NKCC1 in Neonatal Cochlear Support Cells Reloads Ions Necessary for Cochlear Spontaneous Activity.

In the auditory system, the spontaneous activity of cochlear inner hair cells (IHCs) is initiated by the release of ATP from inner supporting cells (ISCs). This ATP release sets off a cascade, activating purinergic autoreceptors, opening of Ca2+-activated Cl- channel TMEM16A, Cl- efflux and osmotic cell shrinkage. Then, the shrunken ISCs efficiently regain their original volume, suggesting the existence of mechanisms for refilling Cland K+, priming them for subsequent activity. This study explores the potential involvement of NKCCs (Na+-K+-Cl- cotransporters) and KCCs (K+-Cl- cotransporters) in ISC spontaneous activity, considering their capability to transport both Cl- and K+ ions across the cell membrane. Employing a combination of immunohistochemistry, pharmacological interventions, and shRNA experiment, we unveiled the pivotal role of NKCC1 in cochlear spontaneous activity. Immunohistochemistry revealed robust NKCC1 expression in ISCs, persisting until the 2nd postnatal week. Intriguingly, we observed a developmental shift in NKCC1 expression from ISCs to synaptophysin-positive efferent terminals at postnatal day 18, hinting at its potential involvement in modulating synaptic transmission during the post-hearing period. Experiments using bumetanide, a well-known NKCC inhibitor, supported the functional significance of NKCC1 in ISC spontaneous activity. Bumetanide significantly reduced the frequency of spontaneous extracellular potentials (sEP) and spontaneous optical changes (sOCs) in ISCs. NKCC1-shRNA experiments conducted in cultured cochlear tissues further supported these findings, demonstrating a substantial decrease in event frequency and area. Taken together, we revealed the role of NKCC1 in shaping the ISC spontaneous activity that govern auditory pathway development.

Low-voltage Activating K+ Channels in Cochlear Afferent Nerve Fiber Dendrites.

Cochlear afferent nerve fibers (ANF) are the first neurons in the ascending auditory pathway. We investigated the low-voltage activating K+ channels expressed in ANF dendrites using isolated rat cochlear segments. Whole cell patch clamp recordings were made from the dendritic terminals of ANFs. Outward currents activating at membrane potentials as low as -64 mV were observed in all dendrites studied. These currents were inhibited by 4-aminopyridine (4-AP), a blocker known to preferentially inhibit low-voltage activating K+ currents (IKL) in CNS auditory neurons and spiral ganglion neurons. When the dendritic IKL was blocked by 4-AP, the EPSP decay time was significantly prolonged, suggesting that dendritic IKL speeds up the decay of EPSPs and likely modulates action potentials of ANFs. To reveal molecular subtype of dendritic IKL, α-dendrotoxin (α-DTX), a selective inhibitor for Kv1.1, Kv1.2, and Kv1.6 containing channels, was tested. α-DTX inhibited 23±9% of dendritic IKL. To identify the α-DTXsensitive and α-DTX-insensitive components of IKL, immunofluorescence labeling was performed. Strong Kv1.1- and Kv1.2-immunoreactivity was found at unmyelinated dendritic segments, nodes of Ranvier, and cell bodies of most ANFs. A small fraction of ANF dendrites showed Kv7.2- immunoreactivity. These data suggest that dendritic IKL is conducted through Kv1.1and Kv1.2 channels, with a minor contribution from Kv7.2 and other as yet unidentified channels.

A Deafness Associated Protein TMEM43 Interacts with KCNK3 (TASK-1) Two-pore Domain K+ (K2P) Channel in the Cochlea.

