GJB2 gene, c.919-2A>G of the SLC26A4 gene, and mitochondrial m.1555A>G of the 12S rRNA gene. The results of the NGS were then correlated to the results of the NHS. Of the 1017 newborns, 16 (1.6%) had unilateral DPOAE screening failure, and 22 (2.2%) had bilateral DPOAE screening failure. A total of 199 (19.6%) babies were found to have at least 1 mutated allele on the NGS for deafness, 11 (1.1%) of whom were homozygous for GJB2 p.V37I, 6 (0.6%) compound heterozygous for GJB2 p.V37I and c.235delC, and 1 (0.1%) homoplasmic for m.1555A>G, who may potentially have hearing loss. Among them, 3 babies, 5 babies, and 1 baby, respectively, passed the NHS at birth. Comprehensive audiological assessments in the 9 babies at 3 months identified 1 with slight hearing loss and 2 with mild hearing loss. NGS for common deafness-associated mutations may identify infants with slight/mild or potentially progressive hearing impairment, thus compensating for the inherent limitations of the conventional UNHS.Universal newborn hearing screening (UNHS) is of paramount importance for early identification and management of hearing impairment in children. However, infants with slight/mild, progressive, or late-onset hearing impairment might be missed in conventional UNHS. To investigate whether genetic screening for common deafness-associated mutations could assist in identifying these infants, 1017 consecutive newborns in a tertiary hospital were subjected to both newborn hearing screening using a two-step distortion-product otoacoustic emissions (DPOAE) screening and newborn genetic screening (NGS) for deafness. The NGS targeted 4 deafness-associated mutations commonly found in the Taiwanese population, including p.V37I (c.109G>A) and c.235delC of the  Hearing loss is one of the most common congenital disorders, with approximately 1 in 1000 newborns affected by bilateral moderate, severe or profound (i.e. >40dBHL) permanent congenital hearing loss (PCHL) http://hereditaryhearingloss.org/), mutations in certain genes, such as GJB2 (or Cx26) (MIM *121011), SLC26A4 (or PDS) (MIM *605646), and the mitochondrial 12S rRNA gene (or MTRNR1) (MIM *561000) have been shown to be much more prevalent than other genes GJB2 mutations, such as p.M34T, p.V37I, and p.L90P are associated with mild-to-moderate SNHI SLC26A4 mutations contribute to Pendred syndrome  or non-syndromic hearing loss , and some affected patients have progressive or fluctuating hearing loss With recent advancement in the molecular genetics of hearing impairment, it has been demonstrated that more than 50% of children with SNHI have attributable genetic factors From 2009 to 2010, 1017 consecutive newborns in the National Taiwan University Hospital were enrolled in the study. All newborns were subjected to both newborn hearing screening (NHS) and newborn genetic screening (NGS) for deafness. The results of the NGS were then correlated to the results of the NHS. Written informed consent for participation in the project was obtained from the parents of all infants, and all procedures were approved by the Research Ethics Committee of the National Taiwan University Hospital.The babies received a two-step hearing screening using distortion-product otoacoustic emissions (DPOAEs) recorded with a GSI 60 DPOAE system . The first step of the screening was performed at 48 hours after birth to prevent debris and vernix in the external ear canal from interfering with the DPOAE measurement A bloodspot was obtained within 1 hour after birth. Sample of blood from the heel stick from each infant was spotted onto QIAcard FTA One Spot (Qiagen). For newborn screening, three 3-mm-diameter bloodspots from each blood spot on FTA paper were punched out and used. All DNA samples were extracted using the Chemagic DNA Blood Kit (Chemagen), according to the manufacturer's instructions.GJB2, c.919-2A>G of SLC26A4, and mitochondrial m.1555A>G, were included in the NGS panel. In terms of the allele frequency in the hearing-impaired population, these 4 mutations amount to >80% of the known deafness-associated mutations in Taiwanese individuals Using our previously generated epidemiologic database 2, 0.25 µM of each primer, 0.25 of µM fluorescein probe, 0.25 µM of LC Red fluorophore probe, and 1× LightCycler FastStart DNA Master Hybridization Probes Mix (Roche), as provided by the manufacturer. The cycling conditions for real-time PCR in LiC480 were as follows: 95°C for 10 min followed by 50 cycles of denaturation at 95°C for 10 s with a temperature transition rate of 4.4°C/s, annealing at 56°C for 10 s with a temperature transition rate of 2.2°C/s, and extension at 72°C for 10 s, with a temperature transition rate of 4.4°C/s.The four mutations were screened using the real-time polymerase chain reaction (PCR) assay with fluorescence resonance energy transfer (FRET) hybridization probes in a LightCycler 480 instrument (Roche) . The reahttp://audiology.unife.it/www.gendeaf.org/index.html) For infants who failed the NHS, another DPOAE test was performed within 1 month after discharge, and those failing to pass the test again were referred to a pediatric otologist for comprehensive audiological assessments at 3 months. The comprehensive audiological assessments included a behavioral observation audiometry (BOA) in a sound field using warble tones, narrow band noise, and live voice; DPOAE testing; and a diagnostic auditory brainstem response  under sedation to determine the hearing thresholds at 0.5, 1, 2, and 4 kHz GJB2 alleles , 2 mutated SLC26A4 alleles , or the m.1555A>G mutation , comprehensive audiological assessments including BOA, DPOAE, and ABR were also performed at 3 months to confirm their audiological phenotypes.For infants who passed the NHS but segregated for 2 mutated GJB2 or SLC26A4 allele), mutation screening of both exons of GJB2, all of the 21 exons of SLC26A4, and the whole mitochondrial 12S rRNA gene was completed by direct sequencing GJB2 mutations, direct sequencing of the coding region of GJB2 was also performed.For hearing-impaired infants who revealed an inconclusive genotype in NGS  had two mutated alleles, 11 (1.1%) of whom were homozygous for p.V37I and 6 (0.6%) compound heterozygous for p.V37I and c.235delC. None (0%) were homozygous for c.235delC. Nineteen subjects (1.9%) were shown to have 1 c.235delC allele, and 156 subjects (15.3%) were heterozygous for p.V37I. The high carrier rate of GJB2 p.V37I in this newborn cohort was compatible to that observed in a normal control population in our previous report SLC26A4 c.919-2A>G mutation, 6 subjects (0.6%) were heterozygous for c.919-2A>G and none (0%) were homozygotes. One subject (0.1%) was found to have the m.1555A>G mutation, with the mutation load detected as “homoplasmy” in hybridization probe testing, which was later confirmed using a restriction enzyme digestion method Of the 1017 subjects, a total of 199 (19.6%) were screened as having at least 1 mutated allele according to the NGS for deafness. GJB2 p.V37I mutation and the 6 babies compound heterozygous for GJB2 p.V37I and c.235delC, 8 (73%) and 1 (17%), respectively, failed to pass the NHS. In other words, 3 babies homozygous for p.V37I and 5 babies compound heterozygous for p.V37I and c.235delC, with potential hearing problems, were not detected by NHS, indicating that NGS could identify an additional subgroup of affected newborns outside the target area of NHS.The results of NHS in subjects with different genotypes in NGS are shown in GJB2 p.V37I heterozygotes and 19 c.235delC heterozygotes were 5% (n = 8) and 5% (n = 1), respectively. Both exons of GJB2 were then sequenced in the 9 heterozygous babies who failed to pass NHS, yet we did not identify any mutation in the second GJB2 allele. Likewise, a second GJB2 mutation was not detected in the other 166 heterozygous babies who passed NHS, indicating that they were probably coincidental carriers of the mutations.The failure rates of NHS in the 156 SLC26A4 c.919-2A>G failed to pass NHS. Probably these babies were only carriers. However, the possibility of Pendred syndrome or non-syndromic DFNB4 could not be completely excluded, because PS or DFNB4 patients might be born with normal hearing or minimal hearing loss, with their hearing deteriorating later during childhood or adolescence None (0%) of the 6 babies heterozygous for GJB2 alleles (including 8 p.V37I homozygotes and 1 p.V37I/c.235delC compound heterozygote), 9 (24%) segregated for 1 mutated GJB2 allele (including 8 p.V37I heterozygotes and 1 c.235delC heterozygote), and 20 (53%) segregated for none of the 4 deafness-associated mutations  segregated for 2 mutated utations . Six of GJB2 alleles or homoplasmic m.1555A>G mutation . However, there is still no efficient way to achieve this goal GJB2 mutations, such as p.M34T GJB2 mutations in identifying infants who are likely to have late-onset or progressive hearing loss, necessitating a close observation of hearing in these affected individuals. The potential benefits of identifying subjects with SLC26A4 mutations or the mitochondrial m.1555A>G mutation might even be greater. For subjects with SLC26A4-mutation-associated Pendred syndrome or non-syndromic DFNB4, attacks of acute sensorineural hearing loss could be prevented by avoiding head trauma or abrupt barometric pressure changes To identify infants and children with late-onset or progressive hearing loss at the earliest possible time is also considered important and has been included in the national Early Hearing Detection and Intervention (EHDI) goals  recipients (29.6%) born in Illinois during or after 2003 passed UNHS, demonstrating the limitation of the current UNHS in early identification of delayed-onset SNHI With regard to the difficulty in approaching and screening specific subgroups of infants such as those born outside of hospitals, NGS might also serve as an alternative solution. Obtaining a few drops of blood from a heel stick within the