Repeated temporary threshold shift and changes in cochlear and neural function.

A robust temporary threshold shift (TTS) can create significant primary damage to the auditory synapse, termed cochlear synaptopathy (CS). The common model applied to examination of this pathology is a single noise exposure or extended duration exposures at relatively high noise dosages. It is unclear if a single noise exposure that does not produce physiological changes consistent with CS (such as suppressed suprathreshold responses) can create evidence consistent with the pathology induced by repeated exposures. Here, we exposed 16-week (wk) old Sprague-Dawley rats to repeated noise exposures (4 consecutive days, 8-16 kHz octave-band of noise, 97 dB SPL for 2 h) and examined measures of cochlear function (distortion product otoacoustic emissions) and auditory neural integrity (auditory brainstem response, wave 1 amplitude). Our results demonstrated a mean maximal threshold shift of 16 dB at 24 h post the initial noise exposure. Subsequent daily repeated exposures (4 consecutive days) resulted in diminished threshold shift at 24 h post repeated TTS. In addition to recovered thresholds, no sustained reduction in suprathreshold responses was observed. The findings are consistent with conditioning literature suggesting diminished TTS with repeated exposures. Repeated TTS that was not individually synaptopathic did not produce physiological evidence consistent with acute CS.

alpha10: a determinant of nicotinic cholinergic receptor function in mammalian vestibular and cochlear mechanosensory hair cells.

We report the cloning and characterization of rat alpha10, a previously unidentified member of the nicotinic acetylcholine receptor (nAChR) subunit gene family. The protein encoded by the alpha10 nAChR subunit gene is most similar to the rat alpha9 nAChR, and both alpha9 and alpha10 subunit genes are transcribed in adult rat mechanosensory hair cells. Injection of Xenopus laevis oocytes with alpha10 cRNA alone or in pairwise combinations with either alpha2-alpha6 or beta2-beta4 subunit cRNAs yielded no detectable ACh-gated currents. However, coinjection of alpha9 and alpha10 cRNAs resulted in the appearance of an unusual nAChR subtype. Compared with homomeric alpha9 channels, the alpha9alpha10 nAChR subtype displays faster and more extensive agonist-mediated desensitization, a distinct current-voltage relationship, and a biphasic response to changes in extracellular Ca(2+) ions. The pharmacological profiles of homomeric alpha9 and heteromeric alpha9alpha10 nAChRs are essentially indistinguishable and closely resemble those reported for endogenous cholinergic eceptors found in vertebrate hair cells. Our data suggest that efferent modulation of hair cell function occurs, at least in part, through heteromeric nAChRs assembled from both alpha9 and alpha10 subunits.

Block of the alpha9 nicotinic receptor by ototoxic aminoglycosides.

In the present study, we report that the alpha9 nicotinic acetylcholine receptor (nAChR) expressed in Xenopus laevis oocytes is reversibly blocked by aminoglycoside antibiotics. The aminoglycosides tested blocked the alpha9 nAChR in a concentration-dependent manner with the following rank order of potency: neomycin>gentamicin>streptomycin>amikacin>kanamycin. The antagonistic effect of gentamicin was not overcome by increasing the concentration of acetylcholine (ACh), indicative of a non-competitive type of block. Blockage of ACh-evoked currents by gentamicin was found to be voltage-dependent, being more potent at hyperpolarized than at depolarized holding potentials. Furthermore, gentamicin blockage was dependent upon the extracellular Ca(2+) concentration, shown by the fact that increments in extracellular Ca(2+) significantly reduced the potency of this aminoglycoside to block the alpha9 nAChR. Possible mechanisms of blockage by the aminoglycosides are discussed. The present results suggest that the initial reversible actions of aminoglycosides at the organ of Corti, such as the elimination of the olivocochlear efferent function, are due in part to the interaction with the native alpha9-containing cholinergic receptor of the outer hair cells.

Behavioral assessments of auditory sensitivity in transgenic mice.

This report summarizes positive reinforcement conditioning procedures for assessing sensory function in transgenic mice. To illustrate these behavioral methods auditory sensitivity was measured in mice lacking alpha9 acetylcholine receptor subunits (alpha9 knock-out mice). These receptors are known to play an important role in the efferent pathways that modify cochlear responses to sound stimuli. The strategies of parameter manipulation that led these subjects through their preliminary training stages to stable threshold performances are described in detail. Techniques for estimating and interpreting sensory thresholds are discussed from the perspective of signal detection analyses. This study found no significant differences between alpha9 knock-out mice and control subjects when hearing thresholds were measured under quiet conditions, as predicted by previous behavioral and electrophysiological evidence.

