Jérôme Ruel

Salicylate Induces Tinnitus through Activation of Cochlear NMDA Receptors

Salicylate, the active component of aspirin, is known to induce tinnitus. However, the site and the mechanism of generation of tinnitus induced by salicylate remains unclear. Here, we developed a behavioral procedure to measure tinnitus in rats. The behavioral model was based on an active avoidance paradigm in which rats had to display a motor task (i.e., to jump on a climbing pole when hearing a sound). Giving salicylate led to a decrease in the percentage of correct responses (score) and a drastic increase in the number of false positive responses (i.e., animals execute the motor task during a silent period). Presentation of the sound at a constant perceptive level prevents decrease of the score, leading to the proposal that score is related to hearing performance. In contrast, the increase of false positive responses remained unchanged. In fact, animals behaved as if they hear a sound, indicating that they are experiencing tinnitus. Mefenamate in place of salicylate also increased the number of false positive responses, suggesting that salicylate-induced tinnitus is related to an inhibition of cyclooxygenase. One physiological basis of salicylate ototoxicity is likely to originate from altered arachidonic acid metabolism. Because arachidonic acid potentiates NMDA receptor currents, we tested the involvement of cochlear NMDA receptors in the occurrence of tinnitus. Application of NMDA antagonists into the perilymphatic fluids of the cochlea blocked the increase in pole-jumping behavior induced by salicylate, suggesting that salicylate induces tinnitus through activation of cochlear NMDA receptors.

Physiology, pharmacology and plasticity at the inner hair cell synaptic complex.

This report summarizes recent neuropharmacological data at the IHC afferent/efferent synaptic complex: the type of Glu receptors and transporter involved and the modulation of this fast synaptic transmission by the lateral efferents. Neuropharmacological data were obtained by coupling the recording of cochlear potentials and single unit of the auditory nerve with intra-cochlear applications of drugs (multi-barrel pipette). We also describe the IHC afferent/efferent functioning in pathological conditions. After acoustic trauma or ischemia, acute disruption of IHC-auditory dendrite synapses are seen. However, a re-growth of the nerve fibres and a re-afferentation of the IHC were completely done 5 days after injury. During this synaptic repair, multiple presynaptic bodies were commonly found, either linked to the membrane or floating in ectopic positions. In the meantime, the lateral efferents directly contact the IHCs. The demonstration that NMDA receptors blockade delayed the re-growth of neurites suggests a neurotrophic role of NMDA receptors in pathological conditions.

Salicylate Enables Cochlear Arachidonic-Acid-Sensitive NMDA Receptor Responses

Currently, many millions of people treated for various ailments receive high doses of salicylate. Consequently, understanding the mechanisms by which salicylate induces tinnitus is an important issue for the research community. Behavioral testing in rats have shown that tinnitus induced by salicylate or mefenamate (both cyclooxygenase blockers) are mediated by cochlear NMDA receptors. Here we report that the synapses between the sensory inner hair cells and the dendrites of the cochlear spiral ganglion neurons express NMDA receptors. Patch-clamp recordings and two-photon calcium imaging demonstrated that salicylate and arachidonate (a substrate of cyclooxygenase) enabled the calcium flux and the neural excitatory effects of NMDA on cochlear spiral ganglion neurons. Salicylate also increased the arachidonate content of the whole cochlea in vivo. Single-unit recordings of auditory nerve fibers in adult guinea pig confirmed the neural excitatory effect of salicylate and the blockade of this effect by NMDA antagonist. These results suggest that salicylate inhibits cochlear cyclooxygenase, which increased levels of arachidonate. The increased levels of arachidonate then act on NMDA receptors to enable NMDA responses to glutamate that inner hair cells spontaneously release. This new pharmacological profile of salicylate provides a molecular mechanism for the generation of tinnitus at the periphery of the auditory system.

Inhibition of the c-Jun N-terminal kinase-mediated mitochondrial cell death pathway restores auditory function in sound-exposed animals.

