ORCID Profile
0000-0002-8413-588X
Current Organisation
UNSW Sydney
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In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Biochemistry and Cell Biology | Receptors and Membrane Biology | Medical Devices | Physiology | Rehabilitation And Therapy: Hearing And Speech | Medical Biotechnology | Pharmaceutical Sciences | Cell Physiology | Structural Biology (incl. Macromolecular Modelling) | Biological Physics | Cellular Interactions (incl. Adhesion, Matrix, Cell Wall) | Animal Physiology - Cell | Nanomaterials | Animal Physiology - Systems | Sensory Systems | Clinical Sciences | Cell Physiology | Polymers and Plastics | Animal Physiology—Biophysics | Neurosciences | Gene Therapy | Nanobiotechnology |
Expanding Knowledge in the Biological Sciences | Hearing, vision, speech and their disorders | Expanding Knowledge in Engineering | Management of Solid Waste from Energy Activities | Respiratory System and Diseases (incl. Asthma) | Nervous system and disorders | Neurodegenerative Disorders Related to Ageing | Human Diagnostics | Medical instrumentation | Expanding Knowledge in the Medical and Health Sciences | Expanding Knowledge in Technology | Plastic Products (incl. Construction Materials) | Medical Instruments | Endocrine Organs and Diseases (excl. Diabetes) | Cancer and Related Disorders | Hearing, Vision, Speech and Their Disorders
Publisher: Elsevier BV
Date: 04-2013
Publisher: Public Library of Science (PLoS)
Date: 14-05-2014
Publisher: Elsevier BV
Date: 08-2012
Publisher: Springer US
Date: 1997
Publisher: Elsevier BV
Date: 09-2019
DOI: 10.1016/J.HEARES.2019.06.002
Abstract: This Review outlines the development of DNA-based therapeutics for treatment of hearing loss, and in particular, considers the potential to utilize the properties of recombinant neurotrophins to improve cochlear auditory (spiral ganglion) neuron survival and repair. This potential to reduce spiral ganglion neuron death and indeed re-grow the auditory nerve fibres has been the subject of considerable pre-clinical evaluation over decades with the view of improving the neural interface with cochlear implants. This provides the context for discussion about the development of a novel means of using cochlear implant electrode arrays for gene electrotransfer. Mesenchymal cells which line the cochlear perilymphatic compartment can be selectively transfected with (naked) plasmid DNA using array - based gene electrotransfer, termed 'close-field electroporation'. This technology is able to drive expression of brain derived neurotrophic factor (BDNF) in the deafened guinea pig model, causing re-growth of the spiral ganglion peripheral neurites towards the mesenchymla cells, and hence into close proximity with cochlear implant electrodes within scala tympani. This was associated with functional enhancement of the cochlear implant neural interface (lower neural recruitment thresholds and expanded dynamic range, measured using electrically - evoked auditory brainstem responses). The basis for the efficiency of close-field electroporation arises from the compression of the electric field in proximity to the ganged cochlear implant electrodes. The regions close to the array with highest field strength corresponded closely to the distribution of bioreporter cells (adherent human embryonic kidney (HEK293)) expressing green fluorescent reporter protein (GFP) following gene electrotransfer. The optimization of the gene electrotransfer parameters using this cell-based model correlated closely with in vitro and in vivo cochlear gene delivery outcomes. The migration of the cochlear implant electrode array-based gene electrotransfer platform towards a clinical trial for neurotrophin-based enhancement of cochlear implants is supported by availability of a novel regulatory compliant mini-plasmid DNA backbone (pFAR4 plasmid Free of Antibiotic Resistance v.4) which could be used to package a 'humanized' neurotrophin expression cassette. A reporter cassette packaged into pFAR4 produced prominent GFP expression in the guinea pig basal turn perilymphatic scalae. More broadly, close-field gene electrotransfer may lend itself to a spectrum of potential DNA therapeutics applications benefitting from titratable, localised, delivery of naked DNA, for gene augmentation, targeted gene regulation, or gene substitution strategies.
Publisher: Springer US
Date: 1997
Publisher: Proceedings of the National Academy of Sciences
Date: 23-01-2013
Abstract: Age-related hearing loss and noise-induced hearing loss are major causes of human morbidity. Here we used genetics and functional studies to show that a shared cause of these disorders may be loss of function of the ATP-gated P2X 2 receptor (ligand-gated ion channel, purinergic receptor 2) that is expressed in sensory and supporting cells of the cochlea. Genomic analysis of dominantly inherited, progressive sensorineural hearing loss DFNA41 in a six-generation kindred revealed a rare heterozygous allele, P2RX2 c.178G T (p.V60L), at chr12:133,196,029, which cosegregated with fully penetrant hearing loss in the index family, and also appeared in a second family with the same phenotype. The mutation was absent from more than 7,000 controls. P2RX2 p.V60L abolishes two hallmark features of P2X 2 receptors: ATP-evoked inward current response and ATP-stimulated macropore permeability, measured as loss of ATP-activated FM1-43 fluorescence labeling. Coexpression of mutant and WT P2X 2 receptor subunits significantly reduced ATP-activated membrane permeability. P2RX2 -null mice developed severe progressive hearing loss, and their early exposure to continuous moderate noise led to high-frequency hearing loss as young adults. Similarly, among family members heterozygous for P2RX2 p.V60L, noise exposure exacerbated high-frequency hearing loss in young adulthood. Our results suggest that P2X 2 function is required for life-long normal hearing and for protection from exposure to noise.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 05-2003
DOI: 10.1097/00001756-200305060-00008
Abstract: Regulation of P2X(2) receptor (P2X(2)R) expression in the rat cochlea in response to noise was analysed. Sustained loud sound (90-120 dB white noise, > 6 h), increased P2X(2)R mRNA and protein levels in rat organ of Corti and spiral ganglion (primary auditory neurones). P2X(2)R expression by the type I spiral ganglion neurones, which innervate the inner hair cells via the inner spiral plexus, was confirmed by confocal immunofluorescence. This also revealed increased P2X(2)R labelling of outer hair cell (OHC) stereocilia and cuticular plates, reflecting trafficking of greater numbers of ATP-gated ion channels assembled with P2X(2)R subunits to the transducer site. Whole-cell voltage cl of OHC confirmed the noise-induced up-regulation of ATP-gated inward currents. These data indicate that regulation of P2X(2) receptor gene expression in the cochlea is adaptive, with sustained loud sound promoting increased transcription and translation specifically at sites regulating hearing sensitivity and auditory neurotrans-mission.