High calcium permeability and calcium block of the alpha9 nicotinic acetylcholine receptor.

At the synapse between olivocochlear efferent fibers and outer hair cells (OHCs) of the cochlea, a non-classical ionotropic cholinergic receptor allows Ca(2+) entry into the hair cell, thus activating a Ca(2+)-sensitive K(+) current which hyperpolarizes the cell's membrane. In the mammalian ear, this leads to a reduction in basilar membrane motion, altering auditory nerve fiber activity and reducing the dynamic range of hearing. The alpha9 nicotinic acetylcholine receptor (nAChR) subunit mediates synaptic transmission between cholinergic olivocochlear fibers and OHCs. Given that Ca(2+) is a key player at this inhibitory synapse, we evaluated the permeability to Ca(2+) of the recombinant alpha9 receptor expressed in Xenopus laevis oocytes and the modulation of its activity by extracellular Ca(2+). Our results show that the alpha9 receptor is highly permeable to Ca(2+) and that this cation potently blocks monovalent currents through this channel (IC(50)=100 microM, at -70 mV) in a voltage-dependent manner. At a Ca(2+) concentration similar to that found in the perilymph bathing the base of the OHCs, approximately 90% of the Na(+) current through the alpha9 receptor is blocked, suggesting that one of the main functions of this channel could be to provide a pathway for Ca(2+) influx.

LFG: an anti-apoptotic gene that provides protection from Fas-mediated cell death.

Programmed cell death regulates a number of biological phenomena, and the apoptotic signal must itself be tightly controlled to avoid inappropriate cell death. We established a genetic screen to search for molecules that inhibit the apoptotic signal from the Fas receptor. Here we report the isolation of a gene, LFG, that protects cells uniquely from Fas but not from the mechanistically related tumor necrosis factor alpha death signal. LFG is widely distributed, but remarkably is highly expressed in the hippocampus. LFG can bind to the Fas receptor, but does not regulate Fas expression or interfere with binding of an agonist antibody. Furthermore LFG does not inhibit binding of FADD to Fas.

Role of alpha9 nicotinic ACh receptor subunits in the development and function of cochlear efferent innervation.

Cochlear outer hair cells (OHCs) express alpha9 nACh receptors and are contacted by descending, predominately cholinergic, efferent fibers originating in the CNS. Mice carrying a null mutation for the nACh alpha9 gene were produced to investigate its role(s) in auditory processing and development of hair cell innervation. In alpha9 knockout mice, most OHCs were innervated by one large terminal instead of multiple smaller terminals as in wild types, suggesting a role for the nACh alpha9 subunit in development of mature synaptic connections. Alpha9 knockout mice also failed to show suppression of cochlear responses (compound action potentials, distortion product otoacoustic emissions) during efferent fiber activation, demonstrating the key role alpha9 receptors play in mediating the only known effects of the olivocochlear system.

Inner ear defects induced by null mutation of the isk gene.

The isk gene is expressed in many tissues. Pharmacological evidence from the inner ear suggests that isk mediates potassium secretion into the endolymph. To examine the consequences of IsK null mutation on inner ear function, and to produce a system useful for examining the role(s) IsK plays elsewhere, we have produced a mouse strain that carries a disrupted isk locus. Knockout mice exhibit classic shaker/waltzer behavior. Hair cells degenerate, but those of different inner ear organs degenerate at different times. Functionally, we show that in mice lacking isk, the strial marginal cells and the vestibular dark cells of the inner ear are unable to generate an equivalent short circuit current in vitro, indicating a lack of transepithelial potassium secretion.

Alpha 9: an acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells.

We report the isolation and functional characterization of a member of the nicotinic acetylcholine receptor subunit gene family, alpha 9. Xenopus oocytes injected with alpha 9 cRNA express a homomeric receptor-channel complex that is activated by acetylcholine. The alpha 9 receptor displays an unusual mixed nicotinic-muscarinic pharmacological profile. The unique properties of the alpha 9 receptor-channel complex closely match those described for the cholinergic receptor present in vertebrate cochlear hair cells. In situ hybridization studies reveal a restricted pattern of alpha 9 gene expression that includes the outer hair cells of the rat cochlea. Our results suggest that the alpha 9 receptor is involved in the cholinergic efferent innervation of cochlear hair cells and thus may modulate the encoding of auditory stimuli.