We tested and characterized the therapeutic value of round window membrane-delivered (RWM) d-JNKI-1 peptide (Bonny et al., 2001) against sound trauma-induced hearing loss. Morphological characteristics of sound-damaged hair cell nuclei labeled by Hoechst staining show that apoptosis is the predominant mode of cell death after sound trauma. Analysis of the events occurring after sound trauma demonstrates that c-Jun N-terminal kinase (JNK)/stress-activated protein kinase activates a mitochondrial cell death pathway (i.e., activation of Bax, release of cytochrome c, activation of procaspases, and cleavage of fodrin). Fluorescein isothiocyanate (FITC)-conjugated d-JNKI-1 peptide applied onto an intact cochlear RWM diffuses through this membrane and penetrates cochlear tissues with the exception of the stria vascularis. A time sequence of fluorescence measurements demonstrates that FITC-labeled d-JNKI-1 remains in cochlear tissues for as long as 3 weeks. In addition to blocking JNK-mediated activation of a mitochondrial cell death pathway, RWM-delivered d-JNKI-1 prevents hair cell death and development of a permanent shift in hearing threshold that is caused by sound trauma in a dose-dependent manner (EC50 = 2.05 μM). The therapeutic window for protection of the cochlea from sound trauma with RWM delivery of d-JNKI-1 extended out to 12 h after sound exposure. These results show that the mitogen-activated protein kinase/JNK signaling pathway plays a crucial role in sound trauma-initiated hair cell death. Blocking this signaling pathway with RWM delivery of d-JNKI-1 may have significant therapeutic value as a therapeutic intervention to protect the human cochlea from the effects of sound trauma.

Oxidative stress, inflammation, and autophagic stress as the key mechanisms of premature age-related hearing loss in SAMP8 mouse Cochlea.

AIMSnIn our aging society, age-related hearing loss (ARHL) or presbycusis is increasingly important. Here, we study the mechanism of ARHL using the senescence-accelerated mouse prone 8 (SAMP8) which is a useful model to probe the effects of aging on biological processes.nnnRESULTSnWe found that the SAMP8 strain displays premature hearing loss and cochlear degeneration recapitulating the processes observed in human presbycusis (i.e., strial, sensory, and neural degeneration). The molecular mechanisms associated with premature ARHL in SAMP8 mice involve oxidative stress, altered levels of antioxidant enzymes, and decreased activity of Complexes I, II, and IV, which in turn lead to chronic inflammation and triggering of apoptotic cell death pathways. In addition, spiral ganglion neurons (SGNs) also undergo autophagic stress and accumulated lipofuscin.nnnINNOVATION AND CONCLUSIONnOur results provide evidence that targeting oxidative stress, chronic inflammation, or apoptotic pathways may have therapeutic potential. Modulation of autophagy may be another strategy. The fact that autophagic stress and protein aggregation occurred specifically in SGNs also offers promising perspectives for the prevention of neural presbycusis.

Deafness and Cochlear Fibrocyte Alterations in Mice Deficient for the Inner Ear Protein Otospiralin

ABSTRACT In the cochlea, the mammalian auditory organ, fibrocytes of the mesenchymal nonsensory regions play important roles in cochlear physiology, including the maintenance of ionic and hydric components in the endolymph. Occurrence of human deafness in fibrocyte alterations underlines their critical roles in auditory function. We recently described a novel gene, Otos, which encodes otospiralin, a small protein of unknown function that is produced by the fibrocytes of the cochlea and vestibule. We now have generated mice with deletion of Otos and found that they show moderate deafness, with no frequency predominance. Histopathology revealed a degeneration of type II and IV fibrocytes, while hair cells and stria vascularis appeared normal. Together, these findings suggest that impairment of fibrocytes caused by the loss in otospiralin leads to abnormal cochlear physiology and auditory function. This moderate dysfunction may predispose to age-related hearing loss.

Transient Ca2+-permeable AMPA receptors in postnatal rat primary auditory neurons.

