Search

International audience; Aging is often considered to affect both the peripheral (i.e. the cochlea) and central (brainstem and thalamus-cortex) auditory systems. We investigated the effects of aging on the cochlea, brainstem and cortex of female Sprague-Dawley rats. The auditory nerve threshold remained stable between the ages of nine and 21 months, as did distortion product otoa-coustic emissions and the number of ribbon synapses between inner hair cells and nerve fibers. The first clear signs of aging appeared in the brainstem, in which response amplitude decreased, with thresholds remaining stable until the age of 15 months, and increasing slightly thereafter. The responses of primary auditory cortex neurons revealed specific effects of aging: at 21 months, receptive fields were spectrally narrower and the temporal reliability of responses to communication sounds was lower. However, aging had a null or even positive effect on neuronal responses in the presence of background noise, responses to amplitude-modulated sounds, and responses in gap-detection protocols. Overall, inter-animal variability remained high relative to the variability across groups of different ages, for all parameters tested. Beha-vioral performance for the modulation depth of amplitude modulation noise was worse in 21-month old animals than in other animals. Age-related alterations of cortical and behavioral responses were thus observed in animals displaying no signs of aging at the peripheral level. These results suggest that intrinsic, central aging effects can affect the perception of acoustic stimuli independently of the effects of aging on peripheral receptors.

Characterizing the microenvironment of a damaged organ of Corti and identifying the basic mechanisms involved in subsequent epithelial reorganization are critical for improving the outcome of clinical therapies. In this context, we studied the expression of a variety of cell markers related to cell shape, cell adhesion and cell plasticity in the rat organ of Corti poisoned with amikacin. Our results indicate that, after severe outer hair cell losses, the cytoarchitectural reorganization of the organ of Corti implicates epithelial-mesenchymal transition mechanisms and involves both collective and individual cell migratory processes. The results also suggest that both root cells and infiltrated fibroblasts participate in the homeostasis of the damaged epithelium, and that the flat epithelium that may emerge offers biological opportunities for late regenerative therapies.

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.

This study was designed to determine whether Coxsackie adenovirus receptor (CAR) and alpha nu beta3/alpha nu beta5 integrin co-receptors are involved in adenovirus gene transfer in the rat cochlea. We find that CAR and integrin co-receptors are expressed in every cell subtype transduced by the adenoviral vector Ad5 DeltaE1-E3/cytomegalovirus/green fluorescent protein (GFP) on cochlear slices in vitro. The spiral ganglion neurons, which do not express CAR, were not transduced by the virus. Blocking these receptors by monoclonal antibodies decreased transgene expression, whereas disrupting tight junctions with ethylenediaminetetraacetic acid led to an increased transgene expression. However, sensory hair cells and strial cells also expressing CAR and alpha nu integrins were not transduced by the vector. GFP expression was also studied in vivo. Perilymphatic perfusion of adenovirus in vivo did not affect hearing and only cells lining the perilymphatic spaces were transduced. Endolymphatic perfusion resulted in low-frequency hearing loss and although some cells of the organ of Corti were efficiently transduced, the sensory and the strial cells were not. Transduced sensory and strial cells were occasionally observed in cochleas after single shot of adenovirus. Pretreatment with anti-CAR and anti-alpha nu antibodies decreases GFP expression in vivo, suggesting that the CAR/alpha nu integrin pathway is involved in adenovirus transduction in the cochlea.

Prenatal cocaine exposure causes alterations in auditory brainstem response in children and experimental animals and has adverse effects on auditory information processing and language skills in children. These effects may result from lesions in the cochlea since this organ is particularly sensitive to chemical insults during the development. We have thus studied here the effect of prenatal cocaine exposure on the maturation of the rat cochlea using the transient non-catecholaminergic expression of tyrosine hydroxylase in spiral ganglion neurons as an index of cochlear maturation and morphometry to evaluate the maturation of primary auditory neurons and the organ of Corti. We showed that prenatal cocaine exposure accelerated the cochlear maturation. In the basal coil of cochleas from PND8 cocaine-treated pups, the Kölliker's organ had disappeared, the tunnel of Corti was opened, and the stria vascularis no longer contained undifferentiated marginal cells. The maximum expression of tyrosine hydroxylase in type I primary auditory neurons occurred at PND8 instead of PND12 in pair-fed controls. On the other hand, the prenatal cocaine exposure had no effect on the width and height of the organ of Corti, spiral ganglion volume and number and size of primary auditory neurons. In conclusion, our data suggest that prenatal cocaine exposure, though not lethal to primary auditory neurons, accelerates aspects of the cochlear sensorineural maturation. This accelerated cochlear maturation in cocaine-treated rat pups could cause auditory dysfunctions by desynchronizing the development of the whole auditory pathway., This work was supported by projectsSTRAD/C/SAV/304/92, PECS/C/SAV/87/95, Praxis XXI/PC/C/CED/157/96 and Bolsa de curta duração da Fundação CalousteGulbenkian.

Fast excitatory transmission in the nervous system is mostly mediated by alpha-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 (100 microM) induced Co2+ accumulation in primary auditory neurons until postnatal day (PND) 10. This accumulation was concentration-dependent, strengthened by cyclothiazide (50 microM) and blocked by GYKI 52466 (80 microM) and Joro spider toxin (1 microM). It was unaffected by D-AP5 (50 microM), and it could not be elicited by 56 mM K+ or 1 mM NMDA + 10 microM 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 postsynaptically 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.

Cysteine-string protein is a vesicle-associated protein that plays a vital function in neurotransmitter release. We have studied its expression and regulation during cochlear maturation. Both the mRNA and the protein were found in primary auditory neurons and the sensory inner hair cells. More importantly, cysteine-string protein was localized on synaptic vesicles associated with the synaptic ribbon in inner hair cells and with presynaptic differentiations in lateral and medial olivocochlear terminals -- the cell bodies of which lie in the auditory brainstem. No cysteine-string protein was expressed by the sensory outer hair cells suggesting that the distinct functions of the two cochlear hair cell types imply different mechanisms of neurotransmitter release. In developmental studies in the rat, we observed that cysteine-string protein was present beneath the inner hair cells at birth and beneath outer hair cells by postnatal day 2 only. We found no expression in the inner hair cells before about postnatal day 12, which corresponds to the period during which the first cochlear action potentials could be recorded. In conclusion, the close association of cysteine-string protein with synaptic vesicles tethered to synaptic ribbons in inner hair cells and its synchronized expression with the appearance and maturation of the cochlear potentials strongly suggest that this protein plays a fundamental role in sound-evoked glutamate release by inner hair cells. This also suggests that this role may be common to ribbon synapses and conventional central nervous system synapses.