The TMEM43 has been studied in human diseases such as arrhythmogenic right ventricular cardiomyopathy type 5 (ARVC5) and auditory neuropathy spectrum disorder (ANSD). In the heart, the p.(Ser358Leu) mutation has been shown to alter intercalated disc protein function and disturb beating rhythms. In the cochlea, the p.(Arg372Ter) mutation has been shown to disrupt connexin-linked function in glia-like supporting cells (GLSs), which maintain inner ear homeostasis for hearing. The TMEM43-p.(Arg372Ter) mutant knock-in mice displayed a significantly reduced passive conductance current in the cochlear GLSs, raising a possibility that TMEM43 is essential for mediating the passive conductance current in GLSs. In the brain, the two-pore-domain potassium (K2P) channels are generally known as the "leak channels" to mediate background conductance current, raising another possibility that K2P channels might contribute to the passive conductance current in GLSs. However, the possible association between TMEM43 and K2P channels has not been investigated yet. In this study, we examined whether TMEM43 physically interacts with one of the K2P channels in the cochlea, KCNK3 (TASK-1). Utilizing co-immunoprecipitation (IP) assay and Duolink proximity ligation assay (PLA), we revealed that TMEM43 and TASK-1 proteins could directly interact. Genetic modifications further delineated that the intracellular loop domain of TMEM43 is responsible for TASK-1 binding. In the end, gene-silencing of Task-1 resulted in significantly reduced passive conductance current in GLSs. Together, our findings demonstrate that TMEM43 and TASK-1 form a protein-protein interaction in the cochlea and provide the possibility that TASK-1 is a potential contributor to the passive conductance current in GLSs.

A nonsense TMEM43 variant leads to disruption of connexin-linked function and autosomal dominant auditory neuropathy spectrum disorder.

Genes that are primarily expressed in cochlear glia-like supporting cells (GLSs) have not been clearly associated with progressive deafness. Herein, we present a deafness locus mapped to chromosome 3p25.1 and an auditory neuropathy spectrum disorder (ANSD) gene, TMEM43, mainly expressed in GLSs. We identify p.(Arg372Ter) of TMEM43 by linkage analysis and exome sequencing in two large Asian families segregating ANSD, which is characterized by inability to discriminate speech despite preserved sensitivity to sound. The knock-in mouse with the p.(Arg372Ter) variant recapitulates a progressive hearing loss with histological abnormalities in GLSs. Mechanistically, TMEM43 interacts with the Connexin26 and Connexin30 gap junction channels, disrupting the passive conductance current in GLSs in a dominant-negative fashion when the p.(Arg372Ter) variant is introduced. Based on these mechanistic insights, cochlear implant was performed on three subjects, and speech discrimination was successfully restored. Our study highlights a pathological role of cochlear GLSs by identifying a deafness gene and its causal relationship with ANSD.

Distribution of Kv3 Subunits in Cochlear Afferent and Efferent Nerve Fibers Implies Distinct Role in Auditory Processing.

Kv3 family K+ channels, by ensuring speedy repolarization of action potential, enable rapid and high frequency neuronal firing and high precision temporal coding of auditory information in various auditory synapses in the brain. Expression of different Kv3 subtypes within the auditory end organ has been reported. Yet, their precise role at the hair cell synaptic transmission has not been fully elucidated. Using immunolabeling and confocal microscopy we examined the expression pattern of different Kv3 family K+ channel subunits in the nerve fibers innervating the cochlear hair cells. Kv3.1b was found in NKA-positive type 1 afferent fibers, exhibiting high signal intensity at the cell body, the unmyelinated dendritic segment, first heminode and nodes of Ranvier. Kv3.3 signal was detected in the cell body and the unmyelinated dendritic segment of NKA-positive type 1 afferent fibers but not in peripherin-positive type 2 afferent. Kv3.4 was found in ChAT-positive LOC and MOC efferent fibers as well as peripherin-positive type 2 afferent fibers. Such segregated expression pattern implies that each Kv3 subunits participate in different auditory tasks, for example, Kv3.1b and Kv3.3 in ascending signaling while Kv3.4 in feedback upon loud noise exposure.

Selective Loss of Calretinin-Poor Cochlear Afferent Nerve Fibers in Streptozotocin-Induced Hyperglycemic Mice.

