Selective deletion of cochlear hair cells causes rapid age-dependent changes in spiral ganglion and cochlear nucleus neurons.

During nervous system development, critical periods are usually defined as early periods during which manipulations dramatically change neuronal structure or function, whereas the same manipulations in mature animals have little or no effect on the same property. Neurons in the ventral cochlear nucleus (CN) are dependent on excitatory afferent input for survival during a critical period of development. Cochlear removal in young mammals and birds results in rapid death of target neurons in the CN. Cochlear removal in older animals results in little or no neuron death. However, the extent to which hair-cell-specific afferent activity prevents neuronal death in the neonatal brain is unknown. We further explore this phenomenon using a new mouse model that allows temporal control of cochlear hair cell deletion. Hair cells express the human diphtheria toxin (DT) receptor behind the Pou4f3 promoter. Injections of DT resulted in nearly complete loss of organ of Corti hair cells within 1 week of injection regardless of the age of injection. Injection of DT did not influence surrounding supporting cells directly in the sensory epithelium or spiral ganglion neurons (SGNs). Loss of hair cells in neonates resulted in rapid and profound neuronal loss in the ventral CN, but not when hair cells were eliminated at a more mature age. In addition, normal survival of SGNs was dependent on hair cell integrity early in development and less so in mature animals. This defines a previously undocumented critical period for SGN survival.

Loss of auditory activity modifies the location of potassium channel KCNQ5 in auditory brainstem neurons.

KCNQ5/Kv7.5, a low-threshold noninactivating voltage-gated potassium channel, is preferentially targeted to excitatory endings of auditory neurons in the adult rat brainstem. Endbulds of Held from auditory nerve axons on the bushy cells of the ventral cochlear nucleus (VCN) and calyces of Held around the principal neurons in the medial nucleus of the trapezoid body (MNTB) are rich in KCNQ5 immunoreactivity. We have previously shown that this synaptic distribution occurs at about the time of hearing onset. The current study tests whether this localization in excitatory endings depends on the peripheral activity carried by the auditory nerve. Auditory nerve activity was abolished by cochlear removal or intracochlear injection of tetrodotoxin (TTX). Presence of KCNQ5 was analyzed by immunocytochemistry, Western blotting, and quantitative reverse transcription polymerase chain reaction. After cochlear removal, KCNQ5 immunoreactivity was virtually undetectable at its usual location in endbulbs and calyces of Held in the anteroventral CN and in the MNTB, respectively, although it was found in cell bodies in the VCN. The results were comparable after intracochlear TTX injection, which drastically reduced KCNQ5 immunostaining in MNTB calyces and increased immunolabeling in VCN cell bodies. Endbulbs of Held in the VCN also showed diminished KCNQ5 labeling after intracochlear TTX injection. These results show that peripheral activity from auditory nerve afferents is necessary to maintain the subcellular distribution of KCNQ5 in synaptic endings of the auditory brainstem. This may contribute to adaptations in the excitability and neurotransmitter release properties of these presynaptic endings under altered input conditions.

TRPC1 and metabotropic glutamate receptor expression in rat auditory midbrain neurons.