Cochlear fibrosis is a common finding following cochlear implantation. Evidence suggests that cochlear fibrosis could be triggered by inflammation and epithelial-to-mesenchymal cell transition (EMT). In this study, we investigate the mechanisms of cochlear fibrosis and the risk/benefit ratio of local administration of the anti-inflammatory drug dexamethasone (DEX) and antimitotic drug aracytine (Ara-C). Cochlear fibrosis was evaluated in cochlear fibrosis models of rat cochlear slices in vitro and in KLH-induced immune labyrinthitis and platinum wire cochlear implantation-induced fibrosis in vivo. Cochleae were invaded with tissue containing fibroblastic cells expressing α-SMA (alpha smooth muscle actin), which along with collagen I, fibronectin, and laminin in the extracellular matrix, suggests the involvement of a fibrotic process triggered by EMT in vitro and in vivo. After perilymphatic injection of an adenoviral vector expressing GFP in vivo, we demonstrated that the fibroblastic cells derived from the mesothelial cells of the scalae tympani and vestibuli. Activation of inflammatory and EMT pathways was further assessed by ELISA analysis of the expression of IL-1β and TGF-β1. Both markers were elevated in vitro and in vivo, and DEX and Ara-C were able to reduce IL-1β and TGF-β1 production. After 5days of culture in vitro, quantification of calcein-positive cells revealed that Ara-C was 30-fold more efficient in preventing fibrosis, and provoked less sensory hair cell loss, than DEX. In KLH-induced immune labyrinthitis and platinum wire-implanted models, Ara-C was more efficient in preventing proliferation of fibrosis with less side effects on hair cells and neurons than DEX. In conclusion, DEX and Ara-C both prevent fibrosis in the cochlea. Analysis of the risk/benefit ratio favors the use of Ara-C for preventing cochlear fibrosis.

Cochlear activity is regulated by the olivo-cochlear bundle, which originates from the brainstem and projects onto the hair cells and auditory nerve fibers. Two efferent components can be distinguished: the medial and lateral olivo-cochlear efferent originating from the medial, and the lateral nuclei of the superior olivary complex. The input of the efferent systems on hair cells occurs during development and persists in the adult cochlea. Recent studies have shown that the efferent innervations are required to set the activity pattern in developing hair cells and auditory nerve fibers and to protect the synaptic structures in adult cochlea. In addition, efferent innervations undergo plasticity during pathological conditions such as noise-trauma or aging. This review discusses the mechanisms underlying the control of the hair cells and afferent fibers excitability by efferent neurons and their putative role in developing adult and pathological conditions.

Efferent feedback systems provide a means for modulating the input to the central nervous system. The lateral olivocochlear efferents modulate auditory nerve activity via synapses with afferent dendrites below sensory inner hair cells. We examined the effects of dopamine, one of the lateral olivocochlear neurotransmitters, by recording compound and single unit activity from the auditory nerve in adult guinea pigs. Intracochlear application of dopamine reduced the compound action potential (CAP) of the auditory nerve, increased the thresholds and decreased the spontaneous and driven discharge rates of the single unit fibres without changing their frequency-tuning properties. Surprisingly, dopamine antagonists SCH-23390 and eticlopride decreased CAP amplitude as did dopamine. In some units, both SCH-23390 and eticlopride increased the basal activity of auditory nerve fibres leading to an improvement of threshold sensitivity and a decrease of the maximum driven discharge rates to sound. In other units, the increase in firing rate was immediately followed by a marked reduction to values below predrug rates. Because CAP reflects the summed activity of auditory nerve fibres discharging in synchrony, both the decrease in sound-driven discharge rate and the postexcitatory reduction account for the reduction in CAP. Ultrastructural examination of the cochleas perfused with eticlopride showed that some of the afferent dendrites were swollen, suggesting that the marked reduction in firing rate may reflect early signs of excitotoxicity. Results suggest that dopamine may exert a tonic inhibition of the auditory nerve activity. Removal of this tonic inhibition results in the development of early signs of excitotoxicity.

This study investigates the morphological and molecular changes that occur in the inner hair cell area of the rat cochlea following aminoglycoside treatment. Rats were injected daily with 500 mg/kg of amikacin between postnatal day 9 (PND9) and PND16. Cochleae were examined at PND16 to PND120 using both scanning and transmission electron microscopy and molecular fluorescent labeling. The inner hair cells showed obvious signs of apoptosis in response to amikacin treatment and most of them were missing by one week after the end of the aminoglycoside exposure period. Concomitantly, the epithelium became scarred as the surrounding supporting cells expanded and filled the space vacated by the missing IHCs. The mid-basolateral region of these modified supporting cells was surrounded by many afferent and efferent terminals. However, these cells expressed neither calbindin nor SNAP25, proteins that are both expressed by IHCs in the normal, untreated organ of Corti in the rat. In addition, these supporting cells remained attached to the basal lamina by a thin cytoplasmic process. The supporting cells surrounding the inner hair cells therefore appear unable to convert directly into inner hair cells following aminoglycoside induced hair-cell loss but may be able to provide trophic support for the remaining afferent and efferent neurites.

Glutamate receptors mediate most excitatory synaptic transmission in the adult vertebrate brain, but their activation in developing neurons also influences developmental processes. However, little is known about the developmental regulation of the subunits composing these receptors. Here we have studied age-dependent changes in the expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) and N-methyl-D-aspartate (NMDA) receptor subunits in the cochlear nucleus complex (CN), the superior olivary complex (SOC), the nuclei of the lateral lemniscus, and the inferior colliculus of the developing rat. In the lateral superior olive, the medial nucleus of the trapezoid body, and the ventral nucleus of the lateral lemniscus, the distribution of AMPA receptor subunits changed drastically with age. While GluR1 and GluR2 subunits were highly expressed in the first 2 postnatal weeks, GluR4 staining was detectable only thereafter. GluR1 and GluR2 immunoreactivities rapidly decreased during the third postnatal week, with the GluR1 subunits disappearing from most neurons. In contrast, the adult pattern of the distribution of AMPA receptor subunits emerged gradually in most of the other auditory nuclei. Thus, progressive as well as regressive events characterized AMPA receptor development in some nuclei, while a monotonically maturation was seen in other regions. In contrast, the staining patterns of NMDA receptor subunits remained stable or only decreased during the same period. Although our data are not consistent with a generalized pattern of AMPA receptor development, the abundance of GluR1 subunits is a distinctive feature of early AMPA receptors. As similar AMPA receptors are present during plasticity periods throughout the brain, neurons undergoing synaptic and structural remodelling might have a particular need for these receptors.