Hearing loss is one of the major complications of diabetes mellitus and significantly lowers the quality of life of diabetic patients. In studies using diabetic animal models hearing loss have been frequently associated with damages to cochlear afferent fibers. Recent studies suggested that cochlear afferent neurons are composed of heterogeneous populations and a subgroup of neurons equipped with low level of calretinin might be more vulnerable to various noxious stimuli such as noise and neurotoxins. Here, we tested if cochlear afferent neurons deficient in the Ca2+-buffering protein calretinin are more vulnerable to hyperglycemic insults. Streptozotocin-induced (50 mg/kg, i.p.) hyperglycemic mice (>250 mg/dl) were tested. The expression patterns of calretinin in peripheral processes and the cell bodies of cochlear afferent nerve fibers were examined using immunohistochemistry and confocal microscopy. The proportion of calretinin-poor cochlear afferent fibers was much lower in hyperglycemic mice compared to the normoglycemic control group. (30.0 vs. 55.5% in the peripheral process; 15.7 vs. 24.4 % in spiral ganglion neuron). The results suggest that calretinin-poor cochlear nerve fibers may be selectively lost after the hyperglycemic insults. The finding also supports a calretinin's neuroprotective role against diabetic neuropathy in cochlear afferent neurons.

Sound exposure dynamically induces dopamine synthesis in cholinergic LOC efferents for feedback to auditory nerve fibers.

Lateral olivocochlear (LOC) efferent neurons modulate auditory nerve fiber (ANF) activity using a large repertoire of neurotransmitters, including dopamine (DA) and acetylcholine (ACh). Little is known about how individual neurotransmitter systems are differentially utilized in response to the ever-changing acoustic environment. Here we present quantitative evidence in rodents that the dopaminergic LOC input to ANFs is dynamically regulated according to the animal's recent acoustic experience. Sound exposure upregulates tyrosine hydroxylase, an enzyme responsible for dopamine synthesis, in cholinergic LOC intrinsic neurons, suggesting that individual LOC neurons might at times co-release ACh and DA. We further demonstrate that dopamine down-regulates ANF firing rates by reducing both the hair cell release rate and the size of synaptic events. Collectively, our results suggest that LOC intrinsic neurons can undergo on-demand neurotransmitter re-specification to re-calibrate ANF activity, adjust the gain at hair cell/ANF synapses, and possibly to protect these synapses from noise damage.

Every day, we hear sounds that might be alarming, distracting, intriguing or calming ­ or simply just too loud. Our hearing system responds to these acoustic changes by fine-tuning sounds before they enter the brain. For example, if a noise is too loud, the volume can be turned down by dampening the signals nerve fibers in the ear send to the brain. This is thought to reduce the damage loud sounds can cause to the sensory organ inside the ear. A set of nerve cells located at the base of the brain called the lateral olivocochlear (LOC) neurons coordinate this adjustment to different volumes and sounds. When these neurons receive information on external sounds, they signal back to the hearing organs and adjust the activity of auditory nerve fibers that communicate this information to the brain. LOC neurons use a diverse range of molecules to modify the activity of auditory nerve fibers, including the 'feel-good' neurotransmitter dopamine. But it is unclear what role dopamine plays in this auditory feedback loop. To find out, Wu et al. studied the hearing system of mice that had been exposed to different levels of sound. This involved imaging LOC neurons stained with a marker for dopamine and measuring the activity of nerve fibers in the inner ear. The experiments showed that LOC neurons in mice that had recently been exposed to sound were covered in an enzyme that is essential for making dopamine. The louder the sound, the more of this enzyme was present, suggesting that the amount of dopamine released depends on the volume of the sound. LOC neurons release another neurotransmitter called acetylcholine, which stimulates activity in auditory nerve fibers. Wu et al. found that dopamine and acetylcholine are released from the same group of LOC neurons. However, dopamine had the opposite effect to acetylcholine and reduced nerve activity. These findings suggest that by controlling the mixture of neurotransmitters released, LOC neurons are able to fine-tune the activity of auditory nerve fibers in response to acoustic changes. This work provides a new insight into how our hearing system is able to perceive and relay changes in the sound environment. A better understanding of this auditory feedback loop could influence the design of implant devices for people with impaired hearing.