Canonical transient receptor potential (TRPC) channels are plasma membrane cation channels included in the TRP superfamily. TRPC1 is expressed widely in the central nervous system and is linked to group I metabotropic glutamate receptors (mGluRs). In the auditory brainstem, TRPC1 expression has never been described, although group I mGluRs are present. In the central nucleus of the inferior colliculus (CIC), activation of group I mGluRs induces an extracellular Ca(2+) influx after store depletion. Therefore, this study examines whether TRPC1 is expressed in this region to establish a correlation with mGluRs. By quantitative reverse transcription-polymerase chain reaction and Western blotting, this study assesses the presence of TRPC1 along with both group I mGluR subtypes mGluR1 and mGluR5 in the rat inferior colliculus (IC). All these molecules present a robust expression in the IC. By confocal double immunofluorescence, this study also demonstrates that TRPC1 colocalizes with parvalbumin, a CIC neuronal marker, in many cells. Conversely, TRPC1 was lacking in glial fibrillary acidic protein-positive glial cells. All the glutamate acid decarboxylase 67 (GAD67)-immunoreactive neurons and many GAD67-negative neurons were positive to TRPC1, which indicates the presence of TRPC1 in γ-aminobutyric acid (GABA)-ergic and non-GABAeregic neurons. With regard to subcellular distribution, TRPC1 was absent in synaptophysin-immunoreactive axonic terminals but colocalized with postsynaptic marker microtubule-associated protein 2 in cell bodies and dendrites. TRPC1 totally overlapped group I mGluRs, which supports the involvement of TRPC1 in the mGluR pathway and, likely, in auditory signal processing at the midbrain level. .

Efficacy and Mechanisms of Antioxidant Compounds and Combinations Thereof against Cisplatin-Induced Hearing Loss in a Rat Model.

Cisplatin is an election chemotherapeutic agent used for many cancer treatments. Its cytotoxicity against neoplastic cells is mirrored by that taking place in healthy cells and tissues, resulting in serious adverse events. A very frequent one is ototoxicity, causing hearing loss which may permanently affect quality of life after successful oncologic treatments. Exacerbated oxidative stress is a main cytotoxic mechanism of cisplatin, including ototoxicity. Previous reports have shown antioxidant protection against cisplatin ototoxicity, but there is a lack of comparative studies on the otoprotectant activity and mechanism of antioxidant formulations. Here, we show evidence that a cocktail of vitamins A, C, and E along with Mg++ (ACEMg), previously shown to protect against noise-induced hearing loss, reverses auditory threshold shifts, promotes outer hair cell survival, and attenuates oxidative stress in the cochlea after cisplatin treatment, thus protecting against extreme cisplatin ototoxicity in rats. The addition of 500 mg N-acetylcysteine (NAC), which, administered individually, also shows significant attenuation of cisplatin ototoxicity, to the ACEMg formulation results in functional degradation of ACEMg otoprotection. Mg++ administered alone, as MgSO4, also prevents cisplatin ototoxicity, but in combination with 500 mg NAC, otoprotection is also greatly degraded. Increasing the dose of NAC to 1000 mg also results in dramatic loss of otoprotection activity compared with 500 mg NAC. These findings support that single antioxidants or antioxidant combinations, particularly ACEMg in this experimental series, have significant otoprotection efficacy against cisplatin ototoxicity. However, an excess of combined antioxidants and/or elevated doses, above a yet-to-be-defined "antioxidation threshold", results in unrecoverable redox imbalance with loss of otoprotectant activity.

Signalling routes and developmental regulation of group I metabotropic glutamate receptors in rat auditory midbrain neurons.

Group I metabotropic glutamate receptors (mGluRs) are linked to intracellular Ca(2+) signalling and play important roles related to synaptic plasticity and development. In neurons from the central nucleus of the inferior colliculus (CIC), the activation of these receptors evokes large [Ca(2+) ](i) responses. By using optical imaging of the fluorescent Ca(2+) -sensitive dye Fura-2, we have explored which [Ca(2+) ](i) routes are triggered by group I mGluR activation in young CIC neurons and whether mGluR-induced [Ca(2+) ](i) responses are regulated during postnatal development. In addition, real-time quantitative RT-PCR was used to study the developmental expression of both group I mGluR subtypes, mGluR1 and mGluR5. Application of DHPG, a specific agonist of group I mGluRs, was used on CIC slices from young rats to elicit [Ca(2+) ](i) responses. A majority of responses consisted of an initial thapsigargin-sensitive Ca(2+) peak, related to store depletion, followed by a plateau phase, sensitive to the store-operated Ca(2+) entry blocker 2-APB. During postnatal development, from P6 to P17, DHPG-induced [Ca(2+) ](i) responses changed. The largest Ca(2+) responses were reached at P6, whereas lower peak and plateau responses were found after hearing onset, at P13-P14 and P17. qRT-PCR analysis also revealed important differences in the expression of both mGluR1 and mGluR5 subtypes during development, with the highest levels of both subtypes at P7 and a developmental decrease of both transcripts. Our results suggest both intra- and extracellular routes for [Ca(2+) ](i) increases linked to group I mGluRs in CIC neurons and a regulation of group I mGluR activity and expression during auditory development.