Auditory brainstem neurons probably depend on afferent input to maintain calcium homeostasis within a narrow range. These neurons are endowed with high concentrations of the calcium-binding proteins parvalbumin, calretinin, and calbindin D28k that are presumed to buffer cytosolic calcium transients. To determine the effects of functional deafferentation on these proteins in the auditory brainstem of adult guinea pigs, we have manipulated the sensory input with an intracochlear perfusion of the glutamate agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), which is known to transiently disconnect inner hair cells and primary auditory dendrites. Semiquantitative measures of immunostaining intensities showed rapid and reversible changes in calcium-binding protein levels. By 24 hours after AMPA treatment, calretinin immunostaining was reduced in deafferented neurons of the cochlear nuclei and their axons in the superior olivary nuclei. In contrast, calbindin D28k immunoreactivity levels by this time were higher in deafferented neurons of the medial nucleus of the trapezoid body and their axons in the lateral superior olivary nucleus (LSO). Parvalbumin immunostaining was also generally increased in deafferented neurons, but changes were less evident and more complex. The changes in all three immunoreactivities disappeared with the progressive restoration of afferent input. Normal levels were reestablished by 5 days after AMPA treatment, when afferent activity had almost completely recovered. These results show that calcium-binding protein immunostaining in auditory neurons is functionally responsive to afferent activity. The increased buffering capacity in deafferented neurons as shown by the rises in parvalbumin and calbindin D28k immunostaining may be part of mechanisms promoting neuronal survival after loss of sensory input. This input, on the other hand, may be necessary for maintaining the high calretinin levels normally present in cochlear nucleus neurons.

Glutamate is thought to act as a neurotransmitter of the sensory hair cells of the organ of Corti. Glutamine synthetase could be involved in a type of glutamate-glutamine cycle in the cochlea which could clear glutamate off the synaptic cleft and replenish the hair cell glutamate neurotransmitter store. Using both light and electron microscopic immunocytochemistry to localize this enzyme in the guinea pig cochlea, we have observed immunoreactive satellite glial cells surrounding parvalbumin-immunoreactive primary auditory neurons in the spiral ganglion. Glutamine synthetase was also detected in Schwann cells of the osseous spiral lamina which form the myelin sheath of nerve fibers. On the contrary, no immunoreactivity could be observed in the cochlear nerve and in the organ of Corti, although this organ contains structures able to take up glutamate. Although they confirm earlier works involving glutamine synthetase in the conversion of L-[3H]glutamate taken up by glial cells, our results suggest that the cochlear glutamate-glutamine cycle is not primarily involved in the recycling and replenishment of hair cell neurotransmitter glutamate. Alternatively, it is proposed that glutamine synthetase functions to limit the perilymphatic glutamate concentrations.

This study investigates the role of dopamine, a putative lateral efferent neurotransmitter/modulator, in cochlear physiology and physiopathology. Cochlear potentials were recorded in guinea pigs after intracochlear perfusion of increasing doses (0.1-1 mM) of piribedil, an agonist of the D2/D3 receptors. A dose-dependent reduction in the amplitude of auditory nerve compound action potential (CAP) was observed, predominantly at high-intensity tone-burst stimulations, and without significant effect on CAP threshold. There was no variation of cochlear microphonic and summating potential. When 1 mM piribedil was perfused into the cochlea during continuous 130 dB SPL pure tone exposure (6 kHz, 15 min), CAP threshold shifts were significantly less than in control animals with artificial perilymph-perfused cochleas. No dendritic damage was observed, although there was evident hair cell damage. Similarly, radial dendrites were clearly protected against ischemia-induced damage when 1 mM piribedil was applied prior to a 10-min ischemia. These results suggest that dopamine modulates the activity of radial afferent fibers via D2/D3 receptors. The protective effect of piribedil during acoustic trauma or ischemia suggests that this modulation corresponds to a prevention of excitotoxicity due to dysfunction of inner hair cell neurotransmission.

Tinnitus, the perception of sound in the absence of external noise, degrades the quality of life, extending from irritability, stress, and anxiety to severe depression and suicide. The increase in human life span in industrialized countries calls for the development of new therapies for tinnitus. Indeed, tinnitus is most often associated with presbycusis (or age-related hearing loss) and noise-induced hearing loss. A distribution histogram shows that almost 12% of men between the ages of 65 and 74 experience tinnitus (see Fig. 1A in Lockwood et al., 2002). Although hearing aids may restore some extend hearing capabilities, they are of little help for tinnitus, which is the major complaint of presbycusis patients. Yet, the exposure of the population to multiple loud noise sources (at work or in public places) leads to an early onset of hearing disorders such as tinnitus. For example, 76% of the disc jockeys who have worked in nightclubs 3 nights a week for 6 years suffer from tinnitus (Potier et al., 2009). However, the highly heterogeneous etiology of tinnitus precludes the generation of a unique “universal” animal model of tinnitus and raises a major biomedical challenge. In addition to presbycusis and noise-induced hearing loss, tinnitus is associated with Meniere’s disease. Other, less frequent origins include exposure to ototoxic drugs (antibiotics, nonsteroidal anti-inflammatory drugs, and chemotherapeutic agents), autoimmune processes, or infectious diseases (Nicolas-Puel et al., 2002). Extremely rare is somatic tinnitus triggered by voluntary or external manipulations of the jaw, movements of the eyes, or pressure applied to head and neck regions (Shore et al., 2007). Finally, the identification of tinnitus generator sites is crucial to (1) decipher the cellular and molecular tinnitus mechanisms and (2) engineer future therapies. Although several studies report that changes in central auditory structures cause tinnitus (for review see Roberts et al., 2010), others argue for peripheral (i.e., cochlea and auditory nerve) origin (Guitton et al., 2003, 2005; Ruel et al., 2008a). This chapter proposes that the auditory nerve is a potential tinnitus generator through recruitment of N-methyl-d-aspartate (NMDA) receptors at the first auditory synapse. In addition, the review proposes alternative mechanisms for tinnitus generation within the cochlea as well as future therapies that can be envisioned to treat tinnitus.

The mammalian cochlea receives efferent innervation from neurons located in the superior olivary complex. This efferent olivocochlear innervation is divided in two separate systems, lateral and medial, which mainly innervate afferent dendrites connected to inner hair cells and the cell body of outer hair cells, respectively. Besides other substances, lateral and medial efferent terminals of the adult cochlea use acetylcholine (ACh) as a neurotransmitter. In this study, we have used immunocytochemistry to detect the presence of choline acetyltransferase (ChAT), the synthesizing enzyme of ACh, in efferent olivocochlear terminals during the development of the rat. The appearance and distribution of immunoreactivity to ChAT has been studied in developing rat cochleas from birth (postnatal day 1, P1) to adulthood. Attention was paid to the temporal relationships between the expression of ChAT, the presence of other putative neuroactive substances, the onset of hearing and other developmental phenomena. Our results indicate that ChAT-like immunoreactivity is already present at birth (P1) in the region of inner hair cells, that it appears at P3 in the outer hair cell area and that it reaches an adult pattern of distribution by P15. ACh may thus be present early in the developing cochlea, before the onset of hearing, as it also occurs with other putative transmitters/modulators such as enkephalins, CGRP or GABA. It is suggested that ACh could be involved in the modulation of sound-evoked potentials as soon as they appear, and in the regulation of other developmental phenomena such as neurite outgrowth or synaptogenesis.

