Deafness induced by Connexin 26 (GJB2) deficiency is not determined by endocochlear potential (EP) reduction but is associated with cochlear developmental disorders

Connexin 26 (Cx26, GJB2) mutations are the major cause of hereditary deafness and are responsible for >50% of nonsyndromic hearing loss. Mouse models show that Cx26 deficiency can cause congenital deafness with cochlear developmental disorders, hair cell degeneration, and the reduction of endocochlear potential (EP) and active cochlear amplification. However, the underlying deafness mechanism still remains undetermined. Our previous studies revealed that hair cell degeneration is not a primary cause of hearing loss. In this study we investigated the role of EP reduction in Cx26 deficiency-induced deafness. We found that the EP reduction is not associated with congenital deafness in Cx26 knockout (KO) mice. The threshold of auditory brainstem response (ABR) in Cx26 KO mice was even greater than 110 dB SPL, demonstrating complete hearing loss. However, the EP in Cx26 KO mice varied and not completely abolished. In some cases, the EP could still remain at higher levels (>70 mV). We further found that the deafness in Cx26 KO mice is associated with cochlear developmental disorders. Deletion of Cx26 in the cochlea before postnatal day 5 (P5) could cause congenital deafness. The cochlea had developmental disorders and the cochlear tunnel was not open. However, no congenital deafness was found when Cx26 was deleted after P5. The cochlea also displayed normal development and the cochlear tunnel was open normally. These data suggest that congenital deafness induced by Cx26 deficiency is not determined by EP reduction and may result from cochlear developmental disorders.     

The effect of PAF in the cochlea of guinea pigs

The influence of 10(-10) and 10(-9) M PAF/animal given into the jugular vein over 30 sec on inner ear potentials, i.e. endolymphatic potential (EP), summating potential (SP) and cochlear microphonics (CM) was investigated. The EP showed the most pronounced changes. When infusing a specific PAF receptor antagonist, ginkgolide B, or the TXA2 receptor antagonist, sulotraban, before the the infusion of PAF, the changes in cochlear potentials could be completely prevented. A second TXA2 receptor antagonist, daltroban, did not effectively prevent PAF actions. It is hypothesized that these PAF effects are due to an interference with ion transport in the non-sensory structures of the inner ear.     

The group delay and suppression pattern of the cochlear microphonic potential recorded at the round window

Background

It is commonly assumed that the cochlear microphonic potential (CM) recorded from the round window (RW) is generated at the cochlear base. Based on this assumption, the low-frequency RW CM has been measured for evaluating the integrity of mechanoelectrical transduction of outer hair cells at the cochlear base and for studying sound propagation inside the cochlea. However, the group delay and the origin of the low-frequency RW CM have not been demonstrated experimentally.

Methodology/Principal Findings

This study quantified the intra-cochlear group delay of the RW CM by measuring RW CM and vibrations at the stapes and basilar membrane in gerbils. At low sound levels, the RW CM showed a significant group delay and a nonlinear growth at frequencies below 2 kHz. However, at high sound levels or at frequencies above 2 kHz, the RW CM magnitude increased proportionally with sound pressure, and the CM phase in respect to the stapes showed no significant group delay. After the local application of tetrodotoxin the RW CM below 2 kHz became linear and showed a negligible group delay. In contrast to RW CM phase, the BM vibration measured at location ∼2.5 mm from the base showed high sensitivity, sharp tuning, and nonlinearity with a frequency-dependent group delay. At low or intermediate sound levels, low-frequency RW CMs were suppressed by an additional tone near the probe-tone frequency while, at high sound levels, they were partially suppressed only at high frequencies.

Conclusions/Significance

We conclude that the group delay of the RW CM provides no temporal information on the wave propagation inside the cochlea, and that significant group delay of low-frequency CMs results from the auditory nerve neurophonic potential. Suppression data demonstrate that the generation site of the low-frequency RW CM shifts from apex to base as the probe-tone level increases.     