Publisher: Springer Science and Business Media LLC
Date: 18-03-2019
DOI: 10.1007/S12311-019-01026-4
Abstract: Canonical transient receptor potential (TRPC) non-selective cation channels are broadly expressed by neurons, glia and the microvasculature of the brain. In neurons and astrocytes, these ion channels are coupled to group I metabotropic glutamate receptors via Gαq-phospholipase C signal transduction. In the mouse cerebellar Purkinje neurons, TRPC channels assembled as tetramers of TRPC3 subunits exclusively mediate this glutamatergic signalling mechanism and regulation of alternative splicing results in dominance of a high Ca
Publisher: Springer Science and Business Media LLC
Date: 07-11-2017
Publisher: Springer Science and Business Media LLC
Date: 13-03-2010
DOI: 10.1007/S00418-010-0686-X
Abstract: Canonical transient receptor potential type 3 (TRPC3) ion channels assemble from TRPC3 subunits and exhibit multiple activation mechanisms. TRPC3 has been proposed to contribute to Ca(2+) entry supporting Ca(2+) homeostasis in cochlear hair cells and to be activated by G protein-coupled receptor (GPCR) signaling in spiral ganglion neurons. The present study was designed to determine the spatiotemporal profile of TRPC3 expression during mouse cochlear ontogeny. TRPC3 immunofluorescence of cryosectioned cochleae was performed using E16-adult tissue. We found that prior to birth, TRPC3 expression was strongest in epithelial cells that form the cochlear partition. In the early postnatal period, to the onset of hearing (~P12), immunofluorescence was strongest in the hair cells, with increased expression in stria vascularis and Reissner's membrane. Afferent neurite labeling in inner spiral plexus and outer spiral bundles developed transiently in the perinatal period, corresponding to the critical period of synaptic consolidation, while signal in the spiral ganglion soma increased from the perinatal period through to adulthood. Compared with the late embryonic/early postnatal levels, hair cell expression was relatively weaker from the third postnatal week, whereas spiral ganglion soma labeling was stronger. In the adult, TRPC3 expression was primarily in the soma of spiral ganglion neurons, the hair cells, and the inner and outer sulcus regions. This spatiotemporal profile of TRPC3 expression was consistent with this ion channel contributing to development of sensory, neural and epithelial cochlear tissues, as well as hair cell Ca(2+) homeostasis and regulation of auditory neurotransmission via GPCR signaling.
Publisher: IEEE
Date: 07-2018
Publisher: Springer Science and Business Media LLC
Date: 2006
DOI: 10.1007/S00232-005-0835-7
Abstract: This review considers the "tween twixt and twain" of hair cell physiology, specifically the signaling elements and membrane conductances which underpin forward and reverse transduction at the input stage of hair cell function and neurotransmitter release at the output stage. Other sections of this review series outline the advances which have been made in understanding the molecular physiology of mechanoelectrical transduction and outer hair cell electromotility. Here we outline the contributions of a considerable array of ion channels and receptor signaling pathways that define the biophysical status of the sensory hair cells, contributing to hair cell development and subsequently defining the operational condition of the hair cells across the broad dynamic range of physiological function.
Publisher: Wiley
Date: 05-2022
Publisher: Bentham Science Publishers Ltd.
Date: 09-2009
Publisher: Elsevier BV
Date: 07-2009
DOI: 10.1016/J.HEARES.2009.03.009
Abstract: Signalling actions of extracellular nucleotides via P2 receptors influence cellular function in most tissues. In the inner ear, P2 receptor signaling is involved in many processes including the regulation of hearing sensitivity and the cochlea's response to noise stress. CD39 (NTPDase1/ENTPD1) is an ectonucleotidase (ecto-nucleoside triphosphate diphosphohydrolase) that can hydrolyse purine and pyrimidine nucleoside tri- and di-phosphates to generate monophosphate nucleosides. Mice null for Cd39 exhibit major alterations in haemostasis and profound alterations in inflammatory and thrombotic reactions. Studies in the cochlea have suggested the involvement of purinergic-type signals that could be modulated by CD39 in regulation of cochlear blood flow and also auditory neurotransmission. This study aimed to determine the auditory phenotype of adult Cd39 null mice on the C57BL6 background. Auditory brainstem responses (ABR) and distortion product otoacoustic emissions (DPOAE) were unaffected in Cd39-deficient mice across the range of test frequencies, suggesting normal neural and outer hair cell function. Mutant mice also showed little difference to wild type mice in vulnerability to acoustic trauma. Gene expression analysis of other membrane-bound NTPDases with comparable hydrolytic activity demonstrated an up-regulation of Entpd2 and Entpd8 in the cochleae of Cd39 deficient mice. These findings suggest that Cd39 deletion alone does not adversely affect cochlear function, possibly as compensatory up-regulation of other surface located NTPDases may offset predicted alterations in cochlear homeostasis.
Publisher: Springer Science and Business Media LLC
Date: 03-2004
Publisher: Elsevier BV
Date: 05-1998
DOI: 10.1016/S0378-5955(97)00206-2
Abstract: The adenosine 5'-triphosphate (ATP)-activated membrane conductance, mediated by P2X receptors, was examined in isolated guinea-pig cochlear inner and outer hair cells. Photo-activated release of caged-ATP elicted a 30-ms latency inwardly rectifying non-selective cation conductance, blocked by the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS 10-100 microM), consistent with the direct activation of ATP-gated ion channels. A K(Ca) conductance in the inner hair cells (IHC), activated by the entry of Ca2+ through the ATP-gated ion channels, was blocked by including 10 mM 1,2-his(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) in the internal solution. Real-time confocal slit-scanning fluorescence imaging of Na+ influx through the ATP-gated ion channels was performed using the dye Sodium Green with simultaneous whole-cell recording of membrane currents. The Na+ entry was localized to the endolymphatic surface, with the increase in [Na+]i detected within approximately 200 ms of the onset of the inward current response. Within 600 ms Na+ had diffused throughout the cell cytoplasm with the exception of the subnuclear region of the outer hair cells. Correlation of voltage-cl measurements of Na+ entry with regional increases in Na+-induced fluorescence demonstrated ATP-induced increases in intracellular Na+ in excess of 45 mM within 4 s. These data provide direct evidence for the Na+ permeability of the ATP-gated ion channels as well as independent evidence for the localization of P2X receptors at the endolymphatic surface of the sensory hair cells. The localization of the ATP-gated ion channels to the apical surface of the hair cells supports an ATP-mediated modulation of 'silent' K+ current across the cochlear partition which could regulate hearing sensitivity by controlling the transcellular driving force for both mechanoelectrical and electromechanical transduction in hair cells.
