃ er than that of other supporting cell types. The lum, nNOS is highly activated 53. NO produced by wave forms recorded in these cells were similar, in⁃ this isoforms can act as a neurotransmitter or neu⁃ dicating qualitative similarities in the frequency re⁃ romodulater. It may be involved in the induction of sponse of supporting cells and hair cells31. Studies long term depression in the central nervous sys⁃ of the basilar papilla of the lizard and the organ of tem54 and of smooth muscle relaxation, gut peristal⁃ Corti in guinea pig suggest electric coupling be⁃ sis, penile erection and dilation of the trachea in tween hair cells and supporting cells32, 35-41. Howev⁃ the peripheral nervous system55. In the cochlea, er this is controversial, as some anatomical works nNOS can be expressed in inner and outer hair suggest the absence of gap junctions between hair cells, ganglion cells, spiral ligament, supporting cells and supporting cells42-46. Research has demon⁃ cells and the nerve endings innervating outer hair strated that the sound induced potentials recorded cells 56, 57. It has also been found in the sensory epi⁃ in the supporting cells reflect the activity of hair thelium of the maculae and cristae ampullares58. cells, that is to say that the hair-cell generated cur⁃ Some studies suggest that neurons in the superior rent flows into adjacent supporting cells 34, 47-51. oliver complex may be additional source of nNOS While there is evidence suggesting the absence of in the cochlea 59. The second principle enzyme in an electronic coupling between inner hair cells the NO/cGMP pathway, soluble guanylate cyclase and abutting supporting cells such as inner pillar （SGC）, localizes in Hensen’s cells, Deiters’cells, cells, inner phalangeal cells 31. Existence of gap vascular pericytes in the lateral wall and sensory junctions between outer hair cells and neighboring neurothelium, but not in inner or outer hair cells. Journal of Otology 2008 Vol. 3 No. 1 ·13·

This is in agreement with the localization of the many places includig the pillar cells, hair cells and cGK-Ⅰ 60. It is indicated that when the NO is pro⁃ small junctional complexes of Deiters’cells. As duced and released from various types of cells in part of a cytoprotective stress response after noise the cochlea, it can diffuse into supporting cells exposure, eNOS can also be upregulated and acti⁃ and lateral wall, act on the sGC, induce the produc⁃ vated by calcium66. tion of cGMP and regulate supporting cell physiol⁃ Regulation of inner ear function by supporting cells ogy and microcirculation in the cochlea 56. As many studies have revealed, ATP can cause free Studies have showed that when exposed to in⁃ calcium concentration rise in several kinds of cells tense sound, the outer region of the organ of the （including supporting cells）in the cochlear 22, 26, Corti including outer hair cells, the third row Deit⁃ 27. Studies show that NO donors such as diethyl⁃ ers’cells and its attached Hensen’s cells move in amine NONOate and sodium nitro prusside or the toward the center of the cochlear turn accompa⁃ cGMP analog such as 8-bromo-cGMP can attenu⁃ nied by sensitivity reduction of the cochlea to the ate the increased calcium concentration induced test sound. The hinge point was located between by ATP. When the NO/cGMP pathway is blocked the third row of outer hair cells and Deiters’cells. by inhibitors of sGC（LY83583）or Protein kinase G The inner part of the cochlea including inner hair （Rp-8- bromo-cGMP or KT5823）, the response cells and the inner