Cochlear efferent innervation originates in two different groups of neurons located in the superior olivary complex. A first group of olivocochlear neurons (lateral efferent neurons) lies in the lateral superior olive. They send axons to the organ of Corti, where they synapse with radial afferent dendrites of primary auditory neurons, postsynaptic to the inner hair cells. The second group of neurons (medial efferent neurons) is found in medial subnuclei of the superior olivary complex and sends axons to synapse with outer hair cells. Subpopulations of both medial and lateral olivocochlear neurons probably use γ-aminobutyric acid (GABA) as a neurotransmitter. We have used an immunoperoxidase technique to detect GABA-like immunoreactivity (GABA-LI) in postnatal maturing rat cochleas. The GABA-LI appeared in the inner hair cell region by P3 (P1 = birth) and reached a mature appearance by P15–P16. In the outer hair cell region, GABA-like immunoreactive fibers and terminals could not be identified until P9 and they were only found in the apical end of the cochlea. There was a dual gradient of maturation of GABA-LI in the cochlea. The GABA-LI appeared first at the cochlear base and then extended towards the apex. It also appeared earlier (about a week) in the inner hair cell region than in the outer hair cell region. This dual gradient of maturation is in close agreement with previous data concerning the maturation of the cochlea.

Central control of cochlear function is mediated by the cholinergic (medial) efferent system and both muscarinic and nicotinic acetylcholine receptors are thought to be present on outer hair cells. All the physiological effects of acetylcholine in the cochlea are blocked by strychnine and we therefore investigated whether strychnine interacts with muscarinic receptors in the cochlea. The effects of strychnine on both (3H)-quinuclidinyl benzylate binding and atropine sensitive carbachol-induced (3H)-inositol phosphate formation were examined. Strychnine (1 to 50 microM) has no effect on either quinuclidinyl benzylate binding or carbachol (1 mM)-induced inositol phosphate synthesis. Moreover, strychnine does not change basal inositol phosphate metabolism. These data indicate that muscarinic receptors are not sensitive to strychnine at concentrations which are known to block the effects of acetylcholine on outer hair cells.

We have used an immunocytochemical approach to gain further data supporting a possible neurotransmitter or neuromodulator function for dopamine at the level of efferent (olivocochlear) innervations of the guinea-pig cochlea. Immunofluorescence screening was first done on cochleas two or seven days after infusion with the neurotoxin 6-hydroxydopamine. Two days after neurotoxin perfusion, the intensity of the tyrosine hydroxylase-like immunoreactivity was decreased in the inner and tunnel spiral bundles of the organ of Corti (and in known noradrenergic sympathetic fibers outside this organ), with respect to the control contralateral cochleas. In cochleas screened seven days after 6-hydroxydopamine infusion, no tyrosine hydroxylase-like immunoreactivity could be found in the organ of Corti. Only occasional faint fluorescence could be detected in sympathetic fibers. In another set of experiments, a pre-embedding immunoperoxidase technique was used to localize tyrosine hydroxylase and aromatic amino acid decarboxylase, another catecholamine-synthesizing enzyme, at the ultrastructural level. With both types of antibody, the same kind of results were observed. Immunoreactivities were only seen in vesiculated fibers within the inner and tunnel spiral bundles, thus are likely in lateral efferent varicosities. These immunostained fibers accounted for approximately half of the efferent profiles in the inner spiral bundle. Within this bundle, the immunoreactive fibers established axodendritic synapses with the radial afferent processes of type I neurons which contacted the inner hair cells. In no case was immunoreactivity to either enzyme observed in the outer hair cell region, at the level of medial efferent terminals. The synaptic localization of tyrosine hydroxylase- and aromatic amino acid decarboxylase-like immunoreactivities in the lateral efferent varicosities of the inner spiral bundle, as well as the effect of 6-hydroxydopamine on the tyrosine hydroxylase-like immunoreactivity in this same bundle, further support the hypothesis that a catecholamine could act as a lateral efferent neurotransmitter or neuromodulator. Based on previous data reporting a lack of dopamine-beta-hydroxylase-like immunoreactivity within the organ of Corti, and the effectiveness of a D2 agonist on the cochlear compound action potential of the auditory nerve, this catecholamine could well be dopamine.

The synapses between the inner hair cells (IHCs) and the radial auditory dendrites are thought to be glutamatergic. Besides its fast excitatory properties, glutamate is known to be neurotoxic when released in excess or incompletely recycled. In the cochlea, this may occur in two pathological conditions: ischemia and noise trauma. We have further investigated the acute excitotoxicity (i.e. the swelling of type I afferent dendrites) by electron microscopy processing on guinea pig cochleas after an ischemic exposure lasting 5 to 40 min. The radial auditory dendrites reacted to ischemia in a time-dependent manner, with the swelling extending when the duration of ischemia increased. The type and the specificity of swelling were comparable to what acutely occurs after an exposure to glutamate analogs such as kainic acid or AMPA. A protection against this swelling was obtained by perfusing the cochlea with glutamate antagonists prior to ischemia. DNQX, an antagonist at AMPA/kainate receptors, had a powerful protective effect, and almost complete protection was obtained by perfusing both DNQX and D-AP5 (a NMDA antagonist). The latter results indicate that the two classes of glutamate receptors (AMPA/kainate and NMDA), both found to be electrophysiologically active at the IHC-auditory nerve synapse, are also involved in the excitotoxic processes. In addition, we also report data involving dopamine (its D2 agonist piribedil) a putative neurotransmitter at the lateral efferent synapses, in a postsynaptic protection of primary auditory neurons during transient ischemia. Altogether, these findings constitute a promising pharmacological approach of cochlear pathologies such as neural presbycusis.

We have investigated the hypothesis that the acute ischemic swelling of the radial dendrites connected to the inner hair cells (IHCs) is mediated by glutamatergic receptors. In control cochleas, after 20 min ischemia all the dendrites were dramatically swollen. Conversely, after a perfusion of 50 microM 6-7-dinitroquinoxaline-2,3-dione (DNQX) before ischemia, most dendrites were protected although those contacting the IHCs on their modiolar side frequently swelled. After 50 microM of D-2-amino-5-phosphonopentanoate (D-AP5), no dendrite protection could be obtained. Finally, after DNQX and D-AP5, no dendrite swelling occurred. These results suggest that, in the cochlea, the acute ischemic swelling of dendrites primarily occurs via non-NMDA receptors. However, in radial dendrites contacting the IHCs on their modiolar side, NMDA receptors may contribute to excitotoxicity.