Kanamycin and Cisplatin Ototoxicity: Differences in Patterns of Oxidative Stress, Antioxidant Enzyme Expression and Hair Cell Loss in the Cochlea.

Kanamycin and cisplatin are ototoxic drugs. The mechanisms are incompletely known. With subcutaneous kanamycin (400 mg/kg, 15 days), auditory threshold shifts were detected at days 12-13 at 16 and 32 kHz, extending to 8 and 4 kHz at days 14-15. The outer hair cell (OHC) loss was concentrated past day 12. The maximum cochlear length showing apoptotic cells, tested with TUNEL, was at day 13. At day 15, 1/5 of the apical cochlea contained preserved OHCs. 3-nitrotyrosine (3-NT) immunolabeling, showing oxidative stress, was found in surviving OHCs and in basal and middle portions of the stria vascularis (SV). The antioxidant Gpx1 gene expression was decreased. The immunocytochemistry showed diminished Gpx1 in OHCs. With intraperitoneal cisplatin (16 mg/kg, single injection), no evoked auditory activity was recorded at the end of treatment, at 72 h. The basal third of the cochlea lacked OHCs. Apoptosis occupied the adjacent 1/3, and the apical third contained preserved OHCs. 3-NT immunolabeling was extensive in OHCs and the SV. Gpx1 and Sod1 gene expression was downregulated. Gpx1 immunostaining diminished in middle and basal SV. More OHCs survived cisplatin than kanamycin towards the apex, despite undetectable evoked activity. Differential regulation of antioxidant enzyme levels suggests differences in the antioxidant response for both drugs.

Age-Related Inflammation and Oxidative Stress in the Cochlea Are Exacerbated by Long-Term, Short-Duration Noise Stimulation.

We have previously reported that young adult rats exposed to daily, short-duration noise for extended time periods, develop accelerated presbycusis starting at 6 months of age. Auditory aging is associated with progressive hearing loss, cell deterioration, dysregulation of the antioxidant defense system, and chronic inflammation, among others. To further characterize cellular and molecular mechanisms at the crossroads between noise and age-related hearing loss (ARHL), 3-month-old rats were exposed to a noise-accelerated presbycusis (NAP) protocol and tested at 6 and 16 months of age, using auditory brainstem responses, Real-Time Reverse Transcription-Quantitative PCR (RT-qPCR) and immunocytochemistry. Chronic noise-exposure leading to permanent auditory threshold shifts in 6-month-old rats, resulted in impaired sodium/potassium activity, degenerative changes in the lateral wall and spiral ganglion, increased lipid peroxidation, and sustained cochlear inflammation with advancing age. Additionally, at 6 months, noise-exposed rats showed significant increases in the gene expression of antioxidant enzymes (superoxide dismutase 1/2, glutathione peroxidase 1, and catalase) and inflammation-associated molecules [ionized calcium binding adaptor molecule 1, interleukin-1 beta (IL-1ß), and tumor necrosis factor-alpha]. The levels of IL-1ß were upregulated in the spiral ganglion and spiral ligament, particularly in type IV fibrocytes; these cells showed decreased levels of connective tissue growth factor and increased levels of 4-hydroxynonenal. These data provide functional, structural and molecular evidence that age-noise interaction contributes to exacerbating presbycusis in young rats by leading to progressive dysfunction and early degeneration of cochlear cells and structures. These findings contribute to a better understanding of NAP etiopathogenesis, which is essential as it affects the life quality of young adults worldwide.