Publisher: Oxford University Press (OUP)
Date: 21-06-2012
DOI: 10.1093/CVR/CVS208
Publisher: Society for Neuroscience
Date: 28-02-2007
DOI: 10.1523/JNEUROSCI.4975-06.2007
Abstract: Deafness is the most common form of sensory impairment in the human population and is frequently caused by recessive mutations. To obtain animal models for recessive forms of deafness and to identify genes that control the development and function of the auditory sense organs, we performed a forward genetics screen in mice. We identified 13 mouse lines with defects in auditory function and six lines with auditory and vestibular defects. We mapped several of the affected genetic loci and identified point mutations in four genes. Interestingly, all identified genes are expressed in mechanosensory hair cells and required for their function. One mutation maps to the pejvakin gene, which encodes a new member of the gasdermin protein family. Previous studies have described two missense mutations in the human pejvakin gene that cause nonsyndromic recessive deafness (DFNB59) by affecting the function of auditory neurons. In contrast, the pejvakin allele described here introduces a premature stop codon, causes outer hair cell defects, and leads to progressive hearing loss. We also identified a novel allele of the human pejvakin gene in an Iranian pedigree that is afflicted with progressive hearing loss. Our findings suggest that the mechanisms of pathogenesis associated with pejvakin mutations are more erse than previously appreciated. More generally, our findings demonstrate that recessive screens in mice are powerful tools for identifying genes that control the development and function of mechanosensory hair cells and cause deafness in humans, as well as generating animal models for disease.
Publisher: Springer Berlin Heidelberg
Date: 2001
Publisher: Wiley
Date: 2007
Publisher: Wiley
Date: 2007
Publisher: S. Karger AG
Date: 2003
DOI: 10.1159/000067891
Abstract: ATP-gated ion channels assembled from P2X sub /sub subunits have been implicated in ontogeny and cellular pathology. Here, the expression of the P2X sub /sub receptor subunit was studied in the embryonic (E14–E18 days) and postnatal (P0–adult) rat cochlea using immunohistochemistry. Strong P2X sub /sub immunolabelling was observed in the primary auditory neurons of the spiral ganglion from E18 to adult and in the fibres innervating the sensory inner and outer hair cells from birth to adult. Strong immunolabelling of P2X sub /sub receptor protein was also observed in the inner and outer hair cells over a limited developmental period, from birth to P6. Weak expression was observed in cochlear duct epithelium on E18 and in the supporting cells (footplates of pillar cells in adult and in Böttcher’s cells after birth). The immunolocalisation of P2X sub /sub receptors further implicates extracellular ATP in signalling process in cochlear ontogeny and in establishment and function of auditory neurotransmission. The P2X sub /sub receptors may be involved in signal transduction and modulation as well as in regulating cell death during development and in pathological conditions.
Publisher: Elsevier BV
Date: 1990
Publisher: Springer Science and Business Media LLC
Date: 18-03-2017
DOI: 10.1007/S11064-017-2228-6
Abstract: The ketone body, β-hydroxybutyrate (βOHB), is metabolised by the brain alongside the mandatory brain fuel glucose. To examine the extent and circumstances by which βOHB can supplement glucose metabolism, we studied guinea pig cortical brain slices using increasing concentrations of [U-
Publisher: The Company of Biologists
Date: 04-2007
DOI: 10.1242/DEV.002279
Abstract: Type I and type II spiral ganglion neurons (SGN) innervate the inner and outer hair cells of the cochlea, respectively. This neural system is established by reorganization of promiscuous innervation of the hair cells,immediately before hearing is established. The mechanism for this synaptic reorganization is unresolved but probably includes regulation of trophic support between the hair cells and the neurons. We provide evidence that P2X receptors (ATP-gated ion channels) contribute such a mechanism in the neonatal rat cochlea. Single-cell quantitative RT-PCR identified the differential expression of two P2X receptor subunits, splice variant P2X2-3 and P2X3, in a 1:2 transcript ratio. Downregulation of this P2X2-3/3 receptor coincided with maturation of the SGN innervation of the hair cells. When the P2X2-3 and P2X3 subunits were co-expressed in Xenopus oocytes, the resultant P2X receptor properties corresponded to the SGN phenotype. This included enhanced sensitivity to ATP and extended agonist action. In P4 spiral ganglion explants, activation of the P2X receptor signaling pathway by ATPγS or α,βMeATP inhibited BDNF-induced neurite outgrowth and branching. These findings indicate that P2X receptor signaling provides a mechanism for inhibiting neurotrophin support of SGN neurites when synaptic reorganization is occurring in the cochlea.
Publisher: Springer Science and Business Media LLC
Date: 06-2008
Publisher: Wiley
Date: 25-02-2005
DOI: 10.1002/CNE.20442
Abstract: ATP-gated ion channels assembled from P2X3 receptor (P2X3R) subunits contribute to neurotransmission and neurotrophic signaling, associated with neurite development and synaptogenesis, particularly in peripheral sensory neurons. Here, P2X3R expression was characterized in the rat cochlea from embryonic day 16 (E16) to adult (P49-56), using RT-PCR and immunohistochemistry. P2X3R mRNA was strongly expressed in the cochlea prior to birth, declined to a minimal level at P14, and was absent in adult tissue. P2X3R protein expression was confined to spiral ganglion neurons (SGN) within Rosenthal's canal of the cochlea. At E16, immunolabeling was detected in the SGN neurites, but not the distal neurite projection within the developing sensory epithelium (greater epithelial ridge). From E18, the immunolabeling was observed in the peripheral neurites innervating the inner hair cells but was reduced by P6. However, from P2-8, immunolabeling of the SGN neurites extended to include the outer spiral bundle fiber tract beneath the outer hair cells. This labeling of type II SGN afferent fiber declined after P8. By P14, all synaptic terminal immunolabeling in the organ of Corti was absent, and SGN cell body labeling was minimal. In adult cochlear tissue, P2X3R immunolabeling was not detected. Noise exposure did not induce P2X3R expression in the adult cochlea. These data indicate that ATP-gated ion channels incorporating P2X3R subunit expression are specifically targeted to the afferent terminals just prior to the onset of hearing, and likely contribute to the neurotrophic signaling which establishes functional auditory neurotransmission.