International audience; Autosomal-dominant sensorineural hearing loss is genetically heterogeneous, with a phenotype closely resembling presbycusis, the most common sensory defect associated with aging in humans. We have identified SLC17A8, which encodes the vesicular glutamate transporter-3 (VGLUT3), as the gene responsible for DFNA25, an autosomal-dominant form of progressive, high-frequency nonsyndromic deafness. In two unrelated families, a heterozygous missense mutation, c.632C-->T (p.A211V), was found to segregate with DFNA25 deafness and was not present in 267 controls. Linkage-disequilibrium analysis suggested that the families have a distant common ancestor. The A211 residue is conserved in VGLUT3 across species and in all human VGLUT subtypes (VGLUT1-3), suggesting an important functional role. In the cochlea, VGLUT3 accumulates glutamate in the synaptic vesicles of the sensory inner hair cells (IHCs) before releasing it onto receptors of auditory-nerve terminals. Null mice with a targeted deletion of Slc17a8 exon 2 lacked auditory-nerve responses to acoustic stimuli, although auditory brainstem responses could be elicited by electrical stimuli, and robust otoacoustic emissions were recorded. Ca(2+)-triggered synaptic-vesicle turnover was normal in IHCs of Slc17a8 null mice when probed by membrane capacitance measurements at 2 weeks of age. Later, the number of afferent synapses, spiral ganglion neurons, and lateral efferent endings below sensory IHCs declined. Ribbon synapses remaining by 3 months of age had a normal ultrastructural appearance. We conclude that deafness in Slc17a8-deficient mice is due to a specific defect of vesicular glutamate uptake and release and that VGLUT3 is essential for auditory coding at the IHC synapse.

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 cochleain 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.

Conclusion: Current neurotransmission models based on animal studies on the mammalian inner ear not always reflect the situation in human. Rodents and primates show significant differences in characteristics of efferent innervation as well as the distribution of neuroactive substances. Objective: Immunohistochemistry demonstrates the mammalian efferent system as neurochemically complex and diverse: several neuroactive substances may co-exist within the same efferent terminal. Using light and electron microscopic immunohistochemistry, this study presents a comparative overview of the distribution patterns of choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme, GABA, CGRP, and enkephalins within the peripheral nerve fiber systems of the human inner ear. Materials and methods: Human temporal bones were obtained post mortem and prepared according to a pre-embedding immunohistochemical technique to detect immunoreactivities to ChAT, GABA, CGRP, leu- and met-enkephalins at the electron microscopic level. Results: Immunoreactivities of all the antigens were present within both the lateral and medial efferent systems of the cochlea, whereas only ChAT, GABA, and CGRP were detected in efferent pathways of the vestibular end organs.

PURPOSE. Mutations in the mitochondrial dynamin-related GTPase OPA1 cause autosomal dominant optic atrophy (ADOA), but the pathophysiology of this disease is unknown. As a first step in functional studies, this study was conducted to evaluate the expression of Opal in whole retina and in isolated retinal ganglion cells (RGCs) and to test the effects of Opal downregulation in cultured RGCs. METHODS. Opal mRNA isoforms from total retina and from RGCs freshly isolated by immunopanning were determined by RT-PCR. Protein expression was examined by immunohistochemistry and Western blot with antibodies against Opal and cytochrome c, and the mitochondrial network was visualized with a mitochondrial marker. Short interfering (si)RNA targeting OPA1 mRNAs were transfected to cultured RGCs and mitochondrial network phenotypes were followed for 15 days, in comparison with those of cerebellar granule cells (CGCs). RESULTS. Opal expression did not predominate in rat postnatal RGCs as found by immunohistochemistry and Western blot analysis. The pattern of mRNA isoforms was similar in whole retina and RGCs. After a few days in culture, isolated RGCs showed line mitochondrial punctiform structures in the soma and neurites that colocalized with cytochrome c and Opal. Opal knockdown in RGCs induced mitochondrial network aggregation at a higher rate than in CGCs. CONCLUSIONS. Results suggest that the level of expression and the mRNA isoforms do not underlie the vulnerability of RGCs to OPA1 mutations. However, aggregation of the mitochondrial network induced by the downregulation of Opal appears more frequent in RGCs than in control CGCs.

Olivocochlear efferent neurons originate in the superior olivary complex of the brainstem and terminate within sensory cell regions of the organ of Corti. Components of this complex include the lateral olivocochlear bundle whose unmyelinated axons synapse with radial afferent dendrites below inner hair cells and the medial olivocochlear bundle, from which myelinated axons form a direct synaptic contact with outer hair cells. γ-Aminobutyric acid (GABA), a major neurotransmitter of the central nervous system believed to be responsible for most fast-inhibitory transmissions, has been demonstrated with interspecies variation between mammal and primate auditory efferents. In the present study, we evaluate the immunocytochemical presence of GABA in 10 human cochleae using light and electron microscopy. GABA-like immunostaining could be observed in inner spiral fibers, tunnel spiral fibers, tunnel-crossing fibers, and at efferent endings synapsing with outer hair cells. To approximate medial efferent fiber quantifications, we counted labeled terminals at the base of each outer hair cell and then compared this sum with the number of tunnel crossing fibers. We found a ‘branching ratio’ of 1:2 implicating a doubling in quantifiable efferent fibers at the level of the outer hair cell. In human, the distribution of GABA-like immunoreactivity showed a consistent presence throughout all turns of the cochlea. A new method for application of immunoelectron microscopy on human cochleae using a pre-embedding technique is also presented and discussed.

Mutations in the gene encoding the gap junction protein connexin26 (Cx26) are responsible for the autosomal recessive isolated deafness, DFNB1, which accounts for half of the cases of prelingual profound hereditary deafness in Caucasian populations [1–5]. To date, in vivo approaches to decipher the role of Cx26 in the inner ear have been hampered by the embryonic lethality of the Cx26 knockout mice [6]. To overcome this difficulty, we performed targeted ablation of Cx26 specifically in one of the two cellular networks that it underlies in the inner ear, namely, the epithelial network. We show that homozygous mutant mice, Cx26OtogCre, have hearing impairment, but no vestibular dysfunction. The inner ear developed normally. However, on postnatal day 14 (P14), i.e., soon after the onset of hearing, cell death appeared and eventually extended to the cochlear epithelial network and sensory hair cells. Cell death initially affected only the supporting cells of the genuine sensory cell (inner hair cell, IHC), thus suggesting that it could be triggered by the IHC response to sound stimulation. Altogether, our results demonstrate that the Cx26-containing epithelial gap junction network is essential for cochlear function and cell survival. We conclude that prevention of cell death in the sensory epithelium is essential for any attempt to restore the auditory function in DFNB1 patients.

Acoustic trauma is the major cause of hearing loss in industrialised nations. We show in guinea-pigs that sound exposure (6 kHz, 120 dB sound pressure level for 30 min) leads to sensory cell death and subsequent permanent hearing loss. Ultrastructural analysis reveals that degeneration of the noise-damaged hair cells involved different mechanisms, including typical apoptosis, autolysis and, to a lesser extent, necrosis. Whatever the mechanisms, a common feature of noise damage to hair cells was mitochondrial alteration. Riluzole (2-amino-6-trifluoromethoxy benzothiazole) is a neuroprotective agent that prevents apoptosis- and necrosis-induced cell death. Perfusion of riluzole into the cochlea via an osmotic minipump prevents mitochondrial damage and subsequent translocation of cytochrome c, DNA fragmentation, and hair cell degeneration. This was confirmed by functional tests showing a clear dose-dependent reduction (ED(50)=16.8 microM) of permanent hearing loss and complete protection at 100 microM. Although less efficient than intracochlear perfusion, intraperitoneal injection of riluzole rescues the cochlea within a therapeutic window of 24 h after acoustic trauma.These results show that riluzole is able to prevent and rescue the cochlea from acoustic trauma. It may thus be an interesting molecule for the treatment of inner ear injuries.