Fine air pollution particles induce endothelial senescence via redox-sensitive activation of local angiotensin system.

Fine dust (FD) is a form of air pollution and is responsible for a wide range of diseases. Specially, FD is associated with several cardiovascular diseases (CVDs); long-term exposure to FD was shown to decrease endothelial function, but the underlying mechanism remains unclear. We investigated whether exposure to FD causes premature senescence-associated endothelial dysfunction in endothelial cells (ECs) isolated from porcine coronary arteries. The cells were treated with different concentrations of FD and senescence associated-beta galactosidase (SA-ß-gal) activity, cell cycle progression, expression of endothelial nitric oxide synthase (eNOS), oxidative stress level, and vascular function were evaluated. We found that FD increased SA-ß-gal activity, caused cell cycle arrest, and increased oxidative stress, suggesting the premature induction of senescence; on the other hand, eNOS expression was downregulated and platelet aggregation was enhanced. FD exposure impaired vasorelaxation in response to bradykinin and activated the local angiotensin system (LAS), which was inhibited by treatment with the antioxidant N-acetyl cysteine (NAC) and angiotensin II receptor type 1 (AT1) antagonist losartan (LOS). NAC and LOS also suppressed FD-induced SA-ß-gal activity, increased EC proliferation and eNOS expression, and improved endothelial function. These results demonstrate that FD induces premature senescence of ECs and is associated with increased oxidative stress and activation of LAS. This study can serve as a pharmacological target for prevention and/or treatment of air pollution-associated CVD.

Citrus junos Fruit Extract Facilitates Anti-Adipogenic Activity of Garcinia cambogia Extract in 3T3-L1 Adipocytes by Reducing Oxidative Stress.

Obesity is a metabolic disorder associated with chronic oxidative stress and inflammation. Recruitment of inflammatory cells to adipose tissue and subsequent production of a large amount of reactive oxygen species (ROS) facilitates adipocyte differentiation and promotes lipid accumulation. The removal of ROS with anti-oxidants appeared an effective strategy against lipid accumulation. Here, we chose Citrus junos, a good dietary source of anti-oxidants and tested the anti-adipogenic potential of Citrus junos extract (CE). CE effectively suppressed the ROS production and lipid accumulation in H2O2-stimulated 3T3-L1 cells. CE also inhibited the expression of CEBP-α and PPAR-γ, the transcription regulators of adipocyte differentiation. These data suggest that CE might suppress the adipocyte differentiation through ROS scavenging action. Also, CE and Garcinia cambogia extract (GE) appeared act additively in reducing ROS and in inhibiting lipid accumulation. It implied a potential usefulness of this combination in the management of obesity related disorders.

Differential Expression of Ca2+-buffering Protein Calretinin in Cochlear Afferent Fibers: A Possible Link to Vulnerability to Traumatic Noise.

The synaptic contacts of cochlear afferent fibers (CAFs) with inner hair cells (IHCs) are spatially segregated according to their firing properties. CAFs also exhibit spatially segregated vulnerabilities to noise. The CAF fibers contacting the modiolar side of IHCs tend to be more vulnerable. Noise vulnerability is thought to be due to the absence of neuroprotective mechanisms in the modiolar side contacting CAFs. In this study, we investigated whether the expression of neuroprotective Ca2+-buffering proteins is spatially segregated in CAFs. The expression patterns of calretinin, parvalbumin, and calbindin were examined in rat CAFs using immunolabeling. Calretinin-rich fibers, which made up ~50% of the neurofilament (NF)-positive fibers, took the pillar side course and contacted all IHC sides. NF-positive and calretinin-poor fibers took the modiolar side pathway and contacted the modiolar side of IHCs. Both fiber categories juxtaposed the C-terminal binding protein 2 (CtBP2) puncta and were contacted by synaptophysin puncta. These results indicated that the calretinin-poor fibers, like the calretinin-rich ones, were afferent fibers and probably formed functional efferent synapses. However, the other Ca2+-buffering proteins did not exhibit CAF subgroup specificity. Most CAFs near IHCs were parvalbumin-positive. Only the pillar-side half of parvalbumin-positive fibers coexpressed calretinin. Calbindin was not detected in any nerve fibers near IHCs. Taken together, of the Ca2+-buffering proteins examined, only calretinin exhibited spatial segregation at IHC-CAF synapses. The absence of calretinin in modiolar-side CAFs might be related to the noise vulnerability of the fibers.