Publisher: Wiley
Date: 06-04-2023
DOI: 10.1111/JNC.15815
Abstract: AMP‐activated protein kinase (AMPK) is a key sensor of energy balance playing important roles in the balancing of anabolic and catabolic activities. The high energy demands of the brain and its limited capacity to store energy indicate that AMPK may play a significant role in brain metabolism. Here, we activated AMPK in guinea pig cortical tissue slices, both directly with A769662 and PF 06409577 and indirectly with AICAR and metformin. We studied the resultant metabolism of [1‐ 13 C]glucose and [1,2‐ 13 C]acetate using NMR spectroscopy. We found distinct activator concentration‐dependent effects on metabolism, which ranged from decreased metabolic pool sizes at EC 50 activator concentrations with no expected stimulation in glycolytic flux to increased aerobic glycolysis and decreased pyruvate metabolism with certain activators. Further, activation with direct versus indirect activators produced distinct metabolic outcomes at both low (EC 50 ) and higher (EC 50 × 10) concentrations. Specific direct activation of β1‐containing AMPK isoforms with PF 06409577 resulted in increased Krebs cycle activity, restoring pyruvate metabolism while A769662 increased lactate and alanine production, as well as labelling of citrate and glutamine. These results reveal a complex metabolic response to AMPK activators in brain beyond increased aerobic glycolysis and indicate that further research is warranted into their concentration‐ and mechanism‐dependent impact. image
Publisher: Springer Science and Business Media LLC
Date: 11-10-2011
Abstract: The mechanisms that consolidate neural circuitry are a major focus of neuroscience. In the mammalian cochlea, the refinement of spiral ganglion neuron (SGN) innervation to the inner hair cells (by type I SGNs) and the outer hair cells (by type II SGNs) is accompanied by a 25% loss of SGNs. We investigated the segregation of neuronal loss in the mouse cochlea using β-tubulin and peripherin antisera to immunolabel all SGNs and selectively type II SGNs, respectively, and discovered that it is the type II SGN population that is predominately lost within the first postnatal week. Developmental neuronal loss has been attributed to the decline in neurotrophin expression by the target hair cells during this period, so we next examined survival of SGN sub-populations using tissue culture of the mid apex-mid turn region of neonatal mouse cochleae. In organotypic culture for 48 hours from postnatal day 1, endogenous trophic support from the organ of Corti proved sufficient to maintain all type II SGNs however, a large proportion of type I SGNs were lost. Culture of the spiral ganglion as an explant, with removal of the organ of Corti, led to loss of the majority of both SGN sub-types. Brain-derived neurotrophic factor (BDNF) added as a supplement to the media rescued a significant proportion of the SGNs, particularly the type II SGNs, which also showed increased neuritogenesis. The known decline in BDNF production by the rodent sensory epithelium after birth is therefore a likely mediator of type II neuron apoptosis. Our study thus indicates that BDNF supply from the organ of Corti supports consolidation of type II innervation in the neonatal mouse cochlea. In contrast, type I SGNs likely rely on additional sources for trophic support.
Publisher: Society for Neuroscience
Date: 31-01-2019
DOI: 10.1523/JNEUROSCI.3948-06.2007
Abstract: ATP is released during hypoxia from the ventrolateral medulla (VLM) and activates purinergic P2 receptors (P2Rs) at unknown loci to offset the secondary hypoxic depression of breathing. In this study, we used rhythmically active medullary slices from neonatal rat to map, in relation to anatomical and molecular markers of the pre-Bötzinger complex (preBötC) (a proposed site of rhythm generation), the effects of ATP on respiratory rhythm and identify the P2R subtypes responsible for these actions. Unilateral microinjections of ATP in a three-dimensional grid within the VLM revealed a “hotspot” where ATP (0.1 m m ) evoked a rapid 2.2 ± 0.1-fold increase in inspiratory frequency followed by a brief reduction to 0.83 ± 0.02 of baseline. The hotspot was identified as the preBötC based on histology, overlap of injection sites with NK1R immunolabeling, and potentiation or inhibition of respiratory frequency by SP ([Sar 9 -Met(O 2 ) 11 ]-substance P) or DAMGO ([ d -Ala 2 , N -MePhe 4 ,Gly-ol 5 ]-enkephalin), respectively. The relative potency of P2R agonists [2MeSADP (2-methylthioadenosine 5′-diphosphate) ≈ 2MeSATP (2-methylthioadenosine 5′-triphosphate) ≈ ATPγs (adenosine 5′-[γ-thio]triphosphate tetralithium salt) ≈ ATP ≫ UTP ≈ αβmeATP (α,β-methylene-adenosine 5′-triphosphate)] and attenuation of the ATP response by MRS2179 (2′-deoxy- N 6 -methyladenosine-3′,5′-bisphosphate) (P2Y 1 antagonist) indicate that the excitation is mediated by P2Y 1 Rs. The post-ATP inhibition, which was never observed in response to ATPγs, is dependent on ATP hydrolysis. These data establish in neonatal rats that respiratory rhythm generating networks in the preBötC are exquisitely sensitive to P2Y 1 R activation, and suggest a role for P2Y 1 Rs in respiratory motor control, particularly in the P2R excitation of rhythm that occurs during hypoxia.
Publisher: S. Karger AG
Date: 2002
DOI: 10.1159/000046853
Publisher: Elsevier
Date: 1992
Publisher: Springer Science and Business Media LLC
Date: 06-2010
Publisher: IEEE
Date: 08-2016
Publisher: Springer Science and Business Media LLC
Date: 12-2001
Abstract: In mammals, sound transduction by inner hair cells (IHC) generates a receptor potential whose litude and phase drive auditory nerve firing. The membrane filter properties that define the input-output function of IHC are derived from membrane conductance and capacitance. These elements of the membrane filter were quantified using whole-cell voltage cl of IHC from the four turns of the guinea pig cochlea. IHC membrane properties were remarkably constant along the cochlea, in contrast with all other auditory hair cell systems, and suggests that extrinsic processes such as the active filter provided by the outer hair cells are matched to a constant transfer function of the IHC. Two outwardly rectifying K+ currents contribute to the IHC membrane conductance. These combined currents activate at approximately -55 mV. IHC mean input resistance was 140 M ohm and capacitance was 10.0 pF, generating a membrane time constant of 1.4 ms or a corner frequency of approximately 115 Hz, which is consistent with reported low-frequency roll-off of the IHC AC receptor potential in vivo. Approximately 40% of the 313-1 nS total K+ conductance about 0 mV was attributed to charybdotoxin-sensitive K(Ca) channels (also sensitive to cell dialysis with the Ca2+ chelator BAPTA or removal of extracellular Ca2+). The only known ligand-activated conductance in mature IHC, the P2X receptor conductance, averaged 31 nS (activated by 400 microM ATP about -75 mV) irrespective of cell origin. Thus, regulation of intracellular Ca2+ and activation of P2X receptors by extracellular ATP provide capacity for local dynamic fine-tuning of the IHC membrane filter.