Cysteine-string protein is a vesicle-associated protein that plays a vital function in neurotransmitter release. We have studied its expression and regulation during cochlear maturation. Both the mRNA and the protein were found in primary auditory neurons and the sensory inner hair cells. More importantly, cysteine-string protein was localized on synaptic vesicles associated with the synaptic ribbon in inner hair cells and with presynaptic differentiations in lateral and medial olivocochlear terminals -- the cell bodies of which lie in the auditory brainstem. No cysteine-string protein was expressed by the sensory outer hair cells suggesting that the distinct functions of the two cochlear hair cell types imply different mechanisms of neurotransmitter release. In developmental studies in the rat, we observed that cysteine-string protein was present beneath the inner hair cells at birth and beneath outer hair cells by postnatal day 2 only. We found no expression in the inner hair cells before about postnatal day 12, which corresponds to the period during which the first cochlear action potentials could be recorded. In conclusion, the close association of cysteine-string protein with synaptic vesicles tethered to synaptic ribbons in inner hair cells and its synchronized expression with the appearance and maturation of the cochlear potentials strongly suggest that this protein plays a fundamental role in sound-evoked glutamate release by inner hair cells. This also suggests that this role may be common to ribbon synapses and conventional central nervous system synapses.

Different neuroactive substances have been found in the efferent pathways of both the olivocochlear and vestibular systems. In the present study, the distribution and role of three neurotransmitters, choline acetyltransferase (ChAT), gamma aminobutyric acid (GABA), and enkephalin were investigated in the human labyrinth of 4 normal-hearing individuals. Immunohistochemical studies in human inner ear research, however, face a problem of procuring well-preserved specimens with maintained neurotransmitter antigenicity and morphology. Methods and findings are reported and discussed.

Different neuroactive substances have been found in the efferent pathways of both the olivocochlear and vestibular systems. In the present study, the distribution and role of three neurotransmitters, choline acetyltransferase (ChAT), gamma aminobutyric acid (GABA), and enkephalin were investigated in the human labyrinth of 4 normal-hearing individuals. Immunohistochemical studies in human inner ear research, however, face a problem of procuring well-preserved specimens with maintained neurotransmitter antigenicity and morphology. Methods and findings are reported and discussed.

A suitable model of sudden deafness occurring after acoustic trauma or ischemia, is obtained in guinea pigs by an acute intracochlear perfusion of 200 microM alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), a glutamate analog. By overloading the AMPA/kainate receptors, located post-synaptically to inner hair cells (IHCs), it induces a massive swelling of primary auditory neuron dendrites, which disconnects the IHCs. This synaptic uncoupling and the resulting hearing loss are followed by a progressive regrowth of dendrites, which make new synapses with IHCs, leading to a functional recovery of auditory responses that is completed after 5 days. Knowing the role of protein kinase C in neuroplastic events, we studied the expression of its isoforms alpha,beta(I,II) and gamma, respectively pre- and post-synaptic, in auditory neurons at various times after AMPA administration. In untreated cochleas, we observed an expression of PKC alpha,beta(I,II) and gamma in cell bodies of primary auditory neurons. After the intracochlear administration of AMPA, both isozymes were transiently overexpressed, with a peak at 3-6 h, followed by a decrease after about 24 h. At this point in time immuno-electron microscopy revealed some regrowing dendrites immunoreactive for PKCgamma. Five days after AMPA, when the auditory responses were restored, PKCgamma levels were still elevated in ganglion cell bodies.

The lateral efferent (olivocochlear) innervation of the cochlea originates in the brainstem lateral superior olive. It is likely to use acetylcholine, gamma-aminobutyric acid, dopamine and various neuropeptides as neurotransmitters and/or neuromodulators. In order to determine the different coexistence patterns of these molecules in lateral efferent perikarya, we have used double and triple immunofluorescence co-localization techniques to colocalize choline acetyltransferase, glutamate decarboxylase, tyrosine hydroxylase, calcitonin gene-related peptide and enkephalins in single sections of the lateral superior olive. We also used a non-radioactive in situ hybridization technique onto serial sections of this nucleus to confirm the immunofluorescence co-localization data at the mRNA level. Whatever the pair or triplet of primary antibodies tested was, a high ratio of coexistence was observed in the immunofluorescence experiments. In triple co-localization experiments, 90-93% of the choline acetyltransferase-like immunoreactive neurons were also immunoreactive to the two other antigens investigated. The in situ hybridization co-localization data, based on the use of biotin-labelled oligoprobes, qualitatively confirmed these immunofluorescence data. In conclusion, it can be postulated that acetylcholine, gamma-aminobutyric acid, dopamine, calcitonin gene-related peptide, enkephalins and dynorphins (whose coexistence with choline acetyltransferase and enkephalins has been previously described immunocytochemically) coexist in lateral efferent neurons. Based on these results, it is tempting to propose the lateral efferent innervation as a useful model with which the functional implications of the coexistence of neurotransmitters/neuromodulators can be investigated in vivo.

This study was intended to provide an overview of the distribution of calcium-binding proteins in the rodent auditory brainstem. We based our observations on immunohistochemical material obtained in the guinea pig, a species widely used in auditory research in which a mapping of calcium-binding proteins in the auditory brainstem is still missing. Differences in the amounts of these proteins throughout the auditory brainstem were further analyzed semiquantitatively. Parvalbumin was present in most neurons and their axon terminals throughout the ascending auditory brainstem. Nuclei that surround the main relay nuclei of the ascending auditory pathway lacked labeling. Calretinin staining was prominent in spherical and globular cells of the cochlear nucleus, in their axon terminals in the superior olivary complex, and in principal cells of the medial superior olive. Measures of optical densities showed that auditory neurons involved in sound localization had the highest calretinin labeling levels. Calbindin D-28k was present in cartwheel cells of the dorsal cochlear nucleus, in almost all neurons of the medial nucleus of the trapezoid body, and in globular cells in the ventral nucleus of the lateral lemniscus. The labeling patterns for calretinin and calbindin D-28k were non-overlapping throughout the auditory brainstem. This was also evident in the ventral nucleus of the lateral lemniscus where calbindin D-28k-immunoreactive terminals were found in the medial portion, while the calretinin-immunoreactive terminals were observed in the lateral portion. This study presents the first direct and comprehensive comparison of these three calcium-binding proteins in the auditory brainstem of a rodent. Each antibody yields a unique staining pattern that provides a basis for further defining neuronal populations. In addition, since their axons are also selectively stained, auditory nuclei can further be compartmentalized based on different terminal fields. These immunoreactivities have provided clues to the complex structure of the auditory brainstem.