first 2 or 3 days of life is a minimally invasive procedure, and can be conducted by a nurse or a midwife with basic training. In addition, the new generation of DNA cards, such as filter blotters, Guthrie cards, and FTA cards, are easy to collect, transport, and store.Offering parents reproductive choices  has been considered a benefit of expanded newborn screening programs GJB2 mutation, and 6 babies were found to carry 1 SLC26A4 mutation; among them, 9 babies and no baby failed the NHS at birth, respectively. These 9 babies were managed as other newborns who failed the NHS according to the national guidelines, i.e., another DPOAE test within 1 month after discharge, followed by comprehensive audiological assessments at 3 months if indicated. Seven of the 9 babies had normal hearing at 3 months  should be added due to its high prevalence in patients with prelingual SNHI Of special note, deafness-associated mutations should be adjusted if NGS for deafness is to be implemented in other populations because different ethnic groups show different mutation spectra for each deafness gene. Although mutations of the connexin 26-encoding GJB2 mutations carry only 1 mutant allele. Although the del(GJB6-D13S1830) mutation, through eliminating a cis-regulatory element of GJB2, provided an explanation for the deafness in as many as 30%–70% of affected GJB2 heterozygotes in some populations GJB2 mutation, or they might be merely coincidental carriers with hearing loss unrelated to GJB2. Similarly, it is also difficult to interpret the genetic results in babies who passed NHS and segregated only 1 mutated GJB2 allele on NGS, although it can be inferred that a greater proportion of these babies are coincidental carriers as compared to “affected” heterozygotes. Nonetheless, NGS for deafness is still useful in identifying babies with 2 mutated GJB2 alleles, for whom the genetic diagnosis is more straightforward. These babies require a close audiological observation or intervention because their genotypes are associated with mild-to-moderate, progressive, or late-onset hearing impairment.Some limitations of the present study deserve discussion. First, only common, known deafness-associated mutations are included in the current screening panel. This might lead to a false assurance in individuals with rare or novel mutations because of normal NGS results. This limitation, however, might be overcome with the advent of the next-generation sequencing technology, as massively parallel sequencing has recently been proven as an effective tool for addressing hereditary hearing loss GJB2 mutations could not be identified with UNHS GJB2 alleles, 2 mutated SLC264 alleles, homoplasmic or heteroplasmic m.1555A>G mutation) who reveal normal hearing at 3 months, comprehensive audiological evaluation will be repeated at 1 year, whereas for normal hearing babies with 1 mutated GJB2 or SLC264 allele, adequate genetic counseling will be implemented, and additional genetic study (such as sequencing of the whole gene) will be provided whenever necessary. Once hearing impairment develops in these babies, the management protocol will be switched to the conventional management protocol for pediatric SNHI regardless of their genotypes. Long-term follow-up of this cohort might provide insight into how and to what extent genetic mutations exert their influence on the development of pediatric hearing impairment, as well as provide important audiological information about the natural history of specific genetic mutations, such as the GJB2 p.V37I mutation.In a retrospective report, it was demonstrated that some patients with 2 In conclusion, the present study revealed that NGS for common deafness-associated mutations, by detecting subjects with mutations associated with mild-to-moderate, progressive, or late-onset hearing impairment, might compensate for the inherent limitations of conventional UNHS . For inf


The active channels were functionally competent with demonstrated modulatory response to H2S and transforming growth factor (TGF)-β1 whereby H2S significantly inhibited the stimulatory effect of TGF-β1 on current density of both BKCa and IKir. Furthermore, H2S attenuated TGF-β1-stimulated KCa1.1/Kv1.1 (encode BKCa) and Kir2.1 (encode IKir) expression in TCs. Our results show that functionally competent K+ channels are present in human atrial and ventricular TCs and their modulation may have significant implications in myocardial physiopathology.Telocytes (TCs) with exceptionally long cellular processes of telopodes have been described in human epicardium to act as structural supporting cells in the heart. We examined myocardial chamber-specific TCs identified in atrial and ventricular fibroblast culture using immunocytochemistry and studied their electrophysiological property by whole-cell patch clamp. Atrial and ventricular TCs with extended telopodes and alternating podoms and podomers that expressed CD34, c-Kit and PDGFR-β were identified. These cells expressed large conductance Ca Telocytes (TC) are newly identified cells that are found in the interstitial space of many organs, including heart 2S) modulated the activity of ion channels in human atrial fibroblasts and suppressed their transformation into myofibroblasts in response to transforming growth factor (TGF)-β1. This may have important implications in TGF-β1-mediated cardiac fibrosis and its associated atrial fibrillation 2S on the ion channel activity.Recently, human myometrial TCs have been reported to express calcium-dependent hyperpolarization-activated chloride inward current n = 10) with mean age of 63 ± 8.7 years old who were scheduled for coronary bypass surgery and were hyperlipidemic, managed with anti-hypertensive and diabetic medications. The protocol was approved by institutional review board of Singapore General Hospital that conformed to the Declaration of Helsinki. Atrial appendages were collected as surgical by-product. Human atrial interstitial cells were isolated by mincing the appendages to less than 1 mm3 and followed by 0.1% trypsin digestion for 20 min. before plating onto tissue culture-treated 60-mm dishes to produce fibroblastic outgrowth from minced tissue pieces. Outgrown fibroblasts were harvested and re-plated onto a fresh tissue culture dish as passage 1 to isolate homogeneous fibroblast culture that expressed collagen I and fibroblast marker as described previously by our group 2. Human cardiac ventricular fibroblasts (cat no. CC-2904) were acquired from Lonza Inc (Singapore). These cells were isolated from normal adult heart and certified to express greater than 90% collagen I and negative for von Willebrand factor VIII-related antigen. After 3 passages of the atrial and ventricular culture, TCs were distinguishable from other interstitial cells and were typically identified by their characteristic ultra-long moniliform cellular processes of Tps under light microscopy and the cultures from passages 3 to 5 were used for subsequent experiments. These TCs containing cultures were seeded on glass coverslips for immunostaining or in 35 mm dish for whole-cell patch-clamp recordings.Informed consent was gathered from male patients  to label Ito channels and anti-Kir2.1  to label IKir channels. Secondary antibodies used were Alexa Fluor555 (1/1000) and Alexa Fluor488 (1/500)-labelled antibodies . Counter staining was performed using DAPI  to visualize nuclei.Telocytes were cultured on coverslips, were fixed with 2% paraformaldehyde/PBS for 15 min., permeabilized by 20 min. incubation in PBS containing 0.1% Triton X-100, and blocked in PBS containing 2.5% bovine serum albumin (BSA). Samples were then incubated overnight at 4°C with primary antibodies in PBS containing 0.1% BSA, after which they were incubated for 1 hr at room temperature with secondary antibody. Coverslips were rinsed and mounted on glass slides and examined by using confocal microscopy. Primary antibodies against CD34 , c-Kit  and PDGF receptor (PDGFR)-β  were used to identify TCs. Primary antibodies used were anti-Kv1.1  to label BK2, 1 MgCl2, 10 HEPES and 10 Glucose (pH adjusted to 7.4 with NaOH). The patch-clamped cell was superfused by means of a temperature-controlled micro-superfusor . Patch pipettes were made from borosilicate glass shanks  and pulled with a Brown–Flaming puller , and had tip resistances of 2–3 MΩ when filled with pipette solution. Pipette tips were polished . The patch pipettes were filled with a standard solution containing (in mM) 140 KCl, 1.2 MgCl2, 0.05 EGTA, 10 HEPES, 0.1 GTP and 5.0 Mg ATP (pH adjusted to 7.2 with KOH). After a gigaohm seal was obtained by negative pressure suction, the cell membrane was ruptured by a gentle suction to establish whole-cell configuration with a seal resistance >800 MΩ. The cell membrane capacitance (40.3 ± 8.2 pF) was electrically compensated with the pulse software. The series resistance  was compensated by 50–70% to minimize voltage errors. Currents were elicited with voltage protocols as described in the following results section for different individual current recordings. Whole-cell voltage-clamp experiments were performed with an Axopatch 200B amplifier  interfaced to a Digidata 1322A data acquisition system controlled by Clampex version 8.1 software (Axon Instruments). Data were analysed with pCLAMP software  and Origin 8.0 .Cell were placed on the stage of a Nikon Diaphot inverted microscope and superfused continuously at 36 ± 1°C with Tyrode solution containing (in mM) 140 NaCl, 5.4 KCl, 1.8 CaClt-test as appropriate. A value of P < 0.05 was considered statistically significant.All data were presented as mean ± SE. Statistical significance of the difference between groups was determined using Student's Telocytes were clearly identifiable in our human atrial and ventricular cardiac fibroblast culture by phase contrast after passage 3. Telocytes displayed characteristic long Tps 100–500 μm) 00–500 μmn = 6 atrial