Publisher: Elsevier BV
Date: 08-1996
Publisher: Wiley
Date: 03-1992
DOI: 10.1113/JPHYSIOL.1992.SP019030
Abstract: 1. Whole-cell currents were measured in outer hair cells isolated from each turn of the organ of Corti of the guinea-pig. 2. The slope input conductances at -70 mV of the cells ranged from 3.6 to 51 nS depending on the length of the cell. Shorter cells from the basal turns of the cochlea had the highest values. The membrane time constant of the cells varied from 3 to 0.2 ms from the apex to the base. 3. Irrespective of the position of the cells along the cochlea, three distinct currents were found. Each type of current was found in approximately the same proportion in all cells. 4. An outward K+ current was present which activated at potentials more positive than -35 mV. The current was sensitive to tetraethylammonium (30 mM), quinidine (100 microM) and nifedipine (50 microM). It could be removed by replacing external Ca2+ with Ba2+ or Mg2+. The current was also removed by substituting Nai+ or Csi+ for Ki+ pipette solution. This outwardly rectifying current appears similar to the calcium-activated K+ current described in other hair cells. 5. The main current present at membrane potentials from -90 mV to -50 mV was a second voltage-activated K+ current. It was 50% activated at -80 mV, and relaxed with a time constant of 20-40 ms on hyperpolarization to -120 mV. Near rest the kinetics were essentially time-dependent , but depended upon the external K+ concentration. The current was blocked by 5 mM external Cs+. 6. This current was highly selective for K+. Measured from reversal of the tail currents, the permeability ratio PK:PNa was approximately 30:1. Depolarization of the cell, presumed to lead to an elevation of intracellular calcium, produced a prolonged activation of the current. 7. A third current found in the cells was a cation current. By external ion replacement, the selectivity sequence was determined to be Ca2+ greater than Na+ approximately equal to K+ greater than choline+ greater than NMDG+ (respective permeabilities relative to Na: 2.9, 1.0, 0.99, 0.63 and 0.37). This current was reduced by external Ba2+ (3 mM) and by nifedipine (50 microM). The activation of this current appeared to depend upon raised levels of Cai2+. 8. These currents account for reported in vivo properties of cochlear outer hair cells as cells permeable to potassium at large negative resting potentials. The consequences for sound detection in the cochlea are briefly discussed.
Publisher: Springer Science and Business Media LLC
Date: 07-02-2007
DOI: 10.1007/S00441-006-0374-2
Abstract: Adenosine is a constitutive cell metabolite that can be released from cells via specific bi-directional transporters and is an end-point for nucleotide hydrolysis. In the extracellular space, adenosine becomes a signalling molecule for P1 (adenosine) receptors that modulate physiological responses in a wide range of mammalian tissues. Whereas adenosine signalling has been implicated in the regulation of cochlear blood flow and in cochlear protection from oxidative damage, the potential roles for adenosine signalling in the modulation of sound transduction and auditory neurotransmission have not been established. We have characterised the expression and distribution of adenosine receptors in the rat cochlea. mRNA transcripts for all four subtypes of adenosine receptors (A(1), A(2A), A(2B) and A(3)) were detected in dissected cochlear tissue by using reverse transcription olymerase chain reaction analysis. The protein distribution for the A(1), A(2A) and A(3) receptor subtypes was identified by immunoperoxidase histochemistry and confocal immunofluorescence labelling. These receptors were differentially expressed in the organ of Corti, spiral ganglion neurones, lateral wall tissues and cochlear blood vessels. The distribution of adenosine receptors in sensory and neural tissues and in the vasculature coincided with other elements of purinergic signalling (P2X and P2Y receptors, ectonucleotidases), consistent with the integrative regulation of many physiological processes in the cochlea by extracellular nucleotides and nucleosides. Our study provides a framework for further investigation of adenosine signalling in the inner ear, including putative roles in oxidative stress responses.
Publisher: Elsevier BV
Date: 10-1995
DOI: 10.1016/0378-5955(95)00152-3
Abstract: There is strong evidence for the presence of P2 purinoceptors on cochlear tissues, but the role of extracellular ATP in cochlear function is still unclear. Our previous studies have determined the presence of ATP in the cochlear fluids and indicated that the purinoceptors are substantially localized to the tissues lining the endolymphatic compartment. This implies that extracellular ATP may have an humoral role confined to the endolymphatic space. In order to study the influence of extracellular ATP in the endolymphatic space, a series of studies were undertaken in which ATP (10 microM to 10 mM) in artificial endolymph (EL) (test solution: 2-12.5 nl) was injected into the scala media and the effect on the cochlear microphonic (CM) and endocochlear potential (EP) evaluated. A double-barrelled pipette, with one barrel containing the test solution and the other artificial EL (control solution) was inserted into scala media of the third turn of the guinea-pig cochlea. A known volume (2-12.5 nl) of test or control solution was then pressure-injected into the space. ATP had a significant dose-dependent suppressive effect on both EP and CM with a threshold of approximately 2 x 10(-14) mol the response was readily reversible, also in a dose-dependent fashion. Artificial EL of the same volume had no effect on EP and CM. The ATP effect on EP was blocked by the P2 purinoceptor antagonists suramin and reactive blue 2 (RB2). Neither adenosine (2 x 10(-13) to 2 x 10(-11) mol) nor suramin or RB2 on their own had any effect on EP and CM. This study provides the first evidence for an effect of extracellular ATP in the endolymphatic compartment on cochlear function which is mediated via P2 purinoceptors. This provides supporting evidence for an humoral role for extracellular ATP in the modulation of cochlear function.
Publisher: Elsevier BV
Date: 03-2002
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 03-2004
DOI: 10.1097/00001756-200403010-00011
Abstract: Developmental expression of two-pore domain potassium (2P K) channels, TASK-1 and TREK-1, was investigated in the rat cochlea at onset of hearing and after maturity using RT-PCR and immunocytochemistry. TASK-1 and TREK-1 mRNAs were detected by RT-PCR at postnatal day (P) 9-12. TASK-1 like immunoreactivity (LIR) in the P13 cochlea was observed in Deiters', pillar, Claudius' and outer sulcus cells, spiral limbus fibrocytes, and neuroglia. At P13, TREK-1-LIR was more wide-spread, and included sensory and supporting cells of the organ of Corti, spiral ganglion, stria vascularis, Reissner's membrane, inner and outer sulcus cells, connective and support tissues surrounding modiolus. By P105 the pattern of TASK-1- and TREK-1-LIR became limited to a subset of the above structures, suggesting developmental regulation. During postnatal development, TASK-1 may be important in the onset (around P11) and maturation (by P22) of endocochlear potential and hearing. The distribution of TASK-1 and TREK-1 suggest a role in K cycling and homeostasis. As TASK-1 and TREK-1 are inhibited by local anesthetics at doses used to treat tinnitus, 2P K channels may also be important in cochlear dysfunction.