International audience; The medial and lateral efferent innervations originate from distinct parts of the superior olivary complex. Both use acetylcholine, respectively, to modulate the activity of outer hair cells (OHC), and spiral ganglion neurons (SGN) which are postsynaptic to the inner hair cells (IHC). Besides predominantly activating nicotinic receptors, acetylcholine recognizes muscarinic M3 receptors, whose the role(s) and cellular localization(s) are not yet firmly established. We used reverse transcription and polymerase chain reaction to amplify the M3 receptor cDNA in the rat and guinea pig organ of Corti and spiral ganglion. Then, we localized the M3 receptor mRNAs in cochleas and superior olivary complex of both species. The M3 receptor cDNA was amplified from samples of brain, organ of Corti and spiral ganglion. Indeed, its corresponding mRNA was localized in SGNs, OHCs and IHCs. However, in the apical turns, OHCs were often found unlabeled. In the superior olivary complex, M3 mRNAs were colocalized with choline acetyltransferase mRNAs in neurons of the lateral superior olive and ventral nucleus of the trapezoid body. These results suggest that the M3 receptor-induced inositol phosphate formation described in previous studies [21] takes place in both postsynaptic (SGNs, OHCs) and presynaptic components of efferent cochlear synapses, and in cells that are not contacted by efferents in the adult cochlea (IHCs).

We have cloned and sequenced an alpha parvalbumin cDNA from the guinea pig cochlea. The deduced amino acid sequence shows greater identity with the rabbit sequence (86.3%) than with other mammalian sequences (82%). Using in situ hybridization and immunohistochemistry, alpha parvalbumin mRNA and protein were found in primary auditory neurons and inner hair cells, in agreement with RT-PCR data showing alpha parvalbumin mRNA expression in the spiral ganglion and the organ or Corti.

We report our findings in the guinea pig involving dopamine in postsynaptic regulation of the activity of glutamatergic inner hair cells (IHCs) and in protection of primary auditory neurons during transient ischemia. Seven days after intracochlear perfusion of 6-hydroxydopamine, no immunoreactivity to tyrosine hydroxylase (TH) was demonstrable within the organ of Corti. TH and aromatic amino acid decarboxylase were immunolocalized at an ultrastructural level within lateral olivocochlear varicosities synapsing with radial auditory dendrites postsynaptic to the IHCs. The D2 agonist piribedil induced a dose-dependent decrease in the amplitude of the compound action potential of the auditory nerve. Piribedil also prevented appearance of ischemia-induced swelling of the radial dendrites.

In the mammalian cochlea acetylcholine has been considered a major neurotransmitter of the lateral and medial efferent fibers. The aims of the present study were to investigate the expression of ChAT in the human cochlea and to develop a new method for immunohistochemical investigations in the human cochlea both at the light and electronmicroscopic level. We thus examined the ChAT-like immunoreactivity in the human inner ear using light and electron microscopy with a pre-embedding technique. Our present results agree with the previously published data acquired in rodent species. The ChAT-like immunostaining could be found in the inner spiral fibers, the inner spiral bundle, tunnel crossing fibers and at the base of the outer hair cells. No staining was noted in the negative controls experiments, while rat cochleas used as positive controls showed the usual ChAT-like immunostaining as described above. The main difference between human and rat cochleas was that the efferent nerve supply seems to be less pronounced in the human cochleas.

Since ischemic damage in the brain is linked to glutamate excitotoxicity, the effects of an acute exposure to glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) or N-methyl-D aspartate (NMDA) on the radial dendrites were compared with those occurring after a severe cochlear ischemia. Glutamate and AMPA, but not NMDA, produced a drastic swelling restricted to the radial dendrites below the inner hair cells (IHCs). At a concentration of 20 microM AMPA, a full electrophysiological recovery could be observed in some cochleas after washing the drug out. A prior perfusion of 6-7-dinitroquinoxaline-2,3-dione (DNQX, 50 microM) prevented the 25 microM AMPA-induced dendritic swelling. No protective effect of D-2-amino-5-phosphonopentanoate (D-AP5) could be observed. In the same way, ischemia (5-40 minutes) resulted in a clear swelling of the radial dendrites. While D-AP5 had no protective effects, 50 microM DNQX protected most of the radial dendrites from the ischemia-induced swelling, excepting those contacting the modiolar side of the IHCs. Finally, 50 microM DNQX + 50 microM D-AP5 resulted in a nearly complete protection of all the radial dendrites. Altogether, these results suggest that the acute swelling of radial dendrites primarily occurs via AMPA/kainate receptors. However, in radial dendrites contacting the inner hair cells on their modiolar side, NMDA receptors may be also involved.

Major topics addressed in this review on neurotransmitters and neuromodulators of the mammalian cochlea are ; 2) neuronal circuity of the organ of Corti ; 2) hair cell neurotransmitter(s) ; efferent neurotransmitters and neuromodulators (acetylcholine, γ-aminobutyric acid, dopamine, enkephalins, dynorphins, calcitonin gene-related peptide, excitatory amino acids ; and 3) other neuroactive substances in the cochlea (adenosine, ATP, taurine, auditory nerve-activating substance, histamine)

The efferent (olivocochlear) nerve supply to the cochlea is subdivided into a lateral and a medial innervation according to several criteria, e.g. locus of origin in the superior olivary complex and type of synaptic connections established in the organ of Corti. We have used a triple immunofluorescence colocalization approach to determine whether putative cholinergic neurons from the lateral innervation contain both metenkephalin and calcitonin gene-related peptide (CGRP), and whether those from the medial innervation also contain CGRP. About 80% of the choline acetyltransferase (ChAT)-like immunostained lateral efferent neurons within the lateral superior olive were CGRP- and metenkephalin-like immunostained. In the organ of Corti, colocalization of the three antigens within the inner spiral bundle was also found. This bundle contains the lateral efferent synapses, with the dendrites of the primary auditory neurons innervating the sensory inner hair cells. Most of the medial efferent neurons in the ventral nucleus of the trapezoid body were only immunoreactive for ChAT. However, in the rostral part of the nucleus, a minority of ChAT-like immunostained neurons were also CGRP-like immunostained. None of the ChAT-like immunostained medial efferent neurons presented metenkephalin-like immunostaining. In agreement with these brainstem data, partial colocalization of the ChAT- and CGRP-like immunostaining and a lack of metenkephalin immunoreactivity was noted below the sensory outer hair cells, which are the synaptic targets of medial efferent terminals in the organ of Corti. This distinction in the coexistence pattern of the two efferent innervations probably reflects distinct modes of action for acetylcholine in the cochlea. In one case, the effects of acetylcholine on the primary auditory neurons innervating the inner hair cells may require balanced modulation by metenkephalin and CGRP. In the other case, modulation of the effects of acetylcholine on the outer hair cells by neuropeptides would be less critical.