TCs; 8.9 ± 2.1 pA/pF, n = 6 ventricular TCs), the activated currents were sensitive to inhibition  at +90 mV by 1 mM Paxilline (a specific BKCa inhibitor) and were responsive  at +90 mV to stimulation by 10 μM (+/−) Naringenin (a specific BKCa opener), confirming the presence of BKCa currents in atrial and ventricular TCs  at +90 mV. However, additional presence of H2S (100 μM) reduced the effect of TGF-β1 , thereby demonstrating functionality of the channels and confirming the inhibitory effect of H2S on BKCa currents in TCs (Ca currents at +100 mV with activation τ (τact) at baseline  was lowered significantly by TGF-β1 , but was significantly reversed  by H2S, confirming its modulation of BKCa channel kinetics in TCs  inhibition, indicating the absence of transient outward currents (Ito) in atrial and ventricular TCs (ATP specific channel enhancer), suggesting ATP-sensitive K current (KATP) was not present in atrial and ventricular TCs  and 3.1 ± 1.2 pS/pF (n = 6) in 5.4 mmol/l [K]o respectively. The baseline current density of IKir at −120 mV  was increased by 1 ng/ml TGF-β1 , confirming the functionality of IKir channel. However, the current density at −120 mV reverted towards baseline  in the presence of H2S  and Kir2.1 (responsible for IKir) expression and reduced proliferation of human atrial fibroblasts 2S for 24 hrs reduced the expression levels and organizational distribution of Kv1.1 and Kir2.1 induced by TGF-β1, supporting its inhibitory role on channel expression in cardiac TCs. Contrary to atrial fibroblasts to) expression in TCs was barely above background noise levels (data not shown), which supported the undetectable Ito current in our electrophysiological findings.Transforming growth factor-β1 is a major mediator of myocardial fibrosis. Our previous study showed that exogenously applied HCa and IKir channels and functionally active currents in human atrial and ventricular TCs.Telocytes have previously been reported in human epicardium to and ATP-sensitive KATP currents were not detected in both the atrial and ventricular TCs in this study, the presence of functionally active BKCa and hyperpolarized-activated inwardly rectifying IKir channels and their close proximity to cardiac fibroblasts and cardiomyocytes may suggest a probable role of cardiac TCs within the multicellular framework that facilitates mechano-electrical coupling of the heart.To date, only very limited is known about the electrophysiological property of isolated TCs. In human myometrium, patch-clamp recordings of TCs revealed a calcium-dependent hyperpolarization-activated chloride inward current, but absence of L-type calcium channels, which was postulated to modulate myometrial smooth muscle contractions 2S effectively suppressed proliferation of atrial fibroblasts by inhibiting BKCa, IKir and Ito channels 2S attenuated TGF-β1-induced BKCa activation and decelerated the transition from closed to open state of the channel, suggesting a role for H2S in regulating BKCa channel kinetic and voltage sensitivity in human cardiac TCs. Furthermore, H2S down-regulated the voltage-dependent relationships of IKir induced by TGF-β1 in the TCs. It is currently unclear about the implications of greater BKCa , the Goh Foundation (Duke-NUS-GCR/2013/008) and Biomedical Research Council Singapore (BMRC 13/1/96/19/86) to W.S.The authors confirm that there are no conflicts of interest.


ESRRB) gene is the underlying cause of autosomal recessive nonsyndromic hearing impairment (ARNSHI) due to the DFNB35 locus which maps to 14q24.3. A genome scan of a large consanguineous Pakistani pedigree with ARNSHI established linkage with a maximum multipoint LOD score of 4.2 to the 14q24 region and the region of homozygosity contained the ESRRB gene. Sequencing of the ESRRB gene using DNA samples from hearing-impaired family members uncovered a novel three-nucleotide deletion c.1018_1020delGAG (p.Glu340del). The deletion segregates with hearing impairment in the pedigree and was not observed in 500 control chromosomes. The deletion of glutamic acid residue occurs in the ligand-binding domain of ESRRB protein. It is expected that the deletion affects the ligand-binding activity of the domain in ESRRB, which leads to the ARNSHI.Mutations in the estrogen-related receptor beta ( ESRRB gene . ESRRB belongs to a subfamily of orphan nuclear receptors that are structurally related to estrogen receptors (ER) but do not directly bind estrogen. The ESRR proteins have DNA-binding and ligand-binding domains, which are both required for transcriptional regulation of ER target genes. The ligand-binding domain (LBD) provides structural stability and influences the binding affinity of the ESRRB protein [ESRRB gene which is responsible for ARNSHI. Hearing impairment (HI) has been associated with estrogen physiology for decades. Turner's syndrome in females and Noonan's syndrome in males are both marked by hypoestrinism and include the HI phenotype. Variations in hearing function have also been found according to gender, menstrual cycle or menopausal state, estrogen therapy, and oral contraceptive use, which implicate estrogen as a primary requirement for hearing preservation . Recentlne MIM 60167; 2. EThe study was approved by the Institutional Review Boards of the Quaid-I-Azam University and the Baylor College of Medicine and Affiliated Hospitals prior to initiation. Informed consent was obtained from all members from family 4243 who participated in the study. Family 4243  is a conGenomic DNA was extracted from venous blood which was provided by five hearing and four HI family members . The ninESRRB (RefSeq NM_004452.3) gene were designed using Primer3 software [Exon primers for software . PCR-ampESRRB-related HI [The audiogram of HI individual IV-4 displays bilateral, severe-to-profound HI affecting all frequencies . This islated HI . θ = 0) was observed at marker rs935340  in exon 8 which segregates with ARNSHI . The glutamic acid residue 340 is located in the ligand-binding domain of the ESRRB protein. The identification of the deletion mutation of ESRRB gene will expand our understanding of hearing impairment due to mutations in the estrogen-related gene ESRRB.A genome-wide linkage scan performed using DNA samples from a large consanguineous ARNSHI pedigree and the subsequent sequencing of the


Nervous system development is dependent on early regional specification to create functionally distinct tissues within an initially undifferentiated zone. Within the retina, photoreceptors are topographically organized with rod free area centrales faithfully generated at the centre of gaze. How does the developing eye regulate this placement? Conventional wisdom indicates that the distal tip of the growing optic vesicle (OV) gives rise to the area centralis/fovea. Ectopic expression and ablation studies do not fully support this view, creating a controversy as to the origin of this region. In this study, the lineage of cells in the chicken OV was traced using DiI. The location of labelled cells was mapped onto the retina in relation to the rod-free zone at embryonic (E) 7 and E17.5. The ability to regenerate a rod free area after OV ablation was determined in conjunction with lineage tracing.Anterior OV gave rise to cells in nasal retina and posterior OV became temporal retina. The OV distal tip gave rise to cells above the optic nerve head. A dorsal and anterior region of the OV correlated with cells in the developing rod free area centralis. Only ablations including the dorsal anterior region gave rise to a retina lacking a rod free zone. DiI application after ablation indicated that cells movements were greater along the anterior/posterior axis compared with the dorsal/ventral axis.Our data support the idea that the chicken rod free area centralis originates from cells located near, but not at the distal tip of the developing OV. Therefore, the hypothesis that the area centralis is derived from cells at the distal tip of the OV is not supported; rather, a region anterior and dorsal to the distal tip gives rise to the rod free region. When compared with other studies of retinal development, our results are supported on molecular, morphological and functional levels. Our data will lead to a better understanding of the mechanisms underlying the topographic organization of the retina, the origin of the rod free zone, and the general issue of compartmentalization of neural tissue before any indication of morphological differentiation. A hallmark of nervous system development is the early fate determination of tissues followed by progressive morphological differentiation of initially featureless regions into cyto-architecturally and functionally distinct organs and systems. The onset of visual system formation begins with the establishment of tissue fated to become the retina, which occurs when the eye fields are inducted by a group of eye field transcription factors in the anterior neural plate ,2. Once By the time the OV invaginates to form an optic cup, the tissues fated to be the neural retina and retina pigment epithelium (RPE) have been established. The retinal axes are further refined, which becomes critically important in the organization of many retinal cell types . FoHow does the developing eye regulate the placement of the rod free area centralis? The perceived wisdom indicates that the first part of the growing OV to make physical contact with the overlying ectoderm, that is the distal tip, establishes the location of the area centralis. Once this region is established as the centre of the visual map, the basics divisions of the retina are formed ,11. HoweIn this paper, the hypothesis that the distal tip of the growing OV gives rise to the rod free area centralis was tested by lineage and ablation analysis. The data indicate that the chicken rod free area centralis originates from cells located near, but not at the distal tip of the developing OV. This