Multiple specializations in the peripheral auditory system of the CF-FM bat,Pteronotus parnellii

Summary Cochlear microphonic (CM) and evoked neural potentials (N₁) were recorded from the cochlear aqueduct of awakePteronotus parnellii. The CM audiograms obtained with continuous sounds had more or less uniform thresholds except for a sharp threshold notch at about 60 kHz (Fig. 1). When brief tone bursts were presented, the envelopes of the CM responses were always similar to the envelopes of the applied signals except when tone bursts having frequencies at or close to the frequency of the tuned sensitivity notch were presented (i.e., 59–63 kHz). The CM rise-decay times for frequencies around 60kHz were much longer than those of the presented signals (Fig. 2). The prolonged decay times are thought to be due to the ringing of the basilar membrane resulting from a mechanical resonance in the cochlea.The evoked neural potential audiograms (N₁-on and N₁-off responses) differed considerably from the CM audiogram. Of particular importance is the N₁-off audiogram which exhibited very sharp tuning in four frequency regions: 31–33 kHz, 60–63 kHz, 71–73 kHz, and 91–92 kHz (Fig. 5). The frequencies evoking the lowest thresholds of the CM and N₁-off (in the 60 kHz region) were either identical or differed by only 100–400 Hz.The sharp tuning in the 60 kHz region of both the CM and N₁ audiograms could be eliminated by presenting 90–100 dB continuous sounds for one min but only if the signal frequency was equal to the tuned frequency of the CM audiogram (Figs. 8–13). Presenting intense sounds having frequencies above or below the tuned 60kHz region had no effect on the audiogram. The overstimulation procedure had remarkably specific effects on the CM and N₁-off audiograms causing the greatest threshold increases at the 60 kHz tuned frequency and progressively smaller threshold changes on the slopes of the tuned notch.Assuming that the sharp changes of the N₁-off thresholds reflect some important underlying mechanism, the N₁-off audiograms demonstrate multiple specializations in the peripheral auditory system ofPteronotus with the bat possessing at least three and possibly four sharply tuned regions. With regard to mechanism, the tuned notch in the CM audiogram, the curious CM rise-decay times evoked by tone bursts, and the ease with which the 60 kHz sensitivity notch can be eliminated all argue strongly in favor of a mechanical resonance in the cochlea which is responsible for the sharp tuning around 60 kHz. On the other hand, the absence of tuned notches in the 30 kHz and 90 kHz regions of the CM audiogram together with the absence of any discernable ringing of the CM potentials evoked by 30 kHz and 90 kHz tone bursts both argue against a resonance mechanism for the tuning at these harmonically related frequency regions. Finally, the fact that overstimulating the 60 kHz region had no discernable effect on the N₁-off tuning at 30 kHz and 90 kHz demonstrates that the mechanism responsible for the tuned regions at 30 kHz and 90 kHz are independent of the resonance feature of the cochlea at 60 kHz.Abbreviations BF best frequency - CF constant frequency - CM cochlear microphonics - CM-aft after-response of the CM - FM frequency modulated - N ₁ evoked neural potentials We thank Professor Alvin Novick for the generous support provided during the conduct of these experiments. We also thank Professor Gerhard Neuweiler and Dr. Gerd Schuller for their helpful comments and suggestions. Supported by PHS Grant NB7616 11.     

Labile cochlear tuning in the mustached bat

Summary Acoustic stimuli near 60 kHz elicit pronounced resonance in the cochlea of the mustached bat (Pteronotus parnellii parnellii). The cochlear resonance frequency (CRF) is near the second harmonic, constant frequency (CF2) component of the bat's biosonar signals. Within narrow bands where CF2 and third harmonic (CF3) echoes are maintained, the cochlea has sharp tuning characteristics that are conserved throughout the central auditory system. The purpose of this study was to examine the effects of temperature-related shifts in the CRF on the tuning properties of neurons in the cochlear nucleus and inferior colliculus.Eighty-two single and multi-unit recordings were characterizedin 6 awake bats with chronically implanted cochlear microphonic electrodes. As the CRF changed with body temperature, the tuning curves of neurons sharply tuned to frequencies near the CF2 and CF3 shifted with the CRF in every case, yielding a change in the unit's best frequency. The results show that cochlear tuning is labile in the mustached bat, and that this lability produces tonotopic shifts in the frequency response of central auditory neurons. Furthermore, results provide evidence of shifts in the frequency-to-place code within the sharply tuned CF2 and CF3 regions of the cochlea. In conjunction with the finding that biosonar emission frequency and the CRF shift concomitantly with temperature and flight, it is concluded that the adjustment of biosonar signals accommodates the shifts in cochlear and neural tuning that occur with active echolocation.Abbreviations BF best frequency - CF characteristic frequency - CF2, CF3 second and third harmonic, constant frequency components of the biosonar signal - CM cochlear microphonic - CN cochlear nucleus - CRF cochlear resonance frequency - IC inferior colliculus - MT minimum threshold - OAE otoacoustic emission - Q_10dB BF (or CF) divided by the response bandwidth at 10 dB above MT     