Input from the inferior colliculus to medial olivocochlear neurons in the rat: a double label study with PHA-L and cholera toxin.

The inferior colliculus provides a strong descending influence capable of modulating the excitability levels of olivocochlear neurons (Rajan, 1990). In an attempt to anatomically demonstrate this pathway in rats, an experimental paradigm was designed by which anterogradely transported Phaseolus vulgaris-leucoagglutinin (PHA-L), which delineates axonal arbors, and retrogradely transported cholera toxin B subunit alone (CT-B) or conjugated to horseradish peroxidase (CT-HRP), which delineate dendritic arbors, are visualized in the same brainstem sections. PHA-L was injected unilaterally into the central nucleus of the inferior colliculus of adult rats 5-9 days prior to injection of CT-B or CT-HRP into either the contralateral or the ipsilateral cochlea. Descending collicular axons labeled with PHA-L densely innervate the ventral nucleus of the trapezoid body (VNTB), which contains neurons of the medial olivocochlear system (MOCS), but do not enter the lateral superior olive, where the neurons of the lateral olivocochlear system (LOCS) are found. The collicular projection to VNTB is largely ipsilateral and supplies mostly the ventral half of the nucleus. Within VNTB, the collicular fibers intermingle with dendrites and, to a lesser extent, cell bodies of MOCS. Collicular boutons, predominantly of the en passant type, are often observed in close apposition to dendrites and, less frequently, cell bodies of both crossed and uncrossed MOCS. These light microscopic results suggest the existence of direct, synaptic contacts between descending collicular axons and ipsilateral crossed and uncrossed MOCS. Numerous collicular boutons were also seen at a distance from MOCS, suggesting that they establish synapses with other neuron types of the VNTB that do not send their axons to the cochlea.

Protein chemical characterization and immunocytochemical localization of the NMDA receptor subunit NMDA R1.

In the rat central nervous system, the mRNA encoding the N-methyl-D-aspartate receptor subunit R1 is the most ubiquitously distributed among the cloned subunit mRNAs of this glutamate receptor subtype. The N-methyl-D-aspartate R1 mRNA is very abundantly expressed and N-methyl-D-aspartate R1 coexpression is necessary for functional expression of all other cloned N-methyl-D-aspartate receptor subunits. Therefore, the R1 subunit is likely to be an essential component of all known N-methyl-D-aspartate receptors in rat brain. By employing sequence specific polyclonal antibodies, we demonstrate that rat brain N-methyl-D-aspartate R1, as well as recombinantly expressed receptor protein, has an apparent molecular mass of 116 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The receptor protein is heavily glycosylated. It is specifically localized to the central nervous system, and it co-enriches with synaptic membranes upon subcellular fractionation of the cerebral cortex. Chemical cross-linking of synaptic membrane proteins shows that the N-methyl-D-aspartate R1 protein is part of a receptor protein complex with a molecular mass of 730 kDa. By using immunocytochemical methods, we demonstrate a widespread but distinct distribution of N-methyl-D-aspartate R1 in neurons of the rat brain, with prominent immunostaining in certain layers of the cerebral cortex, in the hippocampus and dentate gyrus, as well as in the cerebellum.

Distribution and dendritic features of three groups of rat olivocochlear neurons. A study with two retrograde cholera toxin tracers.

Cholera toxin B subunit conjugated to horseradish peroxidase, and unconjugated cholera toxin B subunit are useful tools for retrograde tract tracing. Unilateral injection of either cholera toxin preparation into the cochlea results in excellent labeling of olivocochlear neurons, as judged by the Golgi-like filling of cell bodies, dendrites, and even axons. By this approach, we have studied the light microscopic cytology and topographic distribution of olivocochlear neurons and counted their numbers in Sprague-Dawley rats. The olivocochlear system of rats can be divided into three subgroups. The lateral olivocochlear system, composed of small cells located exclusively within the ipsilateral lateral superior olive (relative to the test cochlea), and a medial olivocochlear system, composed of large cells bilaterally dispersed within the ventral nucleus of the trapezoid body, conformed to previous topographic descriptions. A third subgroup of approximately 110 large cells, herein termed shell neurons, was labeled by both tracers, but was not well recognized in previous studies. Shell neurons and their dendrites surround the ipsilateral, and to a much lesser extent the contralateral, lateral superior olive. Lateral olivocochlear neurons do not project their dendrites outside the gray matter of the lateral superior olive, while dendrites belonging to shell neurons penetrate into that nucleus as well as into other auditory brain stem nuclei and the surrounding reticular formation. Medial olivocochlear neurons usually project dendrites ventrally into the trapezoid body and are always excluded from the lateral superior olive.