and pillar cells are relatively sta⁃ to ATP can be restored 61, suggesting that NO/ ble, which may be because that supporting cells cGMP/protein kinase G Pathway can modulate cal⁃ are more elastic then inner hair cells 67. Given the cium in the supporting cells and supporting cell sensitivity reduction accompanied by structural physiology. Our research in 2003 indicated that contraction, the data may indicate that supporting ATP was involved in the activation of NO/cGMP cells contribute to the control of hearing sensitivi⁃ pathway by elevating Ca2 + concentration in the cy⁃ ty68. Evidence has shown that mechanical and toplasm of the cochlea. Subsequently, NO/cGMP acoustic overstimulation can cause intracellular pathway seemed to exert a negative action on the calcium concentration rise, accompanied by dy⁃ effects of ATP, suggesting the existence of an ATP/ namic contractions of the hearing organ 69 and the Ca-NO/cGMP pathway in the guinea pig cochlea. concentration changes of calcium in outer hair In other words, the NO/cGMP pathway may be ex⁃ cells and Deiters’cells, which can induce motility tended to include ATP and its binding with puriner⁃ of the OHC and flexion of phalangeal process2, 69. gic receptors 62． The mechanism for contraction of the organ of Inducible NOS（iNOS）is another kind of isoform Corti induced by intense sound or mechanical of NOS in the cochlear, although it is not ex⁃ stimulation may be a change of concentration of in⁃ pressed under physiological conditions 63. Endotox⁃ tracellular calcium, leading to motility of outer in or inflammation-causing factors can induce iN⁃ hair cells and Deiters’cells. This phenomenon may OS expression in the wall of blood vessels in the be a protective reflex against sound trauma, since spiral ligament and the modiolus, in supporting a stimulation intensity exceeding the protective ca⁃ cells, the limbus, nerve fibers and some of the peri⁃ pacity of the hearing organ can damage outer hair karya of the spiral ganglion 57, 64, 65. These data indi⁃ cells. Such observations may be helpful in under⁃ cate that large amounts of NO produced by iNOS standing why noise almost always injures outer may have neurotoxic effects on the inner ear dur⁃ hair cells, while inner hair cells are less frequently ing bacterial and viral infections. The endothelial injured. This may due to distortion of the cell skel⁃ NOS（eNOS）can also be found in the in the cells eton in Deiters’cells, which likely involves Ca2+, as of the Organ of Corti. Following acute noise trau⁃ the rise of Ca2 + concentration can stiffen the pha⁃ ma, increased levels of eNOS can be identified in lange 2. ·14· Journal of Otology 2008 Vol. 3 No. 1

Second edition ed. 1984: WILLIAMS&WILKINS. 101-116. Supporting cells and hair cell regeneration 2 Dulon D, Blanchet C, and Laffon E. Photo-released in⁃ 2 + The ability of self-repair and regeneration of the tracellular Ca evokes reversible mechanical responses in supporting cells of the guinea-pig organ of Corti. Biochem. mammalian inner ear is very limited, causing sen⁃ Biophy Res Com, 1994; 201(3): 1263-1269. sory hair cells damage to result in permanent hear⁃ 3 Zhixian W, and Yitong Z. Atlas of the cochlea by light 70 ing loss . In contrast, hair cells in the avian inner and electron microscopy. First edition ed. 1988: Tianjin Sci⁃ 71 ear can regenerate after damage . Some research⁃ ence & Technology publishing House. p:140-141. ers supposed that there might be a population of 4 Mogensen M, Hendersen CG, Mackie JB, et al. Keratine dedicated precursors or stem cells in the avian in⁃ filament deployment and cytosckeletal networking in senso⁃ ner ear, but histological and anatomical studies ry epithelium that vibrates during hearing. Cell Motil Cyto⁃ have failed in identifying such cells. Some studies skeleton, 1998; 41(2): 138-153. 