anterior dorsal region, when ablated, produces a retina lacking the presumptive rod free zone with very little plasticity in the immediately surrounding OV regions observed. Taken together, these data do not support the hypothesis that the distal tip of the OV gives rise to the rod free area centralis; rather the data demonstrate that the origin of the rod free area centralis is in the anterior dorsal OV.The insertion of DiI crystals through the overlying ectoderm into the neural tube at the most distal region of the OV Figure  was tracThe anterior OV gave rise to more nasal retina at both E7 . Although the eye grows significantly between E7 and E17.5, the visinin free spot at E7 was located at the same distance from the optic nerve head as the rod free area centralis at E17.5 Figure . These dSmall distal tip ablations did not disturb the development of the presumptive rod free zone, as indicated by the presence of a visinin free spot in the control and operated eye Figure . When a DiI crystals were placed on the anterior edge of the wound following a posterior ablation , the presumptive rod free zone is maintained, but if more than 20% is ablated, the rod free zone is lost. Ablations removing more than 50% of the OV give rise to pigmented blobs or small eyes. Only when an ablation removes a significant amount of the dorsal anterior region is the rod free zone lost Figure . After aOur data suggests that the distal tip of the OV is important for identifying the location of the developing optic nerve; rather than the formation of the rod free zone. During the transition from OV to optic cup, once the distal tip of the OV reaches the overlying ectoderm, the expanding OV begins to invaginate to form a cup-shaped structure, fold along its center line, and to acquire regionally specific characteristics ,12-14. TThe assumption that the distal tip of the OV gives rise to the rod free area centralis stems from a lineage analysis paper by Dutting and Thanos who demonstrated that the distal tip of the OV gives rise to a large proportion of the central retina, above or surrounding the optic nerve head . This hyThe localization of the rod free progenitor zone to an anterior dorsal region is further supported by changes in regional gene expression in the retina after manipulation of the anterior/posterior and dorsal/ventral axes by ablation or ectopic expression. Anterior OV ablation results in the posterior vesicle remnant reconstituting a normal-sized retina with posterior characteristics, which lacks the rod free area centralis Figure  10]. Si. Si10]. Previous studies using partial ablation of either the anterior or posterior OV indicate a range of resulting eye morphologies, ranging from normal sized eye, to microphthalmic eyes to pigmented blobs ,10,24,25Healing of the OV after ablation requires cell movements and increased proliferation to grow the two edges of the wound together. It is likely that there is a difference in proliferation and a limit to plasticity at the edges of the wound. Dutting and Thanos suggested that the cells located in the peripheral anterior or posterior OV give rise to the larger retinal area in the E6 retina, suggesting that retinal progenitor cells located in most anterior and posterior OVs undergo increased proliferation . The incDuring retinal development, there is a correlation between the location of the visinin free spot at E7 or the rod free area centralis at E17.5 and the optic nerve. This consistent distance has been observed in the human retina, with the fovea located 4.9 mm from the optic nerve head with little variability between eyes and individuals . In addiTaken together, our data indicates that the hypothesis that the area centralis is derived from cells at the distal tip of the OV is not supported; rather, a region anterior and dorsal to the distal tip gives rise to the rod free zone. The location of the rod free zone is specified prior to E7 and its location remains constant in relation to the OD throughout the rest of development. When compared with other studies of retinal development, our results are supported on molecular, morphological and functional levels. Our data will lead to a better understanding of the mechanisms underlying the topographic compartmentalization of the retina and the origin of the rod free zone as well as lay the foundation for additional large scale investigations of the molecules controlling the formation of the rod free zone. These results are applicable to a wider audience in that they show the early compartmentalization of neural tissue before any indication of morphological differentiation. The conserved mechanisms that function to generate these distinct regions should be studied more closely as the results will further the development of an accessible and highly informative model for neural differentiation.Gallus gallus) eggs obtained from a local breeder were incubated at 37.8°C in a humidified incubator for 36-48 hours. A window was made in the top of the egg above the developing embryo. The vitelline membrane covering the embryo was removed and India ink diluted in sterile 0.1 M Phosphate buffered saline (PBS), pH 7.4 was injected beneath the embryos to visualize the embryonic structures and to allow the embryos to be staged according to Hamburger [All experiments were performed and tissue collected with ethical approval from the Australian National University and the University of Auckland Animal Ethics committees. Fertilized chicken  of the OV was performed at HH stage 10-11. Using sharpened tungsten needles, varying amounts of the right OV were ablated. The left OV served as a control. For cell lineage analysis following ablation, a DiI crystal was implanted on the cut edge using sharp tungsten needle. After ablations, and ablations with DiI placements, 250 μL 0.1 M phosphate buffered saline (PBS) was added to the top of each embryo, the embryos were photographed with a Leica camera  attached to a Leica dissecting microscope, the windowed egg was sealed with tape, and all eggs returned to the incubator until either E7 or E17.5. The earlier age, E7, was chosen because this is similar to the age used for previous chicken OV fate mapping studies, therefore allowing a comparison to be made with well established data . The oldin situ hybridization or immunohistochemical processing due to the methanol (MeOH) step, the DiI containing fluorescing cells were located and photographed using a Leica compound microscope . The location of the DiI labelled cells was determined by measuring and recording the distance from known retinal landmarks .At E7 (HH stage 31) or E17.5 (HH stage 44), the embryos were removed from the shell, quickly decapitated and the eyes enucleated. The cornea, lens and vitreous were removed. The retina was then dissected away from the sclera and RPE. The retina was fixed overnight in 4% paraformaldehyde, and then washed twice in 1× PBS for 5 minutes. The retina was flattened by making several incisions along the retinal margins and then whole-mounted on a glass slide and photographed on a Leica  dissecting microscope. Because the DiI labelling was not retained in the cells after The retina was then transferred back into PBS and then dehydrated in an ascending series of PBS containing 0.1% Tween-20 (PBT) and MeOH. Retinas were stored in 100% methanol at -20°C until use.in situ hybridization protocol, the E7 retinas were rehydrated through a descending PBT/MeOH series. Once in PBT, any remaining pigment epithelium was bleached [To begin the bleached ,33. The In situ hybridization was carried out at E7 using digoxigenin-labeled visinin riboprobes, as described previously [eviously ,35. Visieviously . The visWholemount immunocytochemistry on the E17.5 retinas was performed as previously described . Brieflyin situ or immunohistochemistry; however the overall shape of the wholemount remained similar to that recorded when the DiI labelled cells were mapped. A schematic representation of the regional labelling of the DiI cells was mapped onto a sketch of the flat-mounted retina. The location of the visinin free spot or the rod free zone was recorded in relation to the optic nerve, and then this information was included in the map containing the location of the DiI labelled cells. Any slight changes in shape or size of the flatmount that were observed when comparing the map of the DiI labelling and post in situ or immunohistochemistry were noted.Slight changes in the shape of the retina were observed after the tissue processing for in situ hybridization experiments, and drafted the manuscript. KMBO supervised the design and implementation of the project, assisted with the lineage and ablation analysis, performed the immunohistochemistry experiments, analysed the results, critically revised the manuscript, and gave final approval for this version to be published. All authors read and approved the final manuscript.SKS assisted in the design of the project and carried out the lineage analysis and ablation studies, followed by the