Chemically distinct rat olivocochlear neurons.

We have produced a neurochemical map of the cell bodies of origin of the cochlear efferent terminals in rat by combining glutamic acid decarboxylase (GAD), choline acetyltransferase (ChAT), or calcitonin gene-related peptide (CGRP) immunocytochemistry with retrograde transport of horseradish peroxidase. The locations of cochlear efferent cell bodies are in general agreement with the medial and lateral systems described by White and Warr (J. Comp. Neurol. 219:203-214, 1983) with some minor modifications. The lateral system consists of at least two pools of chemically distinct neurons located within the lateral superior olive (LSO) ipsilateral to the injected cochlea. One pool immunostains with an antibody to GAD while the other immunostains with antibodies to ChAT and to CGRP. The medial efferent system consists of periolivary neurons that are almost exclusively large and ChAT-positive but CGRP-negative. They are located both ipsilateral and contralateral to the cochlea they project to. There are a few GAD-positive small neurons in the medioventral and rostral periolivary regions that project ipsilaterally, but these may prove tobe ectopic neurons. The ipsilateral lateroventral periolivary region (LVPO) contains some efferent neurons, all of which are ChAT-positive but CGRP-negative. Additional cochlear efferent neurons, some of which are ChAT-positive and others GAD-positive, are present within and immediately dorsal to the fiber capsule surrounding the medial limb, and to a lesser extent the lateral limb, of the ipsilateral LSO. Not all GAD-positive or ChAT-positive olivary cells project to the cochlea. We have complemented the results in the brainstem by demonstrating two immunocytochemically distinct populations of efferent terminals in the cochlea simultaneously, one CGRP-positive and the other GAD-positive. Approximately equal numbers of boutons immunoreactive for both markers are present beneath inner hair cells throughout the entire length of the cochlea. Surprisingly high numbers of GAD-positive and CGRP-positive boutons are also present on outer hair cells, with each class having its spatially and morphologically distinct features. The lack of CGRP-positive periolivary cells that are retrogradely labeled by cochlear injections of HRP suggests that the lateral olivocochlear system sends projections to outer hair cells. Our results raise questions about species differences in the organization of targets of the lateral and medial olivocochlear systems.

An evaluation of retrograde tracing methods for the identification of chemically distinct cochlear efferent neurons.

We have compared retrograde labelling of rat olivocochlear neurons after unilateral cochlear injections of wheatgerm agglutinin conjugated horseradish peroxidase (WGA-HRP) and free HRP. After cochlear injection of WGA-HRP, labelling of nerve cell bodies in the brainstem can be explained not only as conventional retrograde labelling resulting from uptake by efferent nerve terminals synapsing on or near hair cells, but also as spurious labelling originating from tracer leakage, through the periotic duct and over the eighth nerve sheaths, into the cerebral-spinal fluid. Depending on the length of survival time, spurious labelling can involve small portions of the nucleus of the trapezoid body or the entire auditory brainstem and other non-auditory centers. On the contrary, moderate amounts of free HRP delivered to the cochlea do not lead to spurious labelling. With free HRP as the tracer of choice, we found that cochlear efferent cells were located not only in the ipsilateral LSO body and bilaterally within MVPO and RPO as already described by White and Warr, but also surrounding the ipsilateral LSO and in the ipsilateral LVPO. The allocation of these newly described olivocochlear neurons to the medial large cell or lateral small cell system is uncertain because they are located laterally in the brainstem and project ipsilaterally but are large spherical to fusiform or multipolar cells. A zinc salicylate-formol fixative and a metal intensified DAB reaction were found to be effective in visualizing retrogradely transported HRP in neurons and allowed immunocytochemical staining of the same sections with antisera to glutamic acid decarboxylase and choline acetyltransferase. This double label protocol can be used to produce a neurochemical map of the OC systems.