Adaptation of Cochlear Amplification to Low Endocochlear Potential

Endocochlear potential (EP) is essential for cochlear amplification by providing the voltage source needed to drive outer hair cell (OHC) transducer current, which leads to OHC electromechanical force. An early study using furosemide to reversibly reduce EP showed that distortion product otoacoustic emissions (DPOAEs) recovered before EP. This indicated that cochlear amplification may be able to adjust to a new, lower EP. To investigate the mechanism of this adjustment, the extracellular OHC voltage, which we term local cochlear microphonic (LCM), was measured simultaneously with DPOAE and EP while using intraperitoneal (IP) and intravenous injection of furosemide to reversibly reduce EP. With IP injection, the DPOAEs recovered fully, whereas the EP was reduced, but LCM showed a similar time course as EP. The DPOAEs failed to accurately report the variation of cochlear amplification. With intravenous injection, for which both reduction and recovery of EP are known to occur relatively quickly compared to IP, the cochlear amplification observed in LCM could attain nearly full or even full recovery with reduced EP. This showed the cochlea has an ability to adjust to diminished operating condition. Furthermore, the cochlear amplifier and EP recovered with different time courses: cochlear amplification just started to recover after the EP was nearly fully recovered and stabilized. Using a Boltzmann model and the second harmonic of the LCM to estimate the mechanoelectric transducer channel operating point, we found that the recovery of cochlear amplification occurred with recentering of the operating point.     

Prostaglandin synthesis by the cochlea

The exogenous and endogenous syntheses of prostaglandins (PG's) by the cochlea of adult mongolian gerbils were studied . After incubation of the whole membraneous cochlea with [³H]-arachidonic acid (AA), syntheses of PGF_2α, 6-keto PGF_1α, PGE₂, thromboxane (TX) B₂ and PGD₂ were evidenced in this order. The synthesis of radioactive PG's was almost completely inhibited by incubation with 10⁻⁵ M indomethacin. No significant amounts of those PG's were detected by radioimmunoassay (RIA) in the cochlea obtained from animals killed by microwave irradiation at 5.0 kw for 0.8 sec. However, when the homogenate of the whole membraneous cochlea obtained from animals without microwave irradiation was incubated at 37°C for 0–15 min, PGD₂, PGE₂, PGF₂α and 6-keto PGF₁α were found to be formed from endogenous AA in the cochlea by RIA. PG's were formed already at 0 time to considerable level (PGD₂, PGF₂α and 6-keto PGF₁α, 90–120 pg/cochlea; PGE₂, 370 pg/cochlea), reached to the maximum level (PGD₂, PGF₂α and 6-keto PGF₁α, 170–200 pg/cochlea; PGE₂, 500 pg/cochlea) at a 5-min incubation, and then gradually decreased. On the other hand, the amount of TXB₂ was lower than the detection limit by RIA (<50 pg/cochlea) even after the incubation. The cochlea was dissected into three parts: organ of Corti + modiolus (OC + M), lateral wall (LW), and cochlear nerve (CN), and then PG's formed by these tissues were determined after a 5-min incubation of the homogenates. In the CN and OC + M, PGE₂ was the major PG (100 and 160 pg/tissue, respectively), and the amounts of PGD₂, PGF₂α and 6-keto PGF₁α were about of those of PGE₂. In the LW, the amounts of PGD₂, PGE₂, PGF₂α and 6-keto PGF₁α were about the same level (70–100 pg/LW).     