5 Merchan MA, Merchan JA, and Ludena MD. Morpholo⁃ suggested that there were signs indicating the con⁃ gy of Hensen's Cells J Anat, 1980; 131(3,pp): 519-523. version of supporting cells to hair cells. Studies 6 Glueckert R, Pfaller K, Kinnefors A, et al. Ultrastructure have shown unpaired and immature cells in the of the normal human organ of Corti. New anatomical find⁃ basilar papilla in the damaged inner ear of a bird. ings in surgical specimens. Acta Oto-laryngology, 2005; 125 Some of these cells have the phenotypic features (5): 534-539. of both hair cells and supporting cells, suggesting 7 Zhao H, and Yu N. Distinct and gradient distributions of their being a product of the converting process Connexin26 and Connexin30 in the cochlear sensory epithe⁃ from the supporting cells to hair cells 72, 73. They lium of guinea pigs. Journal of Comparative Neurology, may have undergone a procession of de-differenti⁃ 2006; 499(3): 506-518. 8 Kammen-Jolly K., chiki H, Scholtz AW, et al. Connexin ation and re-entered cell cycle in order to regener⁃ 26 in human fetal development of the inner ear. Hear Res, ate sensory hair cells 74. As for the mammal, cells 2001; 160: 15-21. possessing feature of both supporting cells and 9 ZWISLOCKI J, Nguyen M. Ionic Coupling Among Cells hair cells have also been identified in the presence in the Organ of Corti. Hearing Research, 1992; 57(2): 75 of hair cell damage . Studies have showed that cy⁃ 175-194. clin-dependent kinase（CDKs）are involved in nor⁃ 10 Todt I, Ngezahayo A, Ernst A. Inhibition of gap junc⁃ mal development of the organ of Corti. Inhibition tional coupling in cochlear supporting cells by gentamicin. of CDKs is sufficient to trigger differentiation of Pflugers Archiv-European Journal of Physiology, 1999; 438 hair cells and corresponding supporting cells from (6): 865-867. 11 Sato Y, Handa T, and Matsumura M. Gap junction Hensen’s cell progenitors and/or from progenitors change in supporting cells of the organ of Corti with ryano⁃ located in the greater epithelial ridge area 76. Exper⁃ dine and caffeine. Acta Otolaryngol, 1998; 118(6): 821-825. iment of Math1 over expression has showed that 12 Blodow A, Ngezahayo A, Ernst A, et al. Calmodulin an⁃ nonsensory cells in the mature cochlea may be tagonists suppress gap junction coupling in isolated Hensen able to generate new hair cells 77. Study of the fetal cells of the guinea pig cochlea. Pflugers Archiv-European rat organ of Corti explants also indicates that Journal of Physiology, 2003; 446(1): 36-41. Hensen’s cells retain the capacity to differentiate 13 Forge A, Becker D, Casalotti S, et al. in Novartis. into OHCs, or into Deiters’cells78. Although these Found. Symp, 1999; 219: 134-150; discussion 151-156. 14 Eybalin M, Parnaud C, Geffard M, et al. Immunoelec⁃ observations are preliminary, converting support⁃ tron microscopy identifies several types of GABA contain⁃ ing cells into hair cells provides a potential new ing efferent synapses in the guinea pig organ of Corti. Neu⁃ way to treat hearing loss caused by the loss of in⁃ roscience, 1988; 24: 29-38. ner and outer hair cells. 15 Merchan-Perez A, Gil-Loyzaga P, Lopea-Sanchez J, et al. Ontogeny of aminobutyric acid in efferent fibers to the References rat cochlea. Dev Brain Res, 1993; 76: 33-41. 