Carassius auratus) following exposure to specific frequencies. Subjects were divided into six groups of six fish each (five treatment groups plus control). The treatment groups were each exposed to one of five tones: 100, 400, 800, 2000, and 4000 Hz at 176 dB re 1 μPa root mean squared (RMS) for 48 hours. The saccules of each fish were dissected and labeled with phalloidin in order to visualize hair cell bundles. The hair cell bundles were counted at 19 specific locations in each saccule to determine the extent and location of hair cell damage. In addition to quantification of anatomical injury, hearing tests  were performed on each fish immediately following sound exposure. Threshold shifts were calculated by subtracting control thresholds from post-sound exposure thresholds.Mammalian and avian auditory hair cells display tonotopic mapping of frequency along the length of the cochlea and basilar papilla. It is not known whether the auditory hair cells of fishes possess a similar tonotopic organization in the saccule, which is thought to be the primary auditory receptor in teleosts. To investigate this question, we determined the location of hair cell damage in the saccules of goldfish (All sound-exposed fish exhibited significant hair cell and hearing loss following sound exposure. The location of hair cell loss varied along the length of the saccule in a graded manner with the frequency of sound exposure, with lower and higher frequencies damaging the more caudal and rostral regions of the saccule, respectively. Similarly, fish exposed to lower frequency tones exhibited greater threshold shifts at lower frequencies, while high-frequency tone exposure led to hearing loss at higher frequencies. In general, both hair cell and hearing loss declined as a function of increasing frequency of exposure tone, and there was a significant linear relationship between hair cell loss and hearing loss.The pattern of hair cell loss as a function of exposure tone frequency and saccular rostral-caudal location is similar to the pattern of hearing loss as a function of exposure tone frequency and hearing threshold frequency. This data suggest that the frequency analysis ability of goldfish is at least partially driven by peripheral tonotopy in the saccule. Frequency discrimination is the ability of a listener to discriminate between two pure tones that differ only in frequency. Frequency discrimination in fishes has been established from pyschophysical studies of a limited number of species , but hasIndeed, the basis of frequency discrimination in general has been debated for well over a century. The two main models of frequency discrimination are the place theory and temporal theory of hearing. The complex history of these theories is reviewed elsewhere . In brieMany vertebrate auditory systems have been shown to exhibit tonotopic mapping, which is an orderly arrangement of frequency response in the sensory organ. For example, amphibians, reptiles, birds, and mammals are known to use tonotopic mapping to peripherally discriminate frequencies -9. That Fishes hear using an ear that is similar in many aspects to the inner ear of other vertebrates, and have similar capabilities in performing auditory functions such as discriminating between sounds of varying intensity, frequency, and temporal patterns as other vertebrates . Indeed,Gallus domesticus) exposed to tones, higher frequencies produced a loss of short hair cells in the basal region of the basilar papillae, while lower frequencies produced a broader region of hair cell loss in the apical region [Exposure to loud sounds can damage or destroy macular hair cells and induce hearing threshold shifts in fishes -20. Sounl region ,10,23. Sl region -26.Carassius auratus). We also tested hearing loss in the same fishes to understand the relationship between localized hair cell loss and hearing loss. This is the first study to attempt to correlate patterns of hair cell loss with frequency-specific hearing loss in fishes.In order to better understand the role of peripheral processes involved in frequency discrimination in fishes, we examined the tonotopic pattern of saccular hair cell damage in response to tone exposures in goldfish . Hair cell density was greatest at the far-rostral and far-caudal regions  and decreased toward the central region , but the pattern of hair cell bundle loss varied as a function of exposure tone frequency (P < 0.001) and rostral-caudal axis region (P < 0.001), with a significant interaction between these two variables .All sound-exposed fish had significantly higher hearing thresholds compared to controls (P < 0.01). No significant differences occurred in TTS across frequencies following the 800 Hz tone exposure although the highest mean TTS occurred at 800 and 1000 Hz (25 dB) and the lowest at the extremes of 100 Hz (13 dB) and 4 kHz (15 dB). In fish exposed to the 2 kHz tone, threshold shifts were greater at 1 - 2 kHz compared to 4 kHz and frequencies 500 Hz and lower (P < 0.05).There were also significant differences in TTS between audiogram frequencies within specific tone exposures. For the 100 Hz tone exposure, TTS was significantly smaller at 2 - 4 kHz compared to lower frequencies (P < 0.05). In general, fish exposed to lower frequency tones exhibited greater threshold shifts at lower frequencies, while high-tone exposure led to greater hearing loss at higher frequencies , with maximal hearing loss at 1 kHz, the frequency of greatest hearing sensitivity in the goldfish , TTS at 400 Hz was significantly lower than those of 800-1000 Hz (P < 0.02), and TTS at 1000 Hz was significantly higher than those of 600, 2000, and 4000 Hz (P < 0.02).When TTS data for all treatments were pooled, there was a significant change in TTS across audiogram frequencies  and mean TTS  were calculated for each individual fish, and plotted against the stimulus tone of the acoustic exposure. A similar pattern was evident for both hair cell and hearing loss - a decrease in loss with increased frequency of exposure Figure . When plIn order to compare the relationship between specific locations of hair cell bundle loss and hearing loss at specific frequencies, we made two three-dimensional graphs. In the first graph, we plotted percent hair cell bundle loss at all 19 specified locations for each fish saccule as a function of frequency of the exposure tone and the location along the rostral-caudal axis of the saccule Figure . This grWhile both plots result in smoothed surfaces that resemble a piece of paper with the opposite diagonal corners pulled down, one of the downward slopes is much more dramatic with the hair cell loss data Figure  than witThe similarity between the smoothed surfaces of these two plots is what one would expect if the goldfish saccule is indeed tonotopically organized. Using these plots, and the tonotopic pattern of damage in the saccule Figure , we suggWe found a distinct caudal to rostral shift in hair cell bundle loss with exposure frequency, with the 100 Hz tone producing damage localized to the caudal saccule and the 4 kHz tone producing damage to the rostral saccule. A frequency-dependent spatial pattern of sound-induced damage in the inner ear has been reported for other vertebrates -11,21, bGadus morhua). Cod saccular maculae were examined for damage through scanning electron microscopy following exposure to intense tones (approximately 180 dB re 1 μPa) ranging in frequency from 50 to 400 Hz. Damaged areas of epithelia had an almost complete loss of hair cell stereocilia, leaving only cuticular plates or a few disorganized stereocilia on hair cells. As exposure frequency increased, the areas of damaged epithelia were located in successively more rostral regions of the saccule. It was also observed that all frequencies produced damage in the central region of the saccule.Enger  investigOur data show a similar pattern of tone-induced hair cell loss in goldfish, despite differences in hearing sensitivity and bandwidth. The range of hearing is 50 to 470 Hz in cod, with hearing thresholds recorded at 75-80 dB re 1 μPa at 100 Hz , while gWe found that hair cell loss occurred in the central region of the saccule for most tone-exposed goldfish Figure  and thatAnother pattern that was evident was that areas of hair cell damage became generally smaller with increasing exposure frequency. The frequency-position functions are logarithmic in nature in humans and other mammals ,31. ThatThe high intensities used during the sound exposures in the current study may have been enough to spread the area of damage across the saccule beyond the region that normally responds to a given tone, affecting the sensitive, centrally-located hair cells. Previous studies have demonstrated that very intense sound damages an extended region of the hearing end organ in birds and mammals. For example, in chicks, the damaged area of the basilar papilla varies with exposure frequency, but also with tone intensity; a secondary region of damage is created with increased sound pressure level . In the Intense octave band noise can cause focal lesions that extend beyond the frequency-place range of the exposure sound in the cochlea of chinchillas ,34, and Perca fluviatilis) in response to four frequencies of horizontal movement. They found that the amplitude of vibration varied across the length of the otolith in a frequency-dependent manner, suggesting that the otolith motion had a rotational component and that this might explain observed peripheral frequency analysis in teleost fishes. The goldfish saccular otolith, the sagitta, is long, thin, and delicate with a twist in the middle similar to an airplane propeller [The mechanisms that contribute to tonotopic mapping in the fish saccule have not been fully elucidated, but there are several possibilities. One potential mechanism is that the otolith overlying the saccular epithelium vibrates differentially in response to varying frequency. While the modeling of fluid motion around otolith-like spheroids has recently been conducted , direct ropeller . It has ropeller , but litDanio rerio), a close relative of goldfish, was greatest at the caudal tip, least dense in the middle, and intermediate at the rostral tip of the saccule. Similar patterns have also been found previously in goldfish [It is also likely that characteristics of the sensory cells themselves are at least partially responsible for the tonotopic damage found in this study. Our finding of a shift in hair cell density along the goldfish saccular rostral-caudal axis is consistent with that found by other researchers. Higgs et al.  found thgoldfish ,38,41.Helostoma temmincki [Goldfish auditory hair cells also show ultrastructural differences that vary by location on the saccule . For exaemmincki .It is thought that fish hair cells demonstrate