Publisher: Springer Science and Business Media LLC
Date: 11-02-2015
DOI: 10.1038/NPP.2015.47
Publisher: Springer New York
Date: 2014
Publisher: S. Karger AG
Date: 2002
DOI: 10.1159/000058307
Abstract: Adenosine triphosphate (ATP) is a major intercellular signalling molecule that is involved in neurotransmission in the central and autonomic nervous systems, regulation of blood flow, and neuroendocrine function. It is also a key signalling molecule involved in normal cochlear homoeostasis, regulating hearing sensitivity, controlling vascular tone and acting as a candidate neurotransmitter at the hair cell afferent synapses. It has also been established that extracellular ATP mediates some pathological processes such as inflammation, apoptosis and cell proliferation. Evidence for a profound influence of extracellular ATP on normal cochlear function offers the tantalising possibility that extracellular purine nucleotides may play a role in disease processes in the inner ear. This review draws on the current understanding of the pathophysiological role of extracellular ATP in tissues, and the evidence for the functional expression of purinergic signalling elements in the inner ear, to speculate on the potential role of purine nucleotides in cochlear pathology.
Publisher: Elsevier BV
Date: 04-1995
DOI: 10.1016/0378-5955(95)00024-X
Abstract: Two different radioligands were used to identify extracellular ATP binding sites specific to P2 purinoceptors in guinea-pig cochlear tissue. Deoxyadenosine 5'-(alpha-[35S]thio)triphosphate ([35S]dATP alpha S 10 nM) provided a high activity probe for the P2y purinoceptor subtype on the basis of selective block by 2-methylthio-ATP (2MeSATP 100 microM). [3H]alpha, beta-methylene-ATP (10 nM), a high affinity probe for a P2x purinoceptor subtype was selectively blocked by inclusion of the related compound beta, gamma-methylene-ATP (100 microM). Both probes labelled the organ of Corti, stria vascularis and spiral prominence regions. The P2x purinoceptor probe also bound to lateral wall tissue below the spiral prominence and insertion point of the basilar membrane within the scala tympani compartment, a region which failed to show significant binding using [35S]dATP alpha S. Frozen sections of whole cochlea permitted analysis of radioligand binding to the cell body region (spiral ganglion in Rosenthal's canal) of the primary auditory afferents and the auditory nerve itself, which lies within the central region of the modiolus of the cochlea. Both these regions exhibited 2MeSATP blockable [35S]dATP alpha S binding whereas specific [3H]alpha, beta-methylene-ATP binding was absent from spiral ganglion and minimal in the auditory nerve region. These results demonstrate a mixed P2 purinoceptor distribution in cochlear tissues and suggest that complex purine-mediated neurohumoral mechanisms may influence cochlear function at a number of sites.
Publisher: Frontiers Media SA
Date: 23-02-2016
Publisher: Elsevier BV
Date: 12-1999
DOI: 10.1016/S0378-5955(99)00151-3
Abstract: Our previous studies have determined the presence of adenosine 5'-triphosphate (ATP) in the cochlear fluids and shown that extracellular ATP introduced into the endolymphatic compartment of the guinea pig cochlea has a significant dose-dependent suppressive effect on both endocochlear potential (EP) and cochlear microphonic (CM), which is mediated via P2 receptors. In the present study, the influence of P2 receptor agonists and antagonists on this suppressive effect was investigated to characterise the subtypes of P2 receptor mediating the ATP-induced effect on cochlear function. Using a double-barreled pipette attached to a pressure injector, small volumes (2-10 nl) of ATP (0.01-1 mM) and P2 receptor agonists or P2 receptor antagonists in artificial endolymph were introduced into the scala media of the first (basal) and third turns of the guinea pig cochlea, while the EP and CM were monitored. ATP and P2 receptor agonists (5x10(-14)-1x10(-11)cibacron blue. Neither adenosine nor uridine 5'-triphosphate (2x10(-13)-2x10(-11) moles) nor the P2 receptor antagonists on their own had any effect on EP and CM. The ATP effect on the potentials was greater at the third cochlear turn when compared to the first turn. These results provide evidence that in the endolymphatic compartment of the guinea pig, the extracellular ATP effect on cochlear function is likely mediated through an interaction with P2 receptors which assemble as ATP-gated ion channels.
Publisher: Springer Science and Business Media LLC
Date: 26-09-2007
DOI: 10.1007/S00418-007-0340-4
Abstract: Peripherin is a 57 kDa Type III intermediate filament protein associated with neurite extension, neuropathies such as amyotrophic lateral sclerosis, and cranial nerve and dorsal root projections. However, knowledge of peripherin expression in the CNS is limited. We have used immunoperoxidase histochemistry to characterise peripherin expression in the mouse hindbrain, including the inferior colliculus, pons, medulla and cerebellum. Peripherin immunolabelling was observed in the nerve fibres and nuclei that are associated with all cranial nerves [(CN) V-XII] in the hindbrain. Peripherin expression was prominent in the cell bodies and axons of the mesenchephalic trigeminal nucleus and the pars compacta region of nucleus ambiguus, and in the fibres that comprise the solitary tract, the descending spinal trigeminal tract and the trigeminal and facial nerves. A small proportion of peripherin positive fibres in CN VIII likely arise from cochlear type II spiral ganglion neurons. Peripherin positive fibres were also observed in the inferior cerebellar peduncle and folia in the intermediate zone of the cerebellum. Antibody specificity was confirmed by absence of labelling in hindbrain tissue from peripherin knockout mice. This study shows that in the adult mouse hindbrain, peripherin is expressed in discrete neuronal subpopulations that have sensory, motor and autonomic functions.
Publisher: Frontiers Media SA
Date: 26-01-2021
DOI: 10.3389/FNCEL.2020.626610
Abstract: Aminoacyl-tRNA synthetases (ARSs) accurately charge tRNAs with their respective amino acids. As such, they are vital for the initiation of cytosolic and mitochondrial protein translation. These enzymes have become increasingly scrutinized in recent years for their role in neurodegenerative disorders caused by the mutations of ARS-encoding genes. This review focuses on two such genes— DARS1 and DARS2 —which encode cytosolic and mitochondrial aspartyl-tRNA synthetases, and the clinical conditions associated with mutations of these genes. We also describe attempts made at modeling these conditions in mice, which have both yielded important mechanistic insights. L eukoencephalopathy with b rainstem and s pinal cord involvement and l actate elevation (LBSL) is a disease caused by a range of mutations in the DARS2 gene, initially identified in 2003. Ten years later, h ypomyelination with b rainstem and s pinal cord involvement and l eg spasticity (HBSL), caused by mutations of cytosolic DARS1 , was discovered. Multiple parallels have been drawn between the two conditions. The Magnetic Resonance Imaging (MRI) patterns are strikingly similar, but still set these two conditions apart from other leukodystrophies. Clinically, both conditions are characterized by lower limb spasticity, often associated with other pyramidal signs. However, perhaps due to earlier detection, a wider range of symptoms, including peripheral neuropathy, as well as visual and hearing changes have been described in LBSL patients. Both HBSL and LBSL are spectrum disorders lacking genotype to phenotype correlation. While the fatal phenotype of Dars1 or Dars2 single gene deletion mouse mutants revealed that the two enzymes lack functional redundancy, further pursuit of disease modeling are required to shed light onto the underlying disease mechanism, and enable examination of experimental treatments, including gene therapies.