1 Durrant JD, and Lovrinic Jh, Base of Hearing Science. 16 Burgess B, Adams J, and Nadol J. Morphologic evi⁃ Journal of Otology 2008 Vol. 3 No. 1 ·15· dence for innervation of Deiters' and Hensen's cells in the ol, 1996; 271(6 Pt 1): C1817-1827. guinea pig. Hearing Research, 1997; 108(1-2): 74-82. 30 Somjen GG. Electrophysiology of neuroglis. Ann Rev 17 Smith CA, and Haglan BJ. Golgi stains on the guinea Physiol, 1975; 37: 163-190. pig organ of Corti. Acta Otolaryngo(Stockh.), 1973; 75: 31 Oesterle EC, and Dallos P. Intracellular recordings 203-210. from supporting cells in the guinea-pig cochlea: AC poten⁃ 18 Fechner F, Burgess BJ, Adams JC, et al. Dense innerva⁃ tials. J Acoust Soc Am, 1989; 86(3): 1013-1032. tion of Deiters' and Hensen's cells persists after chronic 32 Santos-Sacchi J. Electrical coupling differs in the in vi⁃ deefferentation of guinea pig cochleas. Journal of Compara⁃ tro and in vivo organ of Corti. Hear Res, 1987; 25:227-232. tive Neurology, 1998; 400(3): 299-309. 33 Ristensen K, and Weiss T. Supporting-cell and extracel⁃ 19 Shigemoto T, and Ohmori H. Muscarinic agonists and lular responses to acoustic clicks in the free-standing re⁃ ATP increase the intracellular Ca2 + concentration in chick gion of the alligator lizard cochlea. Hear-Res, 1982；8(3): cochlear hair cells J Phsiol, 1990; 420: 127-148. 295-315. 20 Munoz DJB, Throne PR, Housley GD, et al. Adenosine 34 Furukawa T. Slow depolarizing response from support⁃ 5'-triphosphate(ATP) concentrations in the endolymph and ing cells in the goldfish saccule. J Physiol Lond, 1985; 366: perilymph of the guinea pig cochlea. Hear Res, 1995; 90: 107-117. 119-125. 35 Santos-Sacchi J. Dye coupling in the organ of Corti. 21 Li XQ, Li JX, Yu H, et al. [Inhibition of nifedipine on in⁃ Cell Tissue Res, 1986; 245(3): 525-529. ward current of Hensen cell induced by ATP]. Zhonghua Er 36 Santos-Sacchi J. A re-evaluation of cell coupling in Bi Yan Hou Tou Jing Wai Ke Za Zhi, 2007; 42(3): 217-21. the organ of Corti. Hear Res, 1984; 14: 203-204. 22 Sugasawa M., Erostegui C., and Blanchet C., ATP acti⁃ 37 Santos-Sacchi J. The effects of cytoplasmic acidifica⁃ vates non-selective cation channels and calcium release in tion upon electrical coupling in the organ of Corti. Hear inner hair cells. J.Physiol. Lond., 1996; 491(Pt-3): 707-718. Res, 1985; 19: 207-215. 23 Nilles R, Jörlebark L., Zenner H.P., et al., ATP induced 38 Santos-Sacchi J. Temperature dependence of electri⁃ cytoplasmic[Ca2 + ] increase in isolated cochlear outer hair cal coupling in the organ of Corti. Assoc Res Otolaryngol cells. Involved receptor and channel mechanisms. Hear Abstr, 1986; 9: 30-31. Res, 1994；73(1): 27-34. 39 Santos-Sacchi J. Whole cell studies of gapo junctional 24 Housely GD, Kanjhan R, Raybould NP, et al. Expres⁃ communication in supporting cells of the organ of Corti. As⁃ sion of the P2X2 receptor subunit of the ATP-Gated ion soc Res Otolaryngol Abstr, 1988; 11: 24-25. channel in the cochlea: implications for sound transduction 40 Santos-Sacchi J, and Dallos P. Intracellular communi⁃ and auditory neurotransmission. J Neurosci, 1999; 19(19): cation in the supporting cells of the organ of Corti. Hear 8377-8388. Res, 1983; 9: 317-326. 25 Van-den-Abbele-T, and Tran-Ba-Huny-P Teulon J, 41 Baird I. Biology of the Reptilia. 