Prostaglandin E2 promotes proliferation of skeletal muscle myoblasts via EP4 receptor activation

We recently demonstrated that conditioned media (CM) from osteocytes enhances myogenic differentiation of myoblasts, suggesting that signaling from bone may be important for skeletal muscle myogenesis. The effect of CM was closely mimicked by prostaglandin E₂ (PGE₂), a bioactive lipid mediator in various physiological or pathological conditions. PGE₂ is secreted at high levels by osteocytes and such secretion is further enhanced under loading conditions. Although four types of receptors, EP1 to EP4, mediate PGE₂ signaling, it is unknown whether these receptors play a role in myogenesis. Therefore, in this study, the expression of EPs in mouse primary myoblasts was characterized, followed by examination of their roles in myoblast proliferation by treating myoblasts with PGE₂ or specific agonists. All four PGE₂ receptor mRNAs were detectable by quantitative real-time PCR (qPCR), but only PGE₂ and EP4 agonist CAY 10598 significantly enhance myoblast proliferation. EP1/EP3 agonist 17-phenyl trinor PGE₂ (17-PT PGE₂) and EP2 agonist butaprost did not have any significant effects. Moreover, treatment with EP4 antagonist L161,982 dose-dependently inhibited myoblast proliferation. These results were confirmed by cell cycle analysis and the gene expression of cell cycle regulators. Concomitant with the inhibition of myoblast proliferation, treatment with L161,982 significantly increased intracellular reactive oxygen species (ROS) levels. Cotreatment with antioxidant N-acetyl cysteine (NAC) or sodium ascorbate (SA) successfully reversed the inhibition of myoblast proliferation and ROS overproduction caused by L161,982. Therefore, PGE₂ signaling via the EP4 receptor regulates myogenesis by promoting myoblast proliferation and blocking this receptor results in increased ROS production in myoblasts.     

Development of an in vivo active,dual EP1 and EP3 selective antagonist based on a novel acyl sulfonamide bioisostere

Recent preclinical studies demonstrate a role for the prostaglandin E₂ (PGE₂) subtype 1 (EP1) receptor in mediating, at least in part, the pathophysiology of hypertension and diabetes mellitus. A series of amide and N-acylsulfonamide analogs of a previously described picolinic acid-based human EP1 receptor antagonist (7) were prepared. Each analog had improved selectivity at the mouse EP1 receptor over the mouse thromboxane receptor (TP). A subset of analogs gained affinity for the mouse PGE₂ subtype 3 (EP3) receptor, another potential therapeutic target. One analog (17) possessed equal selectivity for EP1 and EP3, displayed a sufficient in vivo residence time in mice, and lacked the potential for acyl glucuronide formation common to compound 7. Treatment of mice with 17 significantly attenuated the vasopressor activity resulting from an acute infusion of EP1 and EP3 receptor agonists. Compound 17 represents a potentially novel therapeutic in the treatment of hypertension and diabetes mellitus.     

Reduced P2x<Subscript>2</Subscript> receptor-mediated regulation of endocochlear potential in the ageing mouse cochlea

Extracellular adenosine triphosphate (ATP) has profound effects on the cochlea, including an effect on the regulation of the endocochlear potential (EP). Noise-induced release of ATP into the endolymph activates a shunt conductance mediated by P2X₂ receptors in tissues lining the endolymphatic compartment, which reduces the EP and, consequentially, hearing sensitivity. This may be a mechanism of adaptation or protection from high sound levels. As inaction of such a process could contribute to hearing loss, this study examined whether the action of ATP on EP changes with age and noise exposure in the mouse. The EP and the endolymphatic compartment resistance (CoPR) were measured in mice (CBA/CaJ) aged between 3 and 15 months. The EP and CoPR declined slightly with age with an associated small, but significant, reduction in auditory brainstem response thresholds. ATP (100–1,000 μM) microinjected into the endolymphatic compartment caused a dose-dependent decline in EP correlated to a similar decrease in CoPR. This was blocked by pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonate, consistent with a P2X₂ receptor-mediated shunt conductance. There was no substantial difference in the ATP response with age. Noise exposure (octave-band noise 80–100 decibels sound pressure level (dBSPL), 48 h) in young animals induced an upregulation of the P2X₂ receptor expression in the organ of Corti and spiral limbus, most noticeably with the 90-dB exposure. This did not occur in the aged animals except following exposure at 90 dBSPL. The EP response to ATP was muted in the noise-exposed aged animals except following the 90-dB exposure. These findings provide some evidence that the adaptive response of the cochlea to noise may be reduced in older animals, and it is speculated that this could increase their susceptibility to noise-induced injury.     