Publisher: Elsevier BV
Date: 09-2016
DOI: 10.1016/J.NEULET.2016.08.007
Abstract: Frontotemporal dementia (FTD) presents clinically with behavioral changes including disinhibition. Mutations in the tau-encoding MAPT gene identified in familial cases of FTD have been used to generate transgenic mouse models of the human condition. Here, we report behavioral changes in a recently developed P301S mutant tau transgenic mouse, including disinhibition-like behavior in the elevated plus maze and hyperactivity in the open field arena. Furthermore, histological analysis revealed the amygdala as a primary and early site of pathological tau deposition in these mice. Taken together, neuropathological and behavioral changes in P301S tau transgenic mice resemble features of human FTD.
Publisher: Springer Science and Business Media LLC
Date: 12-2012
Abstract: During development, excess synapses form between the central and peripheral nervous systems that are then eliminated to achieve correct connectivity. In the peripheral auditory system, the developing type I spiral ganglion afferent fibres undergo a dramatic re-organisation, initially forming connections with both sensory inner hair cells (IHCs) and outer hair cells (OHCs). The OHC connections are then selectively eliminated, leaving sparse innervation by type II afferent fibres, whilst the type I afferent synapses with IHCs are consolidated. We examined the molecular makeup of the synaptic contacts formed onto the IHCs and OHCs during this period of afferent fibre remodelling. We observed that presynaptic ribbons initially form at all the afferent neurite contacts, i.e. not only at the expected developing IHC-type I fibre synapses but also at OHCs where type I fibres temporarily contact. Moreover, the transient contacts forming onto OHCs possess a broad set of pre- and postsynaptic proteins, suggesting that functional synaptic connections are formed prior to the removal of type I fibre innervation. AMPA-type glutamate receptor subunits were transiently observed at the base of the OHCs, with their downregulation occurring in parallel with the withdrawal of type I fibres, dispersal of presynaptic ribbons, and downregulation of the anchoring proteins Bassoon and Shank. Conversely, at developing type I afferent IHC synapses, the presence of pre- and postsynaptic scaffold proteins was maintained, with differential plasticity in AMPA receptor subunits observed and AMPA receptor subunit composition changing around hearing onset. Overall our data show a differential balance in the patterns of synaptic proteins at developing afferent IHC versus OHC synapses that likely reflect their stable versus transient fates.
Publisher: Wiley
Date: 14-08-2000
DOI: 10.1046/J.1440-1681.2000.03314.X
Abstract: 1. Electrochemical homeostasis, sound transduction and auditory neurotransmission in the cochlea are influenced by extracellular purines and pyrimidines. 2. Evidence that ATP and related nucleotides influence inner ear function arises from a considerable number of cellular, molecular and physiological studies in vitro and in vivo. 3. With a full understanding of these processes, which include ionotropic (P2X receptor) and metabotropic (P2Y receptor) signal transduction pathways, signal termination involving ecto-nucleotidases and recycling via nucleoside transporters, exciting possibilities emerge for treating hearing disorders, such as Meniere's disease, tinnitus and sensorineural deafness.
Publisher: Cold Spring Harbor Laboratory
Date: 06-11-2021
DOI: 10.1101/2021.11.06.467554
Abstract: The medial olivocochlear (MOC) efferent feedback circuit projecting to the cochlear outer hair cells (OHCs) confers protection from noise-induced hearing loss and is generally thought to be driven by inner hair cell (IHC) - type I spiral ganglion afferent (SGN) input. Knockout of the Prph gene ( Prph KO) encoding the peripherin type III intermediate filament disrupted the OHC - type II SGN innervation and virtually eliminated MOC – mediated contralateral suppression from noise delivered to the opposite ear, measured as a reduction in cubic distortion product otoacoustic emissions. Electrical stimulation of the MOC pathway elicited contralateral suppression indistinguishable between wildtype (WT) and Prph KO mice, indicating that the loss of contralateral suppression was not due to disruption of the efferent arm of the circuit IHC – type I SGN input was also normal, based on auditory brainstem responses. High-intensity, broadband noise (108 dB SPL, 1 hour) produced permanent hearing loss in Prph KO mice, but not in WT littermates. These findings associate OHC-type II input with MOC efferent - based otoprotection at loud sound levels.
Publisher: Springer Science and Business Media LLC
Date: 31-03-2022
DOI: 10.1007/S11064-022-03582-4
Abstract: The leukodystrophy Hypomyelination with Brainstem and Spinal cord involvement and Leg spasticity (HBSL) is caused by recessive mutations of the DARS1 gene, which encodes the cytoplasmic aspartyl-tRNA synthetase. HBSL is a spectrum disorder with disease onset usually during early childhood and no available treatment options. Patients display regression of previously acquired motor milestones, spasticity, ataxia, seizures, nystagmus, and intellectual disabilities. Gene-function studies in mice revealed that homozygous Dars1 deletion is embryonically lethal, suggesting that successful modelling of HBSL requires the generation of disease-causing genocopies in mice. In this study, we introduced the pathogenic DARS1 M256L mutation located on exon nine of the murine Dars1 locus. Despite causing severe illness in humans, homozygous Dars1 M256L mice were only mildly affected. To exacerbate HBSL symptoms, we bred Dars1 M256L mice with Dars1 -null ‘enhancer’ mice. The Dars1 M256L/− offspring displayed increased embryonic lethality, severe developmental delay, reduced body weight and size, hydrocephalus, anophthalmia, and vacuolization of the white matter. Remarkably, the Dars1 M256L/− genotype affected energy metabolism and peripheral organs more profoundly than the nervous system and resulted in reduced body fat, increased respiratory exchange ratio, reduced liver steatosis, and reduced hypocellularity of the bone marrow. In summary, homozygous Dars1 M256L and compound heterozygous Dars1 M256L/− mutation genotypes recapitulate some aspects of HBSL and primarily manifest in developmental delay as well as metabolic and peripheral changes. These aspects of the disease might have been overlooked in HBSL patients with severe neurological deficits but could be included in the differential diagnosis of HBSL in the future.