2 ed, ed. C.G.a.T.S. Par⁃ Modulation by purines of Calcium-activated non-selective sons. 1970; New York. 193. cation channels in the outer hair cells of the guinea pig co⁃ 42 Janke K. The fine structure of freeze-fractured intra⁃ chlea. J Physiol Lond, 1996; 494(Pt-1): 77-89. cellular junctions in the guinea pig inner ear. Acta Otolaryn⁃ 26 Ashmore JF, and Ohmori H. Control of intracellular cal⁃ gol. Suppl, 1975; 336: 1-140. cium by ATP in isolated outer hair cells of the guinea-pig 43 Hama K, and Saito K. Gap junctions between the sup⁃ cochlea. J Physiol Lond, 1990; 428: 109-131. porting cells in some acoustico-vestibular receptors. J Neu⁃ 27 Dulon D, Moataz R, and Mollard P, Characterization of rocytol, 1997; 6: 1-12. Ca(2+) signals generated by extracellular nucleotides in sup⁃ 44 Nadol JB, Intracellular junctions in the organ of Corti. porting cells of the organ of Corti. Cell Calcium, 1993; 14 Ann Otol, 1978; 87: 78-80. (3): 245-254. 45 Gulley RL, and Reese TS. Intracellular junctions in the 28 Slepecky N，and Chamberlain S. Tropomyosin co-lo⁃ reticular lamina of the organ of Corti. J Neurocytol, 1976; 5: calizes with actin microfilaments and microtubules within 479-507. supporting cells in the inner ear. Cell Tissue Res, 1987; 248: 46 Iurato S, Franke K, Luciano L, et al. Intracellular junc⁃ 63-66. tions in the organ of Corti as revealed by freeze fracturing. 29 Sugasawa M, Erostegui C, Blanchet C, et al. ATP acti⁃ Acta Otolaryngol, 1976; 82: 57-69. vates a cation conductance and Ca(2 + )-dependent Cl- con⁃ 47 Baden-Kristensen K, and weiss TF. Supporting cells ductance in Hensen cells of guinea pig cochlea. Am J Physi⁃ and extracellular responses to acoustic clicks in the ·16· Journal of Otology 2008 Vol. 3 No. 1 free-standing region of the alligator lizard cochlea. Hear 62 ZHAO L., Yingli Li, Ning Li, et al. Activating efect of Res, 1982; 8: 295-315. ATP on NO／cGM P pathway in guinea pig cochlea. Acta 48 Goodman DA, Smith RL, and Chamberlian SC. Intracel⁃ Physiologica Sinica, 2003; 55(6): 658-662. lular responses in the organ of Corti of the gerbil. Hear Res, 63 Gosepath K, Gath I, Maurer J, et al. Characterization of 1982，7: 161-179. nitric oxide synthase isoforms expressed in different struc⁃ 49 Goodman DA. Intracellular recordings from inner hair tures of the guinea pig cochlea. Brain Res., 1997; 747(1): cells in the gerbil. 1983; Syracuse University. 26-33. 50 Russel IJ, and Sellick PM. Intracellular studies of hair 64 Watanabea K, et al. Changes of the compound action cells in the mammalian cochlea. J Physiol(London), 1978; potential (CAP) and the expression of inducible nitric oxide 284: 261-290. synthase (iNOS/NOS II) in the cochlea under the inflamma⁃ 51 Weiss TF, Peake WT, Ling A, et al. Which structure s de⁃ tory condition. Hear Res, 2000, 145(12): 149-155. termine frequency selectivity and tonotopic organization of 65 Michel O, Hessb A, Zumegenb C, et al. Expression of in⁃ vertebrate cochlear nerve fibers? Evidence from the alliga⁃ ducible nitric oxide synthase(iNOS/NOS Ⅱ) in the hydropic tor lizard. Evoked Electrical Activity in the Auditory Ner⁃ cochlea of guinea pigs. Hear Res, 2000; 143(1-2). vous System, ed. R.F. Nauton. 1978; New York: Academic, 66 Heinrich U, Selivanova O, Feltens R, et al. Endothelial New York. p. 91-112. nitric oxide synthase upregulation in the guinea pig organ 52 Fessenden JD, and Schacht J. The nitric oxide /cyclic of Corti after acute noise trauma. Brain Research, 2005, GMP pathway: a potential major regulations of cochlear 1047(1): 85-96. physiology. Hear Res, 1998; 118: 168-176. 67 Sugawara M, Ishida Y, and Wada H. Mechanical proper⁃ 53 Bredt DS, Hwang PM, Glatt CE, et al. Cloned and ex⁃ ties of sensory and supporting cells in the organ of Corti of pressed nitric oxide synthase structurally resembles cyto⁃ the guinea pig cochlea - study by atomic force microscopy. chrome P-450 reductase. Nature, 1991; 351: 714-718. Hear Res, 2004; 192(1-2): 57-64. 