Expression and function of pannexins in the inner ear and hearing

Pannexin (Panx) is a gene family encoding gap junction proteins in vertebrates. So far, three isoforms (Panx1, 2 and 3) have been identified. All of three Panx isoforms express in the cochlea with distinct expression patterns. Panx1 expresses in the cochlea extensively, including the spiral limbus, the organ of Corti, and the cochlear lateral wall, whereas Panx2 and Panx3 restrict to the basal cells of the stria vascularis in the lateral wall and the cochlear bony structure, respectively. However, there is no pannexin expression in auditory sensory hair cells. Recent studies demonstrated that like connexin gap junction gene, Panx1 deficiency causes hearing loss. Panx1 channels dominate ATP release in the cochlea. Deletion of Panx1 abolishes ATP release in the cochlea and reduces endocochlear potential (EP), auditory receptor current/potential, and active cochlear amplification. Panx1 deficiency in the cochlea also activates caspase-3 cell apoptotic pathway leading to cell degeneration. These new findings suggest that pannexins have a critical role in the cochlea in regard to hearing. However, detailed information about pannexin function in the cochlea and Panx mutation induced hearing loss still remain largely undetermined. Further studies are required.     

Reactivity of pyruvic acid and its derivatives towards reactive oxygen species

Pyruvic acid and its derivatives occurring in most biological systems are known to exhibit several pharmacological properties, such as anti‐inflammatory, neuroprotective or anticancer, many of which are suggested to originate from their antioxidant and free radical scavenger activity. The therapeutic potential of these compounds is a matter of particular interest, due to their mechanisms of action, particularly their possible antioxidant behaviour. Here, we report the results of a study of the effect of pyruvic acid (PA), ethyl pyruvate (EP) and sodium pyruvate (SP) on reactions generating reactive oxygen species (ROS), such as superoxide anion radicals, hydroxyl radicals and singlet oxygen, and their total antioxidant capacity. Chemiluminescence (CL) and spectrophotometry techniques were employed. The pyruvate analogues studied were found to inhibit the CL signal arising from superoxide anion radicals in a dose‐dependent manner with IC₅₀ = 0.0197 ± 0.002 mM for EP and IC₅₀ = 69.2 ± 5.2 mM for PA. These compounds exhibited a dose‐dependent decrease in the CL signal of the luminol + H₂O₂ system over the range 0.5–10 mM with IC₅₀ values of 1.71 ± 0.12 mM for PA, 3.85 ± 0.21 mM for EP and 22.91 ± 1.21 mM for SP. Furthermore, these compounds also inhibited hydroxyl radical‐dependent deoxyribose degradation in a dose‐dependent manner over the range 0.5–200 mM, with IC₅₀ values of 33.2 ± 0.3 mM for SP, 116.1 ± 6.2 mM for EP and 168.2 ± 6.2 mM for PA. All the examined compounds also showed antioxidant capacity when estimated using the ferric–ferrozine assay. The results suggest that the antioxidant activities of pyruvate derivatives may reflect a direct effect on scavenging ROS and, in part, be responsible for their pharmacological actions. Copyright © 2015 John Wiley & Sons, Ltd.     

Effect of the thromboxane receptor antagonist EP 092 on endotoxin shock in the sheep

The thromboxane receptor antagonist EP 092 inhibits the acute pulmonary vascular response to endotoxin in the anaesthetized, closed-chest sheep. The increase in the TXB₂ level in arterial blood was not suppressed by EP 092. Intravenous infusion of the thromboxane mimetic 11,9-epoxymethano PGH₂, but not PGF_2α, raises pulmonary artery pressure and lowers arterial pO₂ similar to the endotoxin. Isolated strips of lobar pulmonary veins but not lobar arteries are contracted by low concentrations of 11,9-epoxymethano PGH₂ - the effects are potently inhibited by EP 092.     

Auditory cortex basal activity modulates cochlear responses in chinchillas

Background

The auditory efferent system has unique neuroanatomical pathways that connect the cerebral cortex with sensory receptor cells. Pyramidal neurons located in layers V and VI of the primary auditory cortex constitute descending projections to the thalamus, inferior colliculus, and even directly to the superior olivary complex and to the cochlear nucleus. Efferent pathways are connected to the cochlear receptor by the olivocochlear system, which innervates outer hair cells and auditory nerve fibers. The functional role of the cortico-olivocochlear efferent system remains debated. We hypothesized that auditory cortex basal activity modulates cochlear and auditory-nerve afferent responses through the efferent system.