Publisher: Elsevier BV
Date: 03-2017
DOI: 10.1016/J.HEARES.2016.12.015
Abstract: Our previous studies have shown that the stimulation of A
Publisher: Springer Science and Business Media LLC
Date: 07-09-2017
DOI: 10.1038/S41467-017-00618-0
Abstract: Neuronal excitotoxicity induced by aberrant excitation of glutamatergic receptors contributes to brain damage in stroke. Here we show that tau-deficient (tau −/− ) mice are profoundly protected from excitotoxic brain damage and neurological deficits following experimental stroke, using a middle cerebral artery occlusion with reperfusion model. Mechanistically, we show that this protection is due to site-specific inhibition of glutamate-induced and Ras/ERK-mediated toxicity by accumulation of Ras-inhibiting SynGAP1, which resides in a post-synaptic complex with tau. Accordingly, reducing SynGAP1 levels in tau −/− mice abolished the protection from pharmacologically induced excitotoxicity and middle cerebral artery occlusion-induced brain damage. Conversely, over-expression of SynGAP1 prevented excitotoxic ERK activation in wild-type neurons. Our findings suggest that tau mediates excitotoxic Ras/ERK signaling by controlling post-synaptic compartmentalization of SynGAP1.
Publisher: Wiley
Date: 08-2005
DOI: 10.1111/J.1460-9568.2005.04261.X
Abstract: Motoneurons of the compact ision of the nucleus ambiguus (cNA) are the final output neurons of the swallowing pattern generator. Thus, their normal function is critical to neonatal survival. To explore the role of purinergic signaling in modulating the excitability of these motoneurons during development, immunohistochemical and whole-cell recording techniques were used to characterize expression patterns of ionotropic P2X receptors and the effects of ATP on cNA motoneurons. Medullary slices containing the cNA were prepared from neonatal (P0-4) and juvenile (P15-21) rats. In neonatal cNA motoneurons, local application of 1 mM ATP produced a large (-133 +/- 17 pA n = 78), desensitizing, inward current that was mimicked by 1 mM alpha,beta meATP and 2meSATP, and inhibited by the P2 antagonist, PPADS (5 microM), and the P2X3 antagonist, A-317481 (0.1-1 mM). In juvenile cNA motoneurons, 1 mM ATP produced negligible currents, while 10 mM ATP produced small (-59 +/- 14 pA n = 42), primarily non-desensitizing currents. Immunohistochemistry demonstrated that in the neonate, the expression of P2X3 was robust, P2X2 and P2X5 moderate, P2X4 and P2X6 weak, and P2X1 absent. In the juvenile cNA, only low levels of P2X5 and P2X6 labeling were detected. These data indicate that P2X receptors in cNA motoneurons are profoundly downregulated during the first two postnatal weeks, and suggest a role for the purinoceptor system, particularly P2X3 receptors, in the control of esophageal motor networks during early postnatal periods.
Publisher: Elsevier BV
Date: 10-1995
DOI: 10.1016/0378-5955(95)00153-5
Abstract: The concentration of adenosine 5'-triphosphate (ATP) in endolymph (EL), perilymph (PL) and cerebrospinal fluid (CSF), collected from anesthetized guinea pigs was determined using the luciferase-luciferin reaction. The cochlea was exposed by a ventrolateral approach and the bone overlying scala media of the third turn (EL) or scala vestibuli of the first turn (PL) was shaved to a thin layer and a small fenestrum made. For EL s ling, a double-barrelled pipette was inserted through the spiral ligament-stria vascularis complex. One barrel was filled with 150 mM KCl to record the endocochlear potential (EP) and upon the appearance of the positive EP, 0.12-1.22 microliter of fluid was aspirated into the other barrel by gentle negative pressure. For PL s ling, a single-barrelled pipette was advanced into scala vestibuli and 0.3-1.6 microliter of fluid was collected by capillarity. CSF (0.36-1.75 microliter) was obtained from the cisterna magna. The cochleae were removed and processed for light microscopy to determine the extent of tissue damage from the s ling procedure. ATP concentrations (mean +/- SEM, nM) for EL, PL and CSF were 12.95 +/- 2.4 (n = 10), 10.5 +/- 3.9 (n = 11) and 16.1 +/- 5.4 (n = 11) respectively. Differences in ATP concentrations among fluids were not statistically significant. To test the effect of hypoxia on ATP levels, a group of guinea pigs was subjected to a 90 s period of respiratory anoxia prior to s ling of EL, PL or CSF. ATP concentrations were 14.4 +/- 3.5 (n = 11), 20.7 +/- 4.1 (n = 10) and 13.5 +/- 4.6 (n = 4) for EL, PL and CSF, respectively only PL ATP concentrations were statistically different (P = 0.018, Wilcoxon rank sum test) to basal conditions. This is the first study which demonstrates the presence of free ATP in cochlear fluids. The results indicate that ATP is present in cochlear fluids at concentrations close to those known to cause hair cell depolarization in vitro.
Publisher: Springer Science and Business Media LLC
Date: 31-10-2009
DOI: 10.1007/S00418-009-0653-6
Abstract: Canonical transient receptor potential (TRPC) subunits assemble as tetramers to form ion channels with high calcium (Ca(2+)) permeability. Here, we investigated the possibility that TRPC3 ion channels are broadly expressed in the adult guinea pig and mouse cochleae. Using immunofluorescence, pronounced labeling occurred in the spiral ganglion (SG) neurons, inner hair cells (IHC), outer hair cells (OHC) and epithelial cells lining scala media. TRPC3 expression was homogeneous in the SG throughout the cochlea. In contrast, there was marked spatial variation in the immunolabeling in the cochlear hair cells with respect to location. This likely relates to the tonotopy of these cells. TRPC3 immunolabeling was more pronounced in the IHC than OHC. Both basal region IHC and OHC had higher TRPC3 expression levels than the corresponding cells from the apical region of the cochlea. These data suggest that TRPC3 ion channels contribute to Ca(2+) homeostasis associated with the hair cells, with higher ion fluxes in more basal regions of the cochlea, and may also be a significant pathway for Ca(2+) entry associated with auditory neurotransmission via the SG neurons. TRPC3 expression was also identified within the spiral limbus region, inner and outer sulcus, but without evidence for spatial variation in expression level. Expression in these gap junction-coupled epithelial cells lining scala media is indicative of a contribution of TRPC3 channels to cochlear electrochemical homeostasis.
Publisher: Wiley
Date: 12-02-2007
Publisher: Springer Science and Business Media LLC
Date: 12-2014