micromechanical tuning through differences in hair cell bundle stiffness, mode of attachment to the otolithic membrane , and eleGoldfish possess excellent tone discrimination ability, distinguishing tone differences as small as 3.5 Hz at 50 Hz . In addiGoldfish threshold shifts varied with frequency of tone exposure as predicted, with the 100 Hz tone exposure causing greater threshold shifts at lower frequencies and 2000 and 4000 Hz tones causing threshold shifts primarily at higher frequencies. As there was significant overlap in regions of damaged saccular epithelia caused by different tone exposures, there was also considerable overlap in frequencies that were affected by a particular tone exposure. For example, the 100 Hz tone exposure caused significant threshold shifts across all frequencies but this effect was much greater at frequencies below 1.5 kHz, while the 4 kHz tone also caused significant threshold shifts across all frequencies but the largest threshold shifts occurred at frequencies ≥1 kHz Figure .Pimephales promelas) exhibited an 8-11 dB TTS in response to 2 h of 142 dB re 1 μPa narrow bandwidth boat motor noise with a peak frequency near 1.3 kHz [Such overlap has been seen in other studies in which fish were acoustically over-stimulated. For example, Popper and Clarke  found th 1.3 kHz . While t 1.3 kHz ,18. Simiper se. The advantage of using SPDs is that they account for differences in hearing sensitivity across different frequencies in the animal's audiogram. The LINTS model was developed using a white noise stimulus, with equal intensity across the frequencies tested. Since most organisms have U-shaped audiograms that are more sensitive at mid-frequencies and less sensitive at lower and higher frequencies, the SPD between a white noise stimulus and hearing thresholds is greatest at mid-frequencies. Thus, TTS is predicted to be greatest at these mid-frequencies [It is not surprising that the goldfish had the greatest hearing loss at frequencies where their hearing is most sensitive because the difference between the sound pressure level of the exposure tone and the level of hearing threshold is greatest at these frequencies. This concept was formalized as a predictive model of hearing loss called the linear threshold shift (LINTS) hypothesis . In thisquencies .2 = 0.13, P = 0.09). One potential explanation for this observation is that the stimulus intensity used in this study may have been too high for the LINTS model to make effective predictions. As previously discussed, louder exposure stimuli produce greater areas of sensory cell damage, and thus hearing loss, over a greater range of frequencies. A replication of this study at lower stimulation intensities would shed light on the overall validity of the LINTS model for predicting hearing loss in fishes.The current study used tonal stimuli such that the SPD between the tone and the hearing threshold was greatest at the frequency of the exposure tone and should have been minimal at all other frequencies. Thus, according to the LINTS model, TTS would be expected only at or near the frequency of the exposure tone. This obviously did not occur-TTS occurred at multiple, if not all of the frequencies tested, although low frequency tones produced the greatest TTS at low frequencies and high frequency tones produced greatest TTS at high frequencies. We tested the LINTS relationship by plotting TTS against SPD and performing linear regression analysis between these two variables. In this analysis, only TTS at hearing frequencies equal to the exposure tone were used since the maximal sound pressure level measured was that of the exposure tone and not adjacent frequencies . This relationship was not significant (TTS = 0.15 (SPD) + 13.3; RLoss of hair cells is correlated with a loss of hearing in other vertebrates ,69 so weIn the current study, there was considerable overlap in hearing loss across frequencies and hair cell loss across saccular regions for a given exposure tone. While this may be the result of the effect of the high intensity of the exposure stimuli that we used in this study, this overlap could also reflect the coarseness of the tonotopic map in the goldfish saccule, suggesting that the peripheral auditory filters are broadly tuned. Another possible explanation for this overlap is harmonics of our tone signals that were evident in our acoustic exposure and difficult to avoid in our small exposure chamber Figure . While pA similar pattern was evident for both hair cell and hearing loss - a decrease in loss with increased frequency of exposure Figure . A similWe demonstrate that overexposure to pure tones causes tonotopically-organized morphological damage in the goldfish saccule. Damage is characterized as a significant decrease in hair cell bundle density. Areas of damage varied with tone frequency, with damage shifting rostrally across the length of the saccule as frequency increased. In addition, there was a corresponding shift in hearing loss with low and high frequency tones causing threshold shifts at lower and higher frequencies, respectively. These data suggest that goldfish use at least a crude level of tonotopy in the saccule to peripherally discriminate between tones.Carassius auratus L.) were chosen as model species because much is known about their hearing capabilities. They are otophysans and show sophisticated auditory capabilities including tone discrimination and auditory segregation [Goldfish  and were housed in 170-L tanks of recirculated, filtered water maintained at approximately 25°C. All work was done under the supervision of the Institutional Animal Care and Use Committee of Western Kentucky University.Subjects were divided into six groups of six fish each (five treatment groups plus control). The treatment groups were each exposed to one of five tones: 100, 400, 800, 2000, and 4000 Hz at 176 dB re 1 μPa root mean squared (RMS) played continuously for 48 hours. The tone stimuli were generated using a B&K Precision function generator (4017A) and played through an amplifier  to an underwater speaker  placed centrally on the bottom of a 19-L cylindrical chamber. Sound pressure levels were measured via a Type 10CT hydrophone placed 2 cm directly above the center of the speaker and a Type 42AC Pistonphone calibrator . In order to maintain fish health during the 48 h exposure, fish were allowed to swim freely in the exposure chamber as in previous fish sound exposure experiments ,20, but AEP is a non-invasive method of measuring neural responses to auditory stimuli and is commonly used for measuring hearing in fishes and other vertebrates ,75. EachStainless steel subdermal electrodes  were used to record auditory evoked potentials. A reference electrode was inserted approximately 2 mm subdermally into the medial dorsal surface of the head between the anterior portion of the eyes while a recording electrode was placed 2 mm into the dorsal midline surface of the fish approximately halfway between the anterior insertion of the dorsal fin and the posterior edge of the operculae, directly over the brainstem. A ground electrode was placed in the tail musculature of the fish.Sound stimuli were presented and AEP waveforms collected using SigGen and BioSig software running on a TDT physiology apparatus . Sounds were computer generated via TDT software and passed through a Hafler P1000 power amplifier  connected to an underwater speaker (University Sound UW-30). Tone bursts were 15, 10, and 5 ms in total duration for 0.1 and 0.25, 4 and 6, and 0.8-4 kHz tones, respectively. Each tone pip had a 2 ms rise and fall time and were gated through a Hanning window . Following AEP, subjects were euthanized by overdose with tricaine methansulfonate (MS-222). The heads were removed, injected with 4% paraformaldehyde, and placed in 4% paraformaldahyde overnight at 4°C. The heads were washed in 0.1 M phosphate buffer and the inner ears removed under a dissecting microscope. Both right and left saccules were then trimmed and incubated for 30 minutes in Alexa Fluor 488 phalloidin , which stains actin in hair cell bundles and cuticular plates. Saccules were mounted whole under a cover slip with Prolong Gold antifade reagent with 4',6-diamidino-2-phenylindole (DAPI) to stain nuclei.2 boxes) across the length of the saccule using Zeiss Axiovision software  epifluorescent microscope and a Zeiss MRm digital CCD camera. Hair cell bundle counts were obtained in 19 predetermined locations . An overall ANOVA was used to examine the effect of tone exposure on goldfish hearing thresholds. Similarly, ANOVAs were also used to test the main effects and interactions of treatment (tone) and frequency (hearing test frequency) on threshold shifts. Tukey's a priori relationships between the variables. All statistical analysis was performed using SYSTAT Version 11 .The relationship between mean hair cell loss and TTS for each individual fish was analyzed using linear regression analysis. Means were calculated across all 19 hair cell counting locations for hair cell loss and all audiogram frequencies for TTS data. The three-dimensional surfaces shown in Figure AEP: auditory evoked potentials; ANOVA: analysis of variance; DAPI: 4',6-diamidino-2-phenylindole; dB: decibel; FFT: Fast Fourier Transform; h: hours; Hz: hertz; LINTS: linear threshold shift; μPa: microPascal; MS-222: tricaine methanosulfonate; TS: torus semicircularis; TTS: temporary threshold shift: PTS: permanent threshold shift; RMS: root mean squared; SPD: sound pressure difference; SPL: sound pressure levelMES conceived the study, did statistical analysis, and wrote the paper. JBS performed dissections of the goldfish saccules and editing of the manuscript. RRG did measurements and analysis of auditory sensitivity via auditory evoked potentials. BDR did microscope work and hair cell counts. MES, JBS, RRG, and BDR prepared the figures. All authors read and approved the final manuscript.