Methodology/Principal Findings

Cochlear microphonics (CM), auditory-nerve compound action potentials (CAP) and auditory cortex evoked potentials (ACEP) were recorded in twenty anesthetized chinchillas, before, during and after auditory cortex deactivation by two methods: lidocaine microinjections or cortical cooling with cryoloops. Auditory cortex deactivation induced a transient reduction in ACEP amplitudes in fifteen animals (deactivation experiments) and a permanent reduction in five chinchillas (lesion experiments). We found significant changes in the amplitude of CM in both types of experiments, being the most common effect a CM decrease found in fifteen animals. Concomitantly to CM amplitude changes, we found CAP increases in seven chinchillas and CAP reductions in thirteen animals. Although ACEP amplitudes were completely recovered after ninety minutes in deactivation experiments, only partial recovery was observed in the magnitudes of cochlear responses.

Conclusions/Significance

These results show that blocking ongoing auditory cortex activity modulates CM and CAP responses, demonstrating that cortico-olivocochlear circuits regulate auditory nerve and cochlear responses through a basal efferent tone. The diversity of the obtained effects suggests that there are at least two functional pathways from the auditory cortex to the cochlea.     

降低外淋巴钙浓度对耳蜗电位的影响

本实验在人工灌流条件下降低鼓阶外淋巴钙的浓度,观察其对听神经动作电位（ＣＡＰ）、耳蜗微音器电位（ＣＭ）以及蜗内直流电位（ＥＰ）的影响,以分析钙离子的作用机制。无钙液鼓阶灌流使ＣＡＰ、ＣＭ幅度可逆地下降,但不明显地改变ＣＡＰＩ／Ｏ曲线的非线性特征。无钙外淋巴灌流并不改变ＥＰ以及用缺氧法得到的负相ＥＰ（Ｎ－ＥＰ）,但使ＥＰ对强声给－撤时的快速变化趋于消失。本文讨论了这些耳蜗生物电改变所提示的钙离子作用的可能机制。     

Mechanism of the inhibition of platelet aggregation produced by prostaglandin F2α

The inhibition of human platelet aggregation produced by PGF_2α is not specific for thromboxane A₂ mimetics. Aggregation waves induced by PAF and thrombin are also inhibited by PGF_2α (8 μM); ADP is unaffected. These effects are still seen in platelets from aspirin-treated donors and platelets desensitized to thromboxane-like agonists (e.g. 11,9-epoxymethano PGH₂). In contrast the thromboxane receptor antagonist EP 045 (up to 20 μM) had no effect on primary aggregation induced by PAF, thrombin and ADP. We have previously shown that EP 045 (IC₅₀ = 0.5 μM), displaces the specific binding of [³H] 9,11-epoxymethano PGH₂ to washed human platelets.PGF_2α produces small increases in cAMP levels, and both this effect and the anti-aggregation are diminished by the adenyl cyclase inhibitor SQ 22536. The rise in cAMP induced by PGF_2α is inhibited to a greater extent by the presence of ADP than by thrombin, PAF or a thromboxane mimetic. The ability of aggregating agents to inhibit this increase correlates inversely with their sensitivity to inhibition by PGF_2α.We suggest that the very weak effect of PGF_2α on cyclic AMP_ production is sufficient to account for its inhibitory activity, and it is unlikely to be a competitive antagonist at the platelet thromboxane receptor as suggested by others.     

Developmentally regulated expression of ectonucleotidases NTPDase5 and NTPDase6 and UDP-responsive P2Y receptors in the rat cochlea

Ectonucleoside triphosphate diphosphohydrolases (E-NTPDases) regulate complex extracellular P2 receptor signalling pathways in mammalian tissues by hydrolysing extracellular nucleotides to the respective nucleosides. All enzymes from this family (NTPDase1-8) are expressed in the adult rat cochlea. This study reports the changes in expression of NTPDase5 and NTPDase6 in the developing rat cochlea. These two intracellular members of the E-NTPDase family can be released in a soluble form and show preference for nucleoside 5′-diphosphates, such as UDP and GDP. Here, we demonstrate differential spatial and temporal patterns for NTPDase5 and NTPDase6 expression during cochlear development, which are indicative of both cytosolic and extracellular action via pyrimidines. NTPDase5 is noted during the early postnatal period in developing sensory hair cells and supporting Deiters’ cells of the organ of Corti, and primary auditory neurons located in the spiral ganglion. In contrast, NTPDase6 is confined to the embryonic and early postnatal hair cell bundles. NTPDase6 immunolocalisation in the developing cochlea underpins its putative role in hair cell bundle development, probably via cytosolic action, whilst NTPDase5 may have a broader extracellular role in the development of sensory and neural tissues in the rat cochlea. Both NTPDase5 and NTPDase6 colocalize with UDP-preferring P2Y₄, P2Y₆ and P2Y₁₄ receptors during cochlear development, but this strong association was lost in the adult cochlea. Spatiotemporal topographic expression of NTPDase5 and NTPDase6 and P2Y receptors in adult and developing cochlear tissues provide strong support for the role of pyrimidinergic signalling in cochlear development.     