Combination of Garcinia cambogia Extract and Pear Pomace Extract Additively Suppresses Adipogenesis and Enhances Lipolysis in 3T3-L1 Cells.

BACKGROUND: Inhibition of adipogenesis has been a therapeutic target for reducing obesity and obesity-related disorders such as diabetes, hypertension, atherosclerosis, and cancer. For decades, anti-adipogenic potential of many herbal extracts has been investigated. One example is Garcinia cambogia extract (GE) containing (-)-hydroxycitric acid as an active ingredient. GE is currently marketed as a weight loss supplement, used alone or with other ingredients. Pear pomace extract (PE), another natural product, has been also shown to have anti-adipogenic activity in a recent report. OBJECTIVE: It was tested if the mixture of PE and GE (MIX) would produce more effective anti-adipogenic activity than PE or GE alone. MATERIALS AND METHODS: Differentiation of 3T3-L1 preadipocyte was induced by adding insulin, dexamethasone, and isobutylmethylxanthine and lipid accumulation was measured by Oil Red O staining. Cellular markers for adipogenesis and lipolysis such as CCAAT/enhancer binding protein (C/EBP-α), peroxisome proliferator-activated receptor gamma (PPAR-γ), fatty acid synthase (FAS), and hormone-sensitive lipase (HSL) was measured using immunocytochemistry. RESULTS: MIX, compared to PE or GE alone, showed greater inhibition of lipid accumulation. Furthermore, MIX reduced the expression of adipogenesis-related factors C/EBP-α, PPAR-γ, and FAS more than PE or GE alone did. In contrast, the expression of HSL the enzyme required for lipolysis was further enhanced in MIX-treated adipocytes compared to the PE or GE alone treated groups. CONCLUSIONS: Anti-adipogenic effect of PE and GE appears synergistic, and the MIX may be a useful therapeutic combination for the treatment of obesity and obesity-related diseases. SUMMARY: PE and GE efficiently inhibited adipocyte differentiation by suppressing the expression of adipogenic transcription factor CEBP-α and PPAR-γ.PE and GE significantly decreased the expression of adipogenic enzyme FAS.PE and GE increased the expression of lipid degrading enzyme HSL.Mixture of PE and GE exhibited additive or moderately synergistic effect on adipocyte differentiation and lipid accumulation. Abbreviations used: CEBP-a: CCAT/enhancer binding protein alpha, CI: Combination Index, FAS: Fatty acid synthase, GE: Garcinia cambogia extract, HSL: Hormone sensitive lipase, PE: Pear pomace extract, PPAR-γ: Peroxisome proliferator-activated receptor gamma.

Preparation and In Vitro Evaluation of Elastic Nanoliposomes for Topical Delivery of Highly Skin-Permeable Growth Factors.

Percutaneous delivery of growth factors is often used to treat wounds, and for cosmetic purposes, as a way of accelerating healing and skin regeneration, respectively. However, the therapeutic effects of growth factors are diminished by their poor absorption when delivered percutaneously, in addition to their rapid degradation by proteinases. To overcome these obstacles, we constructed two skin-permeable compounds. Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor-A (VEGF-A) were both genetically paired with low-molecular-weight protamine (LMWP), to yield the compounds LMWP-bFGF and LMWP-VEGF-A, respectively. The molecular weights and N-terminal amino acid sequences of LMWP-bFGF and LMWP-VEGF-A confirmed that the N-terminus-specific conjugation of LMWP with bFGF and VEGF-A had been successful. The biological abilities of the native factors to stimulate human fibroblast (CCD-986sk) and endothelial cell proliferation were preserved. Both compounds significantly promoted wound (scratch) recovery and enhanced procollagen type I C-peptide synthesis in CCD-986sk cells (to levels 184 and 133% those of the native compounds, respectively). The LMWP-conjugated growth factors were significantly more permeable than the native forms (by 7.29- and 29.22-fold, respectively). Finally, encapsulation of the compounds in positively charged elastic nanoliposomes (115 ± 1.54 nm in diameter with a zeta potential of 57.2 ± 3.05 mV) further improved both permeation and stability. Thus, nanoliposomes loaded with LMWP-conjugated growth factors are expected to enhance skin regeneration; the materials will find applications in wound-healing therapies and anti-wrinkle cosmetics.