Fast excitatory transmission in the nervous system is mostly mediated by α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA) receptors whose subunit composition governs physiological characteristics such as ligand affinity and ion conductance properties. Here, we report that AMPA receptors at inner hair cell (IHC) synapses lack the GluR2 subunit and are transiently Ca2+‐permeable before hearing onset as evidenced using agonist‐induced Co2+ accumulation, Western blots and GluR2 confocal microscopy in the rat cochlea. AMPA (100u2003µm) induced Co2+ accumulation in primary auditory neurons until postnatal day (PND) 10. This accumulation was concentration‐dependent, strengthened by cyclothiazide (50u2003µm) and blocked by GYKI 52466 (80u2003µm) and Joro spider toxin (1u2003µm). It was unaffected by D‐AP5 (50u2003µm), and it could not be elicited by 56u2003mm K+ or 1u2003mm NMDAu2003+u200310u2003µm glycine. Western blots showed that GluR1 immunoreactivity, present in homogenates of immature cochleas, had disappeared by PND12. GluR2 immunoreactivity was not detected until PND10 and GluR3 and GluR4 immunoreactivities were detected at all the ages examined. Confocal microscopy confirmed that the GluR2 immunofluorescence was not located postsynatically to IHCs before PND10. In conclusion, AMPA receptors on maturing primary auditory neurons differ from those on adult neurons. They are probably composed of GluR1, GluR3 and GluR4 subunits and have a high Ca2+ permeability. The postsynaptic expression of GluR2 subunits may be continuously regulated by the presynaptic activity allowing for variations in the Ca2+ permeability and physiological properties of the receptor.

Degeneration of sensory outer hair cells following pharmacological blockade of cochlear KCNQ channels in the adult guinea pig

In the inner ear, hair cell function is inextricably linked with intracellular potassium homeostasis. KCNQ potassium channels may play an important role by preventing accumulation of potassium in the hair cells. Linopirdine, a tool useful in targeting native or heterologous KCNQ channels, was used to study the role of KCNQ channels in the guinea pig cochlea. When perfused into intact cochlea, linopirdine transiently increases the summating potential and endocochlear potential, suggesting that it alters K+ homeostasis. The concomitant decrease in cochlear microphonic potential and distortion product otoacoustic emission amplitude indicates that linopirdine has an effect on the outer hair cells (OHCs). To determine the pathological consequences of the inhibition of cochlear KCNQ channels, we developed a hearing loss model based on a chronic intracochlear perfusion of linopirdine via an osmotic minipump. Ultrastructural analysis reveals that KCNQ channel blockade leads to OHC degeneration. Together, these results demonstrate that KCNQ channels, most probably of the KCNQ4 subtype, are crucial for the function and survival of sensory OHCs. Clinically, KCNQ4 channel dysfunction is known to be associated with the DFNA2 form of nonsyndromic dominant deafness. Our study shows that OHC KCNQ4 dysfunction could contribute to the early (40dB) hearing loss, but not for the profound deafness observed at the final stage of this disease.

Dopamine transporter is essential for the maintenance of spontaneous activity of auditory nerve neurones and their responsiveness to sound stimulation

Dopamine, a neurotransmitter released by the lateral olivocochlear efferents, has been shown tonically to inhibit the spontaneous and sound‐evoked activity of auditory nerve fibres. This permanent inhibition probably requires the presence of an efficient transporter to remove dopamine from the synaptic cleft. Here, we report that the dopamine transporter is located in the lateral efferent fibres both below the inner hair cells and in the inner spiral bundle. Perilymphatic perfusion of the dopamine transporter inhibitors nomifensine and N‐[1‐(2‐benzo[b]thiophenyl)cyclohexyl]piperidine into the cochlea reduced the spontaneous neural noise and the sound‐evoked compound action potential of the auditory nerve in a dose‐dependent manner, leading to both neural responses being completely abolished. We observed no significant change in cochlear responses generated by sensory hair cells (cochlear microphonic, summating potential, distortion products otoacoustic emissions) or in the endocochlear potential reflecting the functional state of the stria vascularis. This is consistent with a selective action of dopamine transporter inhibitors on auditory nerve activity. Capillary electrophoresis with laser‐induced fluorescence (EC‐LIF) measurements showed that nomifensine‐induced inhibition of auditory nerve responses was due to increased extracellular dopamine levels in the cochlea. Altogether, these results show that the dopamine transporter is essential for maintaining the spontaneous activity of auditory nerve neurones and their responsiveness to sound stimulation.

Microtubule-associated protein 2 (MAP2) expression during synaptic plasticity in the guinea pig cochlea

The expression of different isoforms of microtubule-associated proteins 2 (MAP2), including the low molecular weight form MAP2c present mainly in developing neurons, was investigated in the primary auditory neurons after alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) perfusion in the guinea pig cochlea. MAP2 expression appeared to be tightly regulated in the repairing neurons. Neurite regrowth seems to involve the MAP2c isoform. In cochlear neurons, mechanisms involved in the period of development might be reactivated after excitotoxic injury in the mature cochlea.