Gastric type H+,K+-ATPase in the cochlear lateral wall is critically involved in formation of the endocochlear potential

Cochlear endolymph has a highly positive potential of approximately +80 mV known as the endocochlear potential (EP). The EP is essential for hearing and is maintained by K⁺ circulation from perilymph to endolymph through the cochlear lateral wall. Various K⁺ transport apparatuses such as the Na⁺,K⁺-ATPase, the Na⁺-K⁺-2Cl^– cotransporter, and the K⁺ channels Kir4.1 and KCNQ1/KCNE1 are expressed in the lateral wall and are known to play indispensable roles in cochlear K⁺ circulation. The gastric type of the H⁺,K⁺-ATPase was also shown to be expressed in the cochlear lateral wall (Lecain E, Robert JC, Thomas A, and Tran Ba Huy P. Hear Res 149: 147–154, 2000), but its functional role has not been well studied. In this study we examined the precise localization of H⁺,K⁺-ATPase in the cochlea and its involvement in formation of EP. RT-PCR analysis showed that the cochlea expressed mRNAs of gastric ₁-, but not colonic ₂-, and -subunits of H⁺,K⁺-ATPase. Immunolabeling of an antibody specific to the ₁ subunit was detected in type II, IV, and V fibrocytes distributed in the spiral ligament of the lateral wall and in the spiral limbus. Strong immunoreactivity was also found in the stria vascularis. Immunoelectron microscopic examination exhibited that the H⁺,K⁺-ATPase was localized exclusively at the basolateral site of strial marginal cells. Application of Sch-28080, a specific inhibitor of gastric H⁺,K⁺-ATPase, to the spiral ligament as well as to the stria vascularis caused prominent reduction of EP. These results may imply that the H⁺,K⁺-ATPase in the cochlear lateral wall is crucial for K⁺ circulation and thus plays a critical role in generation of EP. hydrogen, potassium-adenosine triphosphatase; stria vascularis; spiral ligament     

Resonance phenomena in the cochlea of the mustache bat and their contribution to neuronal response characteristics in the cochlear nucleus

Summary The cochlea of the mustache bat, Pteronotus parnellii, is very sensitive and sharply tuned to the frequency range of the dominant second harmonic of the echolocation call around 61 kHz. About 900 Hz above this frequency the cochlear microphonic potential (CM) reaches its maximum amplitude and lowest threshold. At exactly the same frequency, pronounced evoked otoacoustic emissions (OAE) can be measured in the outer ear canal, indicating mechanical resonance. The CM amplitude maximum and the OAE are most severely masked by simultaneous exposure to tones within the range from about 61–62 kHz up to about 70 kHz. The data suggest that the mechanism of mechanical resonance involves cochlear loci basal to the 61 kHz position.The resonance contributes to auditory sensitivity and sharp tuning: At the frequency of the OAE, single unit responses in the cochlear nucleus have the lowest thresholds. Maximum tuning sharpness occurs at frequencies about 300 Hz below the OAE-frequency, where the threshold is about 10 dB less sensitive than at the OAE-frequency. In addition, in the frequency range around the OAE-frequency several specialized neuronal response features can be related to mechanical resonance: Long lasting excitation after the end of the stimulus, asymmetrical tuning curves with a shallow high frequency slope and phasic on-off neuronal response patterns. In particular the latter phenomenon indicates the occurrence of local mechanical cancellations in the cochlea.Abbreviations CF constant frequency component of echolocation calls - CM cochlear microphonic potential - FM frequency modulated component of echolocation calls - N1 compound action potential of the auditory nerve - OAE octoacoustic emission - SEOAE synchronous evoked OAE