Noise most frequently affects hearing system, as it may typically cause a bilateral, progressive sensorineural hearing loss at high frequencies.This study was designed to compare three different methods to evaluate noise-induced hearing loss .This was a cross-sectional study. Data was analyzed by SPSS (ver. 19) using chi square, T test and repeated measures analysis. Study samples were workers from tile and ceramic industry.We found that conventional audiometry, extended high-frequency audiometry, low-tone distortion product otoacoustic emission and high-tone distortion product otoacoustic emission had abnormal findings in 29 %, 69 %, 22 %, and 52 % of participants. Most frequently affected frequencies were 4000 and 6000Hz in conventional audiometry, and 14000 and 16000 in extended high-frequency audiometry.Extended high-frequency audiometry was the most sensitive test for detection of hearing loss in workers exposed to hazardous noise compared with conventional audiometry and distortion product otoacoustic. Noise is among the physical exposures originated from fluctuations in air pressure  and is aThe most common method for assessment of hearing loss is pure-tone audiometry which is performed at frequencies of 250 to 8000 Hz. This method evaluates the whole hearing pathway from external ear to the hearing cortex -8. OAE iFor the first time in 1978, Kemp recognized acoustic emissions due to the movement of outer hair cells in the cochlea . Porto eOeken et al. found a decrease in amplitude of DPOAE at 2-5 KHz frequencies after exposure to noise which was reversed after a recovery time . PlinkerIn this study we aimed to compare three different tests for early diagnosis of NIHL  among noise-induced workers.This was a prospective cross-sectional study conducted in Yazd, Iran from 15/10/2011 to 30/5/2012. Sample size was calculated based on a power of 80% and a two-sided significance level of 5%. It was calculated to be 240 subjects (120 in each group). Workers of 3 tile and ceramic factories were assessed. The subjects in the first group (exposure to noise level higher than 85 dBA) were randomly selected, and subjects in the second group (without exposure to noise) were matched to the first group regarding their age and work experience. Those older than 50 years old, with a history of acoustic trauma, history of exposure to ototoxic substances or consuming ototoxic drugs, and history of smoking were excluded from the study.A questionnaire containing medical and occupational history was filled for each subject. Otoscopic examination was performed for all subjects, and those with abnormal tympanic membrane were excluded from the study. Conventional PTA was performed for each subject  in an acoustic chamber meeting criteria of ANSI 2004 at the following frequencies: 500, 1000, 2000, 3000, 4000, 6000, and 8000Hz . Then exIn this study, 263 subjects entered the study and 16 subjects were excluded ; so at last 526 ears were assessed. All subjects were males. Subjects were divided into two groups: with exposure to hazardous noise (142 subjects), and 121 subjects without any exposure. Mean exposure to noise in the first group was 91.97 ± 4.15 dBA (time weighted average-8 hours). Mean age and work experience were 35.00 ± 6.33 and 10.76 ± 5.52 years, and 34.15 ± 5.76 and 11.14 ± 6.12 years in the first and second groups, respectively. The difference between the two groups regarding age (P = 0.81) and work experience (P = 0.71) was not significant. DPOAE was significantly different in the two groups at all frequencies. NIHL is among the most common occupational diseases. Workers exposed to noise are regularly evaluated by conventional audiometry to find cases of NIHL, an irreversible disease. Early diagnosis of NIHL or early detection of ears susceptible to the effects of noise can prevent hearing loss from extension to speech frequencies. Recently, HFA and DPOAE have been proposed as predictors of NIHL in workers exposed to noise. In this study, we compared hearing loss detected by three methods . In the current study, the frequency of hearing loss at extended HFA was significantly higher than conventional frequencies among individuals with exposure to hazardous noise, which was consistent with the results of some previous studies -15. AlthTurkkahraman et al. consistent with the current study found 14000 and 16000 Hz to be more sensitive to noise than other frequencies , but WanNow, hearing conservation programs for case finding are based on PTA. PTA is subjective, time-consuming and sensitive to surrounding noise. Therefore, there is a need for more sensitive and specific methods for early detection of NIHL. Recently, OAE has been introduced as a better predictor of occupational hearing loss, especially NIHL in workers. Attias et al. compared the results of click-evoked OAE, DPOAE and PTA for the early diagnosis of NIHL in three groups of subjects . They showed a higher sensitivity and accuracy for OAE in the diagnosis and monitoring of cochlear status following noise exposure, which was consistent with our study .To the best of our knowledge few studies have compared conventional PTA, HFA, and DPOAE. In the present study, the frequency of abnormal thresholds at high-tone frequencies of audiometry was significantly higher than DPOAE, although our cases mostly had mild hearing loss, and it is believed that the sensitivity of OAE between 10 and 50 dB SPL is low . Han et  Most of the subjects had mild hearing loss in conventional audiometry in which OAE results are not reliable. This study has the inherent limitation of all cross-sectional studies. To reach to this conclusion that a test would become abnormal sooner than other tests, prospective studies are required. Our cases were only males, so the results cannot be extrapolated to females. We could not assess 18000 and 20000 Hz frequencies because of our equipment limitations.We concluded from the results of this study that among three different methods for assessing hearing status of noise-exposed workers, HFA is the most useful one for the early diagnosis of NIHL. To recommend performing this test for workers screening, longitudinal studies should be performed to show the decrease in hearing thresholds at high frequencies.


This case report highlights an unusual case of sudden sensorineural hearing loss related to superficial siderosis (SS). Our patient had a craniotomy for medulloblastoma 23 years earlier, and this may represent a delayed complication related to this procedure. Magnetic resonance imaging (MRI) remains the key diagnostic investigation to illustrate the imaging features of superficial siderosis and exclude other pathologies. Increased awareness of progressive and sudden hearing complications caused by SS is important in the otolaryngologic community to expedite management and better counsel patients during the consent process. Superficial siderosis (SS) classically affects the leptomeninges, brain, brainstem, cerebellum, cranial nerves and spinal cord secondary to hemorrhage and hemosiderin deposition . Hemosid It is an uncommon condition with only approximately 100 cases reported . Early c This case report provides an account of a young male who presented with sudden sensorineural hearing loss. A 30-year-old Caucasian male patient presented to our clinic with sudden-onset left-sided sensorineural hearing loss and nonpulsatile tinnitus. His medical background was remarkable for a medulloblastoma treated with a suboccipital craniotomy and tumour resection with adjuvant radiotherapy at the age of seven. The patient had no postoperative complications or evidence of tumour recurrence on long-term followup. Examination revealed bilateral sensorineural hearing loss on tuning fork testing with normal otoscopic findings. Dysdiadochokinesia, past pointing, and intention tremor were also noted affecting the patient's left upper limb. Audiometry showed bilateral down-sloping moderate-severe sensorineural hearing loss, which was slightly worse on the left side. No prior audiometry was available for comparison.  The pathogenesis of SS results from chronic hemorrhage into the subpial layers with a predilection for the superior vermis, crests of the cerebellar folia, basal frontal lobe, temporal cortex, brainstem, spinal cord, nerve roots, and cranial nerves one and eight. The sources of hemorrhage are cerebrospinal fluid (CSF) cavity lesions, tumors, and vascular abnormalities .  The exhaustion of ferritin biosynthesis by the microglial cells as a result of iron overload subsequently causes neuronal injury . Cerebel The patient's history of previous intradural cranial surgery for medulloblastoma is a risk factor for later development of SS  and may  T2-weighted fast imaging employing steady-state acquisition (FIESTA) magnetic resonance imaging (MRI) revealed hypointense thickened bands along the vestibulocochlear nerves bilaterally which represent classic diagnostic features of SS . FIESTA  SS is a well-described delayed complication following craniotomy. Patients and their carers must be counseled regarding its association with sensorineural hearing loss, both sudden and progressive, during the consent process. Increased awareness of SS amongst the otolaryngologic community is important so that appropriate audiometric testing, imaging, and management may be expedited.