54 Shibuki K, and Okada D. Endogenous nitric oxide re⁃ 68 Flock A, Flock B, Fridberger A, et al. Supporting cells lease required for long-term synaptic depression in the cer⁃ contribute to control of hearing sensitivity. Journal of Neu⁃ ebellum. Nature, 1991; 349: 326-328. roscience, 1999; 19(11): 4498-4507. 55 Brenman JE, and Bredt DS. Nitric oxide signaling in the 69 Fridberger A, Flock Å., Ulfendahl M, et al. Acoustic nervous system. Methods Enzymol, 1996; 269: 119-129. overstimulation increases outer hair cell Ca2 + concentra⁃ 56 Fessenden J, and Schacht J. Localization of soluble gua⁃ tions and causes dynamic contractions of the hearing or⁃ nylate cyclase activity in the guinea pig cochlea suggests in⁃ gan. Proc. Natl. Acad Sci USA, 1998; 95: 7127-7132. volvement in regulation of blood flow and supporting cell 70 Girod D. and Rubel E. Hair cell regeneration in the avi⁃ physiology. Journal of Histochemistry & Cytochemistry, an cochlea: if it works in birds, why not in man? ENT J, 1997; 45(10): 1401-1408. 1991; 70(6): 343-353. 57 Hess A, Bloch W, Huverstuhl J, et al. Expression of in⁃ 71 Tsue T, Osterle E, and Rubel E. Hair cell regeneration ducible nitric oxide synthase(iNOS/NOS Ⅱ) in the cochlea in the inner ear. Otolaryngol. Head Neck Surg, 1994; 111: of guinea pigs after intratympanical endotoxin treatment. 281-301. Brain Res, 1999; 830(1): 113-122. 72 Adler H, Komeda M, and Rapheal Y. Further evidence 58 Hess A, BlochW, Arnhold S, et al. Nitric oxide synthase for supporting cell conversion in the damage avian basilar in the vestibulocochlear system of mice. Brain Res, 1998; papilla. Int. J Dev Neurosci, 1997; 15(4-5): 375-385. 813(1): 97-102. 73 Sun J, Wang J, and Li G. DNA replication and cellular 59 Riemann R, and Reuss S. Nitric oxide synthase in iden⁃ proliferation in the noise damaged basilar papillar of tified olivocochlear projection neurons in rat and guinea chicks. Chung Hua Erh Pi Yen Hou Ko Tsa Chih. 1996；31 pig. Hear Res, 1999; 135(1-2): 181-189. (6): 344-347. 60 Tian F, Fessenden J, and Schacht J. Cyclic GMP-de⁃ 74 Warchol ME. Characterization of supporting cell pheno⁃ pendent protein kinase-I in the guinea pig cochlea. Hear type in the avian inner ear: Implications for sensory regener⁃ Res, 1999; 131(1-2): 63-70. ation. Hearing Research, 2007; 227: 11-18. 61 Takeshi M, and Schacht J. Nitric oxide/cyclic GMP 75 Li L and Forge A. Morphological evidence for support⁃ pathway attenuates ATP-evoked intracellular calcium in⁃ ing cell to hair cell conversion in the mammalian utricular crease in supporting cells of the guinea pig cochlea. J Com⁃ macula. Int J Dev Neurosci, 1997; 15(4-5): 433-446. par Neurol, 2000; 423: 452-461. 76 Malgrange B, Knockaert M, Belachew S, et al. Inhibi⁃ Journal of Otology 2008 Vol. 3 No. 1 ·17· tion of cyclin-dependent kinases induces differentiation of 78 Malgrange B., Thiry M., Van De Water TR., et al., Epi⁃ supernumerary hair cells and Deiters' cells in the develop⁃ thelial supporting cells can differentiate into outer hair cells ing organ of Corti. Faseb Journal, 2003; 17(12): 2136-2161. and Deiters' cells in the cultured organ of Corti. Cellular 77 Kawamoto K, Ishimoto S Minoda R, et al., Math1 gene and Molecular Life Sciences, 2002; 59(10): 1744-1757. transfer generates new cochlear hair cells in mature guinea pigs in vivo. Journal of Neuroscience, 2003; 23(11): （Received July 9, 2008） 4395-4400.