Frailty Syndrome and Oxidative Stress as Possible Links Between Age-Related Hearing Loss and Alzheimer's Disease.

As it is well known, a worldwide improvement in life expectancy has taken place. This has brought an increase in chronic pathologies associated with aging. Cardiovascular, musculoskeletal, psychiatric, and neurodegenerative conditions are common in elderly subjects. As far as neurodegenerative diseases are concerned dementias and particularly, Alzheimer's disease (AD) occupy a central epidemiological position given their high prevalence and their profound negative impact on the quality of life and life expectancy. The amyloid cascade hypothesis partly explains the immediate cause of AD. However, limited therapeutical success based on this hypothesis suggests more complex remote mechanisms underlying its genesis and development. For instance, the strong association of AD with another irreversible neurodegenerative pathology, without curative treatment and complex etiology such as presbycusis, reaffirms the intricate nature of the etiopathogenesis of AD. Recently, oxidative stress and frailty syndrome have been proposed, independently, as key factors underlying the onset and/or development of AD and presbycusis. Therefore, the present review summarizes recent findings about the etiology of the above-mentioned neurodegenerative diseases, providing a critical view of the possible interplay among oxidative stress, frailty syndrome, AD and presbycusis, that may help to unravel the common mechanisms shared by both pathologies. This knowledge would help to design new possible therapeutic strategies that in turn, will improve the quality of life of these patients.

Antioxidants and Vasodilators for the Treatment of Noise-Induced Hearing Loss: Are They Really Effective?

We live in a world continuously immersed in noise, an environmental, recreational, and occupational factor present in almost every daily human activity. Exposure to high-level noise could affect the auditory function of individuals at any age, resulting in a condition called noise-induced hearing loss (NIHL). Given that by 2018, more than 400 million people worldwide were suffering from disabling hearing loss and that about one-third involved noise over-exposure, which represents more than 100 million people, this hearing impairment represents a serious health problem. As of today, there are no therapeutic measures available to treat NIHL. Conventional preventive measures, including public awareness and education and physical barriers to noise, do not seem to suffice, as the population is still being affected by damaging noise levels. Therefore, it is necessary to develop or test pharmacological agents that may prevent and/or diminish the impact of noise on hearing. Data availability about the pathophysiological processes involved in triggering NIHL has allowed researchers to use compounds, that could act as effective therapies, by targeting specific mechanisms such as the excess generation of free radicals and blood flow restriction to the cochlea. In this review, we summarize the advantages/disadvantages of these therapeutic agents, providing a critical view of whether they could be effective in the human clinic.

Expression and Localization of Kv1.1 and Kv3.1b Potassium Channels in the Cochlear Nucleus and Inferior Colliculus after Long-Term Auditory Deafferentation.

Deafness affects the expression and distribution of voltage-dependent potassium channels (Kvs) of central auditory neurons in the short-term, i.e., hours to days, but the consequences in the expression of Kvs after long-term deafness remain unknown. We tested expression and distribution of Kv1.1 and Kv3.1b, key for auditory processing, in the rat cochlear nucleus (CN), and in the inferior colliculus (IC), at 1, 15 and 90 days after mechanical lesion of the cochlea, using a combination of qRT-PCR and Western blot in the whole CN, along with semi-quantitative immunocytochemistry in the AVCN, where the role of both Kvs in excitability control for accurate auditory timing signal processing is well established. Neither Kv1.1/Kv3.1b mRNA or protein expression changed significantly in the CN between 1 and 15 days after deafness. At 90 days post-lesion, however, mRNA and protein expression for both Kvs increased, suggesting that expression regulation of Kv1.1 and Kv3.1b is part of cellular mechanisms for long-term adaptation to auditory input deprivation in the CN. Consistent with these findings, immunocytochemical localization showed increased labeling intensity for both Kvs in the AVCN at day 90 after cochlear lesion, further supporting that up-regulation of Kv1.1 and Kv3.1b in neurons of this CN division, over a long term after auditory deprivation, may be required to adapt intrinsic excitability to altered input. Contrary to findings in the CN, in the IC, expression levels of Kv1.1 and Kv3.1b did not undergo major changes after cochlear lesion. In particular, there was no evidence of long-term up-regulation of neither Kv1.1 or Kv3.1b, supporting that such post-lesion adaptive mechanism may not be needed in the IC. This suggests that post-lesion plastic adaptations to auditory input deprivation are not stereotypical along the auditory pathway.