Different mechanisms have been proposed to be involved in tinnitus generation, among them reduced lateral inhibition and homeostatic plasticity. On a perceptual level these different mechanisms should be reflected by the relationship between the individual audiometric slope and the perceived tinnitus pitch. Whereas some studies found the tinnitus pitch corresponding to the maximum hearing loss, others stressed the relevance of the edge frequency. This study investigates the relationship between tinnitus pitch and audiometric slope in a large sample.This retrospective observational study analyzed 286 patients. The matched tinnitus pitch was compared to the frequency of maximum hearing loss and the edge of the audiogram (steepest hearing loss) by t-tests and correlation coefficients. These analyses were performed for the whole group and for sub-groups , pure-tone vs. narrow-band (340 vs. 115 ears), and low and high audiometric slope (114 vs. 113 ears)).For the right ear, tinnitus pitch was in the same range and correlated significantly with the frequency of maximum hearing loss, but differed from and did not correlate with the edge frequency. For the left ear, similar results were found but the correlation between tinnitus pitch and maximum hearing loss did not reach significance. Sub-group analyses  revealed identical results except for the sub-group with high audiometric slope which revealed a higher frequency of maximum hearing loss as compared to the tinnitus pitch.The study-results confirm a relationship between tinnitus pitch and maximum hearing loss but not to the edge frequency, suggesting that tinnitus is rather a fill-in-phenomenon resulting from homeostatic mechanisms, than the result of deficient lateral inhibition. Sub-group analyses suggest that audiometric steepness and the side of affected ear affect this relationship. Future studies should control for these potential confounding factors. There is a lot of evidence that tinnitus, the phantom perception of sound, is a consequence of neuroplastic alterations in the central auditory pathways One theory proposes that tinnitus results from an edge effect caused by an imbalance of lateral inhibition at the boundary of the region of normal and impaired hearing, the “edge frequency” Alternatively, it has been suggested that tinnitus is caused by homeostatic mechanisms, which aim to compensate reduced sensory input by reduction of inhibitory and/or increase of facilitatory mechanisms. This model predicts that changes in the processing of neuronal activity occur predominantly in the frequency range of reduced sensory input, which finally results in ongoing increased neuronal activity and/or synchrony in the respective central auditory pathways The relation between the individual audiometric slope and the perceived tinnitus pitch has been subject to different studies which have demonstrated somewhat inconsistent results. While some authors found a clear relationship between the tinnitus pitch and the edge frequency In this study we investigated in a large sample of 286 patients with tinnitus the relationship between the perceived tinnitus pitch and parameters derived from the pure tone audiogram such as the lower edge frequency and the maximum hearing loss frequency. The number of participants enabled us to perform sub-group analyses for bilateral vs. unilateral tinnitus, pure-tone vs. noise-like tinnitus, and patients with low vs. high steepness at the audiometric slope.All participating subjects gave written informed consent for analysis of their data and inclusion of their data in the Tinnitus Research Initiative (TRI) database project. The TRI database project has been approved by the ethical committee of the University of Regensburg.We analyzed patient data of the TRI database. The TRI database is a collaborative project of specialized tinnitus clinics following the approach to pool data in one international database 1st 2011 included 1392 subjects. First datasets from the Regensburg center with complete data of audiometry (0.125–8 kHz) and tinnitus matching at screening/baseline visits were selected reducing the sample to 484 subjects. In the following step patients with broad-band-noise-like tinnitus and with a tinnitus pitch above 8 kHz were excluded resulting in a data set of 286 patients and 455 ears respectively . The exclusion of patients with a tinnitus pitch above 8 kHz was motivated by the fact that audiometric data were only available up to 8 kHz. Distribution of patients with uni- and bilateral and with pure-tone and narrow-band tinnitus is shown in the bottom part of The flow of data selection is displayed in The 286 patients which were included in the analyses were 52.9±13.7 (17.1–86.5) years old; 92 (32.4%) subjects were female. Patients suffered for 90.7±93.9 (1–456.6) months from tinnitus. Ratings and questionnaires were 48.8±22.7 (4–94) for the German version of the Tinnitus Handicap Inventory Audiometry and tinnitus matching were done with a Madsen Itera  audiometer with Sennheiser HDA-200 supra-aural headphones . The hearing threshold for nine frequencies  was determined by a standard Hughson-Westlake procedure . In a second step, the frequency/pitch of the tinnitus was determined. In a first step, the pulsed tones of the audiogram where used to roughly match the tinnitus pitch and the patients were asked, whether the tinnitus sounds like a pure tone as just perceived during the audiometry, or does it sound like a broad band or a narrow band noise. To assist the comparison of the tinnitus pitch or sound quality either a pure tone or a white noise or a narrow band octave or 1/3-octave noise was presented to the ear with the tinnitus at nearly the same loudness as the tinnitus. If the pure tone threshold was too high to perceive a test signal at the side of the tinnitus, the contralateral better ear was used to present the sound. In case of a pure tone, a software sinus generator with a 1 Hz frequency resolution was then used in a bracketing procedure to match the pure tone pitch as exactly as possible. This technique is recommended for routine clinical use and seems to produce fewer octave confusions than others In case of a narrow band noise an octave or 1/3-octave filter bank with standard mid frequencies was used. The centre frequency was changed in the same way (1 Hz steps) as the bracketing procedure with pure tones until the narrow band noise matches the tinnitus pitch best. The center frequency of the matching signal was recorded as the pitch of the tinnitus.The audiogram edge was defined as the lower frequency of two neighboring frequency pairs in the audiogram with the largest steepness, calculated as L(f2)−L(f1)/log2(f2/f1) In addition, the frequency of the maximum hearing loss was evaluated for each subject. If there were several frequencies with maximum hearing loss the lowest one was used. Averaging across individuals was done accordingly to the method used for the edge. All calculations were done with MatLab® .th 2011). Data analysis was conducted according to the Standard Operating Procedure , thereby following a study-specific Statistical Analysis Plan (SAP) that was written according to the SAP template  published on http://database.tinnitusresearch.org.The data analysis was based on data of the Tinnitus Research Initiative Database. Data management was conducted according to the Data Handling Plan , tinnitus character (pure-tone vs. narrow-band tinnitus), and steepness of the slope at the audiometric edge  and repeated the analyses in these sub-groups. Thereafter, we contrasted the sub-groups  for tinnitus pitch, and frequency of maximum hearing loss. As analyses were performed in logarithmic scale, we just provide back-transformed mean data of frequency space in text and abstained from presenting standard deviations.Numeric values for the frequencies/pitches for the whole group and sub-group analyses are given in In the whole group analysis tinnitus pitch had a significantly higher frequency than the audiometric edge, but did not differ significantly from the frequency of maximum hearing loss for both ears. In a second analyzes of the sample with narrow band noise like tinnitus we replaced the center frequency of the narrow band noise with the lower bound frequency which is closer to the audiometric edge. This did not cause any changes of the result as there was again only significant correlation of the tinnitus pitch with the maximum hearing loss frequency but not with the edge frequency. Tinnitus pitch correlated positively with the frequency of maximum hearing loss, but not with the audiogram edge for the right ear. For the left ear no significant associations of pitch with edge and frequency of maximum hearing loss was found.Sub-group analyses for tinnitus laterality  and tinnitus character (pure-tone vs. narrow-band) showed results similar to the whole group analyses, i.e., tinnitus pitch had a significant higher frequency than the edge for both ears, frequency of maximum hearing loss did not differ from the pitch frequency for both ears, and frequency of maximum hearing loss was positively correlated with the tinnitus pitch for the right ear. Contrasts between unilateral and bilateral tinnitus did not reveal a significant difference for tinnitus pitch and frequency of maximum hearing loss. Also pure-tone and narrow-band tinnitus did not differ in these variables except for tinnitus pitch with a near-threshold p-value.Dividing the sample in two halves by median split according to the slope at the audiometric edge showed that tinnitus pitch and frequency of maximum hearing loss was higher for the high slope in contrast to the low slope. Again the findings were comparable to the whole group analyses. In addition, frequency of maximum hearing loss was significantly higher than tinnitus pitch for the high slope group.We did not find an association between tinnitus pitch and audiometric edge in a sample of 286 patients (455 ears), but between pitch and frequency of maximum hearing loss, as indicated by t-tests and correlations. Frequency of maximum hearing loss and tinnitus pitch was in the same frequency range and tinnitus pitch had a higher frequency than the audiometric edge. In addition, there was a significant positive correlation between tinnitus pitch and frequency of maximum hearing loss for the right ear. For the left ear, pitch and frequency of maximum hearing loss was not correlated, although both were in the same frequency range. Edge and pitch frequency were not correlated. These findings were replicated in all analyzed sub-groups, i.e., bi- and unilateral tinnitus, pure-tone vs. narrow-band tinnitus, and tinnitus with low slope at the audiometric edge. The group with a high slope showed also a correlation between pitch and frequency of maximum hearing loss for the right ear, but in addition the frequency of maximum hearing loss was higher than tinnitus pitch which was higher than the edge frequency. Thus, our results do not support a relationship between edge frequency and tinnitus pitch, but rather confirm previous studies demonstrating that the tinnitus corresponds to the frequency region of hearing loss With respect to mechanisms involved in tinnitus generation our data favours homeostatic plasticity as the decisive mechanism of tinnitus generation Hitherto, antecedent literature indicated ambiguous results regarding the relationship of audiometric edge and tinnitus pitch . Lying in the same frequency range without correlating depicts only a coarse relationship suggesting that the neurophysiological basis of tinnitus may depend on its laterality. Further support for a lateralization effect comes from epidemiological studies showing a higher proportion of left-sided unilateral tinnitus especially in tinnitus patients with hearing loss Furthermore, for the high slope tinnitus group frequency of maximum hearing loss was higher than tinnitus pitch although both measures were correlated. This is in line with animal models of noise induced tinnitus which show a tinnitus pitch above the frequency of the presented noise In summary, our findings are in line with most previous publications by indicating a relationship between tinnitus pitch and audiogram variables. The observed association of tinnitus pitch with the frequency of maximum hearing loss provides further support for the hypothesis of homeostatic plasticity as the relevant mechanism for tinnitus generation