ATP1A3 mutations can cause progressive auditory neuropathy: a new gene of auditory synaptopathy.

The etiologies and prevalence of sporadic, postlingual-onset, progressive auditory neuropathy spectrum disorder (ANSD) have rarely been documented. Thus, we aimed to evaluate the prevalence and molecular etiologies of these cases. Three out of 106 sporadic progressive hearing losses turned out to manifest ANSD. Through whole exome sequencing and subsequent bioinformatics analysis, two out of the three were found to share a de novo variant, p.E818K of ATP1A3, which had been reported to cause exclusively CAPOS (cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss) syndrome. However, hearing loss induced by CAPOS has never been characterized to date. Interestingly, the first proband did not manifest any features of CAPOS, except subclinical areflexia; however, the phenotypes of second proband was compatible with that of CAPOS, making this the first reported CAPOS allele in Koreans. This ANSD phenotype was compatible with known expression of ATP1A3 mainly in the synapse between afferent nerve and inner hair cells. Based on this, cochlear implantation (CI) was performed in the first proband, leading to remarkable benefits. Collectively, the de novo ATP1A3 variant can cause postlingual-onset auditory synaptopathy, making this gene a significant contributor to sporadic progressive ANSD and a biomarker ensuring favorable short-term CI outcomes.

Rice Bran Extract Inhibits TMEM16A-Involved Activity in the Neonatal Rat Cochlea.

TMEM16A is a Ca²âº-activated Cl⁻ channel found in secretory glands, GI and respiratory tracts, and sensory organs, playing a major physiological role in fluid secretion, autonomous GI motility, and sensory transduction. In addition, overexpression of TMEM16A has been associated with cancer cell proliferation and invasion. Suppression of upregulated TMEM16A has been proposed as an effective anti-cancer strategy. While searching for a potential TMEM16A inhibitor, components of rice bran attracted our attention due to their anti-cancer potential in colon cancer cells, a type of cells known to overexpressing TMEM16A. Here, it was tested whether rice bran extract exhibits anti-TMEM16A activity. Rice bran extract was tested in the neonatal rat cochlear tissues where TMEM16A-involved spontaneous activity is generated as a part of normal development of the auditory pathway. Rice bran extract readily inhibited the TMEM16A-involved activity in the cochlear tissues and the effect was reversible upon washout. Taken together, rice bran extract appears to contain a putative TMEM16A inhibitor and the rice byproduct might serve as a source of a new anti-cancer agent.

The Effect of Quercus salicina Leaf Extracts on Vascular Endothelial Function: Role of Nitric Oxide.

Dysfunction of the vascular endothelium is reported as a hallmark of cardiovascular diseases. Many evidences suggest that polyphenols are associated with a decreased global mortality and might be involved in protection against cardiovascular risk. This beneficial effect of polyphenol may be due to many actions as antioxidant that increases bioavailability of nitric oxide, vasodilation or anti-hypertensive properties. To identify new natural medicine candidate for cardiovascular protection, plant extracts used in traditional medicine were evaluated by vascular reactivity system. Porcine coronary artery rings were suspended in organ chambers for the measurement of changes in isometric tension. Screening results indicated that the ethanolic extract of leaf from Quercus salicina (QSE) has been found to exhibit potent vasorelaxant activity. QSE dose-dependently induced endothelium-dependent relaxations, which were abolished by inhibitors of nitric oxide synthase (Nomega-nitro-L-arginine). In addition, QSE strongly and dose-dependently activate endothelial nitric oxide synthase (eNOS) in porcine coronary artery endothelial cell. Taken together, the present study has demonstrated that QSE is a powerful endothelium-dependentvasodilator and that this effect involves increased nitric oxide bioavailability. In conclusion, QSE could be a cardiovascular protective herbal medicine candidate associated with cardiovascular diseases and endothelial dysfunction.