We have recorded cochlear potentials after perilymphatic perfusion of cumulative doses of the excitatory amino acid alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) which selectively recognizes the non-N-methyl-D-aspartate ionotropic receptor formerly known as the quisqualate receptor. Our results show that AMPA (1-80 microM) caused a significant suppression of the amplitude of the compound action potential evoked by acoustic stimulation. A total elimination of this potential at the 100 microM concentration was observed in all animals. In no case was the cochlear microphonic potential, a hair cell receptor potential, affected by AMPA. Histological examinations were performed either at the end of the physiological studies or on cochleas perfused for 10 min with a single dose of AMPA (50 or 100 microM). In both experimental conditions, a selective dendritic swelling or radial afferent nerve endings under the sensory inner hair cells was observed. No damage was found in both types of hair cells supporting cells, lateral and medial efferent fibers and spiral afferent nerve ending on the outer hair cells. The occurrence of the radial dendrite swelling was prevented when 6,7-dinitroquinoxaline-2,3-dione (500 microM) was perfused in the cochlea 10 min prior, then concomitantly with AMPA. The present study strongly suggests that non-N-methyl-D-aspartate receptors, possibly of the AMPA subtype, are involved in the synaptic transmission between the inner hair cells and the primary auditory neurons. They provide further support for the hypothesis that L-glutamate, or another excitatory amino acid, acts as an inner hair cell neurotransmitter.

This paper presents the works and methods of our respective laboratories using electron microscopic immunocytochemistry to identify and localize cochlear neurotransmitters. Antibodies to various prospective neurotransmitters and associated enzymes have been used to study the ultrastructural localization of several candidates for olivocochlear efferent neurotransmitters previously suggested by light microscopic immunocytochemistry. Antibodies against enkephalins label lateral olivocochlear efferent fibers. Antibodies against choline acetyltransferase (ChAT) (an enzyme marker for acetylcholine) label a major population of both lateral and medial efferent fibers and terminals, whereas antibodies to gamma-aminobutyric acid (GABA) label what might be a small subpopulation of both the lateral and medial efferent systems. The GABA-like immunostained medial efferent fibers are preferentially located in the upper turns of the guinea pig cochlea, particularly the third turn. Immunoelectron microscopy shows that neither GABA nor ChAT immunolabels all medial efferent terminals, regardless of cochlear turn. All the different types of immunolabeled efferent terminals have been observed to make characteristic synaptic contacts; lateral efferent terminals on afferent dendrites and medial efferent terminals on outer hair cells and occasionally on type II afferent dendrites. Other types of contacts involving GABA-like, and sometimes met-enkephalin-like, immunostained fibers are occasionally seen particularly in the upper turns of the cochlea. Immunoelectron microscopic results suggest that both medial and lateral efferent systems might be further subdivided on the basis of differences in neurotransmitters. Future trends of immunocytochemical research on cochlear neurotransmitters are proposed, particularly colocalization studies, which show a complex pattern of coexistence of neurotransmitters in the lateral efferent system.

Using the immunofluorescence technique, three populations of fibers staining for calcitonin gene-related peptide are identified in postnatal developing cochleas and in adult rat cochleas. During the maturation of the cochlea, the immunostaining first appears in the basal turn and then extends toward the apex of the cochlea. The first population of immunostained fibers belongs to the lateral olivocochlear innervation. It is observed at postnatal day 4 within the inner spiral bundle of the organ of Corti. The second population of calcitonin gene-related peptide-like immunostained fibers belongs to the medial olivocochlear innervation. It is first identified at postnatal day 6 under the outer hair cells, generally in the first row. In the older stages, this population of fibers progressively extends toward the external row of outer hair cells. Finally, the third population of immunostained fibers belongs to the sympathetic supply of the cochlea. They can be identified at postnatal day 6 around the cochlear artery and its branches, and also within the entire modiolus. Our results confirm the presence of calcitonin gene-related peptide in fibers of the lateral and medial efferent innervations of the cochlea. They indicate an early appearance of calcitonin gene-related peptide in the developing cochlea, before the onset of the cochlear function, suggesting for this peptide either a neurotrophic function or a regulation of the early cochlear potentials at the level of the lateral efferent synapses.

gamma-Aminobutyric acid (GABA)-ergic efferent nerve fibers were studied during the postnatal development of the rat cochlea, using light microscopic immunocytochemical techniques. Antibodies against GABA and its synthesizing enzyme, glutamate decarboxylase (GAD), were used. Immunoreactivity to GAD is already present at birth (postnatal day 1) and could be found below the inner hair cells of the basal turn. Immunoreactivity progressively extends toward the apical turn until day 3. GAD-like immunoreactivity appears under the outer hair cells on postnatal day 15 and is only found in the upper part of the second turn and in the apical turn. The distribution of GABA-like immunoreactivity closely corresponds to that observed with the anti-glutamate decarboxylase antibody. However, the GABA-like immunoreactivity appears about 1-2 days after GAD-like immunoreactivity. At the beginning of the 3rd postnatal week, an adult pattern of GABA- and GAD-like immunoreactivity is established. These results suggest that GABA, which appears under the inner hair cells largely before the onset of hearing, may play a neurotrophic function during cochlear maturation and participate in the regulation of the first cochlear potentials as soon as they appear.

We have used the retrograde axonal transport of Fast Blue, injected intra-cochlearly, to identify in the rat lateral superior olive (LSO) neurons which belong to the lateral olivocochlear system (LOCS). Using immunohistofluorescence technique, we have localized within Fast Blue-labeled neurons immunostainings for enkephalins (Met-enkephalin, Met-enkephalin-Arg6-Gly7-Leu8), dynorphins (alpha-neo-endorphin, dynorphin 1-17) or choline acetyltransferase (ChAT). Many Fast Blue-labeled neurons did not show any immunostaining, but all the immunostained neurons found in the LSO were Fast Blue-labeled. In immunohistofluorescence colocalization experiments of two antigens, we could colocalize within the same neurons of the rat LSO immunostainings for ChAT and enkephalins and for ChAT and dynorphins. In each case, neurons only immunostained for ChAT, enkephalins or dynorphins could also be observed. A colocalization of the immunostainings for Met-enkephalin and dynorphins within neurons of the guinea pig and rat LSO was also found. However, in this case, neurons which did not show colocalization were only Met-enkephalin-immunoreactive, thus suggesting that all the dynorphins immunoreactive LSO neurons also contain enkephalins. These findings support the idea that the neurons of the LSO which contain ChAT-, enkephalin- or dynorphin-immunostainings project to the cochlea and belong to the LOCS. It can also be concluded that acetylcholine, enkephalins and dynorphins coexist within a same population of neurons of the LOCS, although other patterns of co-containment of neuroactive substances within LOCS neurons may also exist.

We have reviewed the experiments done in our laboratory concerning various cochlear neuroactive substances. Data using chemical neuroanatomy and neurochemical techniques are described. They allow the identification and localization of neuroactive substances which could act as neurotransmitters and/ or neuromodulators at the different types of synapses in the organ of Corti. Three hypotheses are presented: (1) the inner hair cells use glutamate as a neurotransmitter, but in addition to its excitatory properties, glutamate may also be involved in pathophysiological events affecting afferent auditory dendrites; (2) subpopulations of both the lateral and medial olivocochlear efferent systems can be differentiated by the neuroactive substances they may use; (3) the base and the apex of the cochlea can be distinguished on the basis of neurochemical data.