Oral Antioxidant Vitamins and Magnesium Limit Noise-Induced Hearing Loss by Promoting Sensory Hair Cell Survival: Role of Antioxidant Enzymes and Apoptosis Genes.

Noise induces oxidative stress in the cochlea followed by sensory cell death and hearing loss. The proof of principle that injections of antioxidant vitamins and Mg2+ prevent noise-induced hearing loss (NIHL) has been established. However, effectiveness of oral administration remains controversial and otoprotection mechanisms are unclear. Using auditory evoked potentials, quantitative PCR, and immunocytochemistry, we explored effects of oral administration of vitamins A, C, E, and Mg2+ (ACEMg) on auditory function and sensory cell survival following NIHL in rats. Oral ACEMg reduced auditory thresholds shifts after NIHL. Improved auditory function correlated with increased survival of sensory outer hair cells. In parallel, oral ACEMg modulated the expression timeline of antioxidant enzymes in the cochlea after NIHL. There was increased expression of glutathione peroxidase-1 and catalase at 1 and 10 days, respectively. Also, pro-apoptotic caspase-3 and Bax levels were diminished in ACEMg-treated rats, at 10 and 30 days, respectively, following noise overstimulation, whereas, at day 10 after noise exposure, the levels of anti-apoptotic Bcl-2, were significantly increased. Therefore, oral ACEMg improves auditory function by limiting sensory hair cell death in the auditory receptor following NIHL. Regulation of the expression of antioxidant enzymes and apoptosis-related proteins in cochlear structures is involved in such an otoprotective mechanism.

Development of glutamate receptors in auditory neurons from long-term organotypic cultures of the embryonic chick hindbrain.

We used long-range organotypic cultures of auditory nuclei in the chick hindbrain to test the development of glutamate receptor activity in auditory neurons growing in a tissue environment that includes early deprivation of peripheral glutamatergic input, subsequent to removal of the otocyst. Cultures started at embryonic day (E)5, and lasted from 6 h to 15 days. Neuronal migration, clustering and axonal extension from the nucleus magnocellularis (NM) to the nucleus laminaris (NL) partially resembled events in vivo. However, the distinctive laminar organization of the NL was not observed. Glutamate receptor (GluR) activity was tested with optical recordings of intracellular Ca2+ in the NM. alpha-Amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)/kainate receptors had Ca2+ responses with a time course similar to that in control slices. Peak amplitude, however, was significantly lower. N-methyl-D-aspartate (NMDA)-mediated Ca2+ responses were higher in 2-day cultures (E5 + 2d) than in E7 explant controls, returning later to control values. Metabotropic GluRs did not elicit Ca2+ responses at standard agonist doses. Blocking NMDA or AMPA/kainate receptors with specific antagonists for 10 days in culture did not limit neuronal survival. Blocking metabotropic GluRs resulted in complete neuronal loss. Thus, ionotropic GluRs are not required for NM neuronal survival. However, their activity during development is affected when neurons grow in an in vitro environment that includes prevention of arrival of peripheral glutamatergic input.

Age-Related Hearing Loss Is Accelerated by Repeated Short-Duration Loud Sound Stimulation.

Both age-related hearing loss (ARHL) and noise-induced hearing loss (NIHL) may share pathophysiological mechanisms in that they are associated with excess free radical formation and cochlear blood flow reduction, leading to cochlear damage. Therefore, it is possible that short, but repeated exposures to relatively loud noise during extended time periods, like in leisure (i.e., musical devices and concerts) or occupational noise exposures, may add to cochlear aging mechanisms, having an impact on the onset and/or progression of ARHL. Consequently, the aim of the present study was to determine if repeated short-duration overexposure to a long-term noise could accelerate permanent auditory threshold shifts associated with auditory aging in an animal model of ARHL. Toward this goal, young adult, 3-month-old Wistar rats were divided into two groups: one exposed (E) and the other non-exposed (NE) to noise overstimulation. The stimulation protocol consisted of 1 h continuous white noise at 110 dB sound pressure level (SPL), 5 days a week, allowing 2 days for threshold recovery before initiating another stimulation round, until the animals reached an age of 18 months. Auditory brainstem response (ABR) recordings at 0.5, 1, 2, 4, 8, 16, and 32 kHz were performed at 3, 6, 12, and 18 months of age. The results demonstrate that in the E group there were significant increases in auditory thresholds at all tested frequencies starting already at 6 months of age, which extended at 12 and 18 months. However, in NE animals threshold shifts were not evident until 12 months, extending to 18 months of age. Threshold shifts observed in the E animals at 6 and 12 months were significantly larger than those observed in the NE group at the same ages. Threshold shifts at 6 and 12 months in E animals resembled those at 12 and 18 months in NE animals, respectively. This suggests that repeated noise overstimulation in short-duration episodes accelerates the time-course of hearing loss in this animal model of ARHL.

Neuroglial Involvement in Abnormal Glutamate Transport in the Cochlear Nuclei of the Igf1 -/- Mouse.

Insulin-like growth factor 1 (IGF-1) is a powerful regulator of synaptic activity and a deficit in this protein has a profound impact on neurotransmission, mostly on excitatory synapses in both the developing and mature auditory system. Adult Igf1 -/- mice are animal models for the study of human syndromic deafness; they show altered cochlear projection patterns into abnormally developed auditory neurons along with impaired glutamate uptake in the cochlear nuclei, phenomena that probably reflect disruptions in neuronal circuits. To determine the cellular mechanisms that might be involved in regulating excitatory synaptic plasticity in 4-month-old Igf1 -/- mice, modifications to neuroglia, astroglial glutamate transporters (GLTs) and metabotropic glutamate receptors (mGluRs) were assessed in the cochlear nuclei. The Igf1 -/- mice show significant decreases in IBA1 (an ionized calcium-binding adapter) and glial fibrillary acidic protein (GFAP) mRNA expression and protein accumulation, as well as dampened mGluR expression in conjunction with enhanced glutamate transporter 1 (GLT1) expression. By contrast, no differences were observed in the expression of glutamate aspartate transporter (GLAST) between these Igf1 -/- mice and their heterozygous or wildtype littermates. These observations suggest that congenital IGF-1 deficiency may lead to alterations in microglia and astrocytes, an upregulation of GLT1, and the downregulation of groups I, II and III mGluRs. Understanding the molecular, biochemical and morphological mechanisms underlying neuronal plasticity in a mouse model of hearing deficits will give us insight into new therapeutic strategies that could help to maintain or even improve residual hearing when human deafness is related to IGF-1 deficiency.

An Oral Combination of Vitamins A, C, E, and Mg++ Improves Auditory Thresholds in Age-Related Hearing Loss.

The increasing rate of age-related hearing loss (ARHL), with its subsequent reduction in quality of life and increase in health care costs, requires new therapeutic strategies to reduce and delay its impact. The goal of this study was to determine if ARHL could be reduced in a rat model by administering a combination of antioxidant vitamins A, C, and E acting as free radical scavengers along with Mg++, a known powerful cochlear vasodilator (ACEMg). Toward this goal, young adult, 3 month-old Wistar rats were divided into two groups: one was fed with a diet composed of regular chow ("normal diet," ND); the other received a diet based on chow enriched in ACEMg ("enhanced diet," ED). The ED feeding began 10 days before the noise stimulation. Auditory brainstem recordings (ABR) were performed at 0.5, 1, 2, 4, 8, 16, and 32 kHz at 3, 6-8, and 12-14 months of age. No differences were observed at 3 months of age, in both ND and ED animals. At 6-8 and 12-14 months of age there were significant increases in auditory thresholds and a reduction in the wave amplitudes at all frequencies tested, compatible with progressive development of ARHL. However, at 6-8 months threshold shifts in ED rats were significantly lower in low and medium frequencies, and wave amplitudes were significantly larger at all frequencies when compared to ND rats. In the oldest animals, differences in the threshold shift persisted, as well as in the amplitude of the wave II, suggesting a protective effect of ACEMg on auditory function during aging. These findings indicate that oral ACEMg may provide an effective adjuvant therapeutic intervention for the treatment of ARHL, delaying the progression of hearing impairment associated with age.

The potassium channel KCNQ5/Kv7.5 is localized in synaptic endings of auditory brainstem nuclei of the rat.

KCNQ, also called Kv7, is a family of voltage-dependent potassium channels with important roles in excitability regulation. Of its five known subunits, KCNQ5/Kv7.5 is extensively expressed in the central nervous system and it contributes to the generation of M-currents. The distribution of KCNQ5 was analyzed in auditory nuclei of the rat brainstem by high-resolution immunocytochemistry. Double labeling with anti-KCNQ5 antibodies and anti-synaptophysin or anti-syntaxin, which mark synaptic endings, or anti-microtubule-associated protein 2 (MAP2) antibodies, which mark dendrites, were used to analyze the subcellular distribution of KCNQ5 in neurons in the cochlear nucleus, superior olivary complex, nuclei of the lateral lemniscus, and inferior colliculus. An abundance of KCNQ5 labeling in punctate structures throughout auditory brainstem nuclei along with colocalization with such synaptic markers suggests that a preferred localization of KCNQ5 is in synaptic endings in these auditory nuclei. Punctate KCNQ5 immunoreactivity virtually disappeared from the cochlear nucleus after cochlea removal, which strongly supports localization of this channel in excitatory endings of the auditory nerve. Actually, neither glycinergic endings, labeled with an anti-glycine transporter 2 (GlyT2) antibody, nor gamma-aminobutyric acid (GABA)ergic endings, labeled with an anti-glutamic acid decarboxylase (GAD65) antibody, contained KCNQ5 immunoreactivity, suggesting that KCNQ5 is mostly in excitatory endings throughout the auditory brainstem. Overlap of KCNQ5 and MAP2 labeling indicates that KCNQ5 is also targeted to dendritic compartments. These findings predict pre- and postsynaptic roles for KCNQ5 in excitability regulation in auditory brainstem nuclei, at the level of glutamatergic excitatory endings and in dendrites.

Auditory nerve input is not an absolute requirement for the expression, distribution and calcium permeability of AMPA receptors in the adult rat ventral cochlear nucleus.

In order to understand whether glutamatergic excitatory presynaptic input is an absolute requirement for the adult regulation of postsynaptic glutamate receptors we analyzed if a period of 11 days of excitatory deprivation affects the expression, distribution and Ca(2+) permeability of AMPA receptor subunits in the ventral cochlear nucleus of the rat. Bilateral cochlear ablations were performed in 30-day-old rats. After 11 days of survival, immunohistochemistry for GluR1, GluR2/3 and GluR4 AMPA receptor subunits showed no changes in the normal pattern of distribution, with GluR2/3 and GluR4 immunoreactivity predominating, and little GluR1. No changes in the amount of these AMPA receptor subunits were found between normal and cochleotomized rats in Western blots. AMPA receptors lacking the GluR2 subunit are Ca(2+) permeable. Kainate-induced Co(2+) uptake histochemistry, which labels AMPA Ca(2+) permeable receptors, demonstrated no changes in somatic labeling intensity for Co(2+), 11 days after cochleotomy. Therefore, our data indicate that excitatory input is not an absolute requirement to maintain AMPA receptor subunit expression, distribution and functional properties such as Ca(2+) permeability in VCN neurons. Nevertheless, subtle changes in AMPA receptors through regulatory post-transductional mechanisms cannot be ruled out.

Ablation of the auditory cortex results in changes in the expression of neurotransmission-related mRNAs in the cochlea.

The auditory cortex (AC) dynamically regulates responses of the Organ of Corti to sound through descending connections to both the medial (MOC) and lateral (LOC) olivocochlear efferent systems. We have recently provided evidence that AC has a reinforcement role in the responses to sound of the auditory brainstem nuclei. In a molecular level, we have shown that descending inputs from AC are needed to regulate the expression of molecules involved in outer hair cell (OHC) electromotility control, such as prestin and the α10 nicotinic acetylcholine receptor (nAchR). In this report, we show that descending connections from AC to olivocochlear neurons are necessary to regulate the expression of molecules involved in cochlear afferent signaling. RT-qPCR was performed in rats at 1, 7 and 15 days after unilateral ablation of the AC, and analyzed the time course changes in gene transcripts involved in neurotransmission at the first auditory synapse. This included the glutamate metabolism enzyme glutamate decarboxylase 1 (glud1) and AMPA glutamate receptor subunits GluA2-4. In addition, gene transcripts involved in efferent regulation of type I spiral ganglion neuron (SGN) excitability mediated by LOC, such as the α7 nAchR, the D2 dopamine receptor, and the α1, and γ2 GABAA receptor subunits, were also investigated. Unilateral AC ablation induced up-regulation of GluA3 receptor subunit transcripts, whereas both GluA2 and GluA4 mRNA receptors were down-regulated already at 1 day after the ablation. Unilateral removal of the AC also resulted in up-regulation of the transcripts for α7 nAchR subunit, D2 dopamine receptor, and α1 GABAA receptor subunit at 1 day after the ablation. Fifteen days after the injury, AC ablations induced an up-regulation of glud1 transcripts.

The Role of Glia in the Peripheral and Central Auditory System Following Noise Overexposure: Contribution of TNF-α and IL-1ß to the Pathogenesis of Hearing Loss.

Repeated noise exposure induces inflammation and cellular adaptations in the peripheral and central auditory system resulting in pathophysiology of hearing loss. In this study, we analyzed the mechanisms by which noise-induced inflammatory-related events in the cochlea activate glial-mediated cellular responses in the cochlear nucleus (CN), the first relay station of the auditory pathway. The auditory function, glial activation, modifications in gene expression and protein levels of inflammatory mediators and ultrastructural changes in glial-neuronal interactions were assessed in rats exposed to broadband noise (0.5-32 kHz, 118 dB SPL) for 4 h/day during 4 consecutive days to induce long-lasting hearing damage. Noise-exposed rats developed a permanent threshold shift which was associated with hair cell loss and reactive glia. Noise-induced microglial activation peaked in the cochlea between 1 and 10D post-lesion; their activation in the CN was more prolonged reaching maximum levels at 30D post-exposure. RT-PCR analyses of inflammatory-related genes expression in the cochlea demonstrated significant increases in the mRNA expression levels of pro- and anti-inflammatory cytokines, inducible nitric oxide synthase, intercellular adhesion molecule and tissue inhibitor of metalloproteinase-1 at 1 and 10D post-exposure. In noise-exposed cochleae, interleukin-1ß (IL-1ß), and tumor necrosis factor α (TNF-α) were upregulated by reactive microglia, fibrocytes, and neurons at all time points examined. In the CN, however, neurons were the sole source of these cytokines. These observations suggest that noise exposure causes peripheral and central inflammatory reactions in which TNF-α and IL-1ß are implicated in regulating the initiation and progression of noise-induced hearing loss.