Voltage-gated K(+) channels contributing to temporal precision at the inner hair cell-auditory afferent nerve fiber synapses in the mammalian cochlea.

To perform auditory tasks such as sound localization in the space, auditory neurons in the brain must distinguish sub-millisecond temporal differences in signals from two ears. Such high temporal resolution is possible when each neuron in the ascending auditory pathway fires brief action potential at very accurate timing. Various pre- and postsynaptic machineries ensuring such high temporal precision of auditory synaptic transmission have been identified. Of particular, in this review, the role of K(+) channels in shortening the duration of synaptic potentials will be discussed. First, the contribution of K(+) channels to AP firing of general auditory neurons will be discussed. Then, the focus will be moved to the inner hair cell (IHC)-auditory afferent nerve fiber (ANF) synapses, the first synapses of ascending auditory pathway. Molecular and immunohistological techniques have revealed various K(+) channels in the cell bodies and their processes of ANFs. Since the development of patch-clamp recordings from the ANF dendrites in 2002, it became possible to monitor the IHC-ANF synaptic transmission in greater detail. As revealed in brain auditory synapses, several different K(+) channels appear to participate in reducing the duration of synaptic potentials at the IHC-ANF synapses. In addition, K(+) channels at the ANF dendrites might act as potential targets of efferent feedback from the brain. The hypothesis is that, upon loud sound exposure, efferent neurotransmitters released onto the ANF dendrites activate certain K(+) channels and prevent excitotoxicity of ANFs. Therefore, K(+) channels of the ANF dendrites might provide potential sites of pharmacological actions to prevent noise-induced hearing loss.

Developmental Role of Anoctamin-1/TMEM16A in Ca(2+)-Dependent Volume Change in Supporting Cells of the Mouse Cochlea.

Mammalian cochlea undergoes morphological and functional changes during the postnatal period, around the hearing onset. Major changes during the initial 2 postnatal weeks of mouse include maturation of sensory hair cells and supporting cells, and acquisition of afferent and efferent innervations. During this period, supporting cells in the greater epithelial ridge (GER) of the cochlea exhibit spontaneous and periodic activities which involves ATP, increase in intracellular Ca(2+), and cell volume change. This Ca(2+)-dependent volume change has been proposed to involve chloride channels or transporters. We found that the spontaneous volume changes were eliminated by anion channel blocker, 100 µM NPPB. Among candidates, expression of Anoctamin-1 (Ano1 or TMEM16A), bestriphin-1 and NKCC1 were investigated in whole-mount cochlea of P9-10 mice. Immunolabeling indicated high level of Ano1 expression in the GER, but not of betrophin-1 or NKCC1. Double-labeling with calretinin and confocal image analysis further elucidated the cellular localization of Ano1 immunoreactivity in supporting cells. It was tested if the Ano1 expression exhibits similar time course to the spontaneous activities in postnatal cochlear supporting cells. Cochlear preparations from P2-3, P5-6, P9-10, P15-16 mice were subjected to immunolabeling. High level of Ano1 immunoreactivity was observed in the GER of P2-3, P5-6, P9-10 cochleae, but not of P15-17 cochleae. Taken together, the localization and time course in Ano1 expression pattern correlates with the spontaneous, periodic volume changes recorded in postnatal cochlear supporting cells. From these results we propose that Ano1 is the pacemaker of spontaneous activities in postnatal cochlea.

Postsynaptic recordings at afferent dendrites contacting cochlear inner hair cells: monitoring multivesicular release at a ribbon synapse.

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the postsynaptic activity of a single ribbon synapse. Ribbon synapses of sensory cells release neurotransmitter continuously, the rate of which is modulated in response to graded changes in IHC membrane potential. Ribbon synapses have been shown to operate by multivesicular release, where multiple vesicles can be released simultaneously to evoke excitatory postsynaptic currents (EPSCs) of varying amplitudes. Neither the role of the presynaptic ribbon, nor the mechanism underlying multivesicular release is currently well understood. The IHC is innervated by 10-20 auditory nerve fibers, and every fiber contacts the IHC with a unmyelinated single ending to form a single ribbon synapse. The small size of the afferent boutons contacting IHCs (approximately 1 µm in diameter) enables recordings with exceptional temporal resolution to be made. Furthermore, the technique can be adapted to record from both pre- and postsynaptic cells simultaneously, allowing the transfer function at the synapse to be studied directly. This method therefore provides a means by which fundamental aspects of neurotransmission can be studied, from multivesicular release to the elusive function of the ribbon in sensory cells.

Dendritic HCN channels shape excitatory postsynaptic potentials at the inner hair cell afferent synapse in the mammalian cochlea.

Synaptic transmission at the inner hair cell (IHC) afferent synapse, the first synapse in the auditory pathway, is specialized for rapid and reliable signaling. Here we investigated the properties of a hyperpolarization-activated current (I(h)), expressed in the afferent dendrite of auditory nerve fibers, and its role in shaping postsynaptic activity. We used whole cell patch-clamp recordings from afferent dendrites directly where they contact the IHC in excised postnatal rat cochlear turns. Excitatory postsynaptic potentials (EPSPs) of variable amplitude (1-35 mV) were found with 10-90% rise times of about 1 ms and time constants of decay of about 5 ms at room temperature. Current-voltage relations recorded in afferent dendrites revealed I(h). The pharmacological profile and reversal potential (-45 mV) indicated that I(h) is mediated by hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels. The HCN channel subunits HCN1, HCN2, and HCN4 were found to be expressed in afferent dendrites using immunolabeling. Raising intracellular cAMP levels sped up the activation kinetics, increased the magnitude of I(h) and shifted the half activation voltage (V(half)) to more positive values (-104 +/- 3 to -91 +/- 2 mV). Blocking I(h) with 50 microM ZD7288 resulted in hyperpolarization of the resting membrane potential (approximately 4 mV) and slowing the decay of the EPSP by 47%, suggesting that I(h) is active at rest and shortens EPSPs, thereby potentially improving rapid and reliable signaling at this first synapse in the auditory pathway.

Two modes of release shape the postsynaptic response at the inner hair cell ribbon synapse.

Cochlear inner hair cells (IHCs) convert sounds into receptor potentials and via their ribbon synapses into firing rates in auditory nerve fibers. Multivesicular release at individual IHC ribbon synapses activates AMPA-mediated EPSCs with widely ranging amplitudes. The underlying mechanisms and specific role for multivesicular release in encoding sound are not well understood. Here we characterize the waveforms of individual EPSCs recorded from afferent boutons contacting IHCs and compare their characteristics in immature rats (postnatal days 8-11) and hearing rats (postnatal days 19-21). Two types of EPSC waveforms were found in every recording: monophasic EPSCs, with sharp rising phases and monoexponential decays, and multiphasic EPSCs, exhibiting inflections on rising and decaying phases. Multiphasic EPSCs exhibited slower rise times and smaller amplitudes than monophasic EPSCs. Both types of EPSCs had comparable charge transfers, suggesting that they were activated by the release of similar numbers of vesicles, which for multiphasic EPSCs occurred in a less coordinated manner. On average, a higher proportion of larger, monophasic EPSCs was found in hearing compared to immature rats. In addition, EPSCs became significantly faster with age. The developmental increase in size and speed could improve auditory signaling acuity. Multiphasic EPSCs persisted in hearing animals, in some fibers constituting half of the EPSCs. The proportion of monophasic versus multiphasic EPSCs varied widely across fibers, resulting in marked heterogeneity of amplitude distributions. We propose that the relative contribution of two modes of multivesicular release, generating monophasic and multiphasic EPSCs, may underlie fundamental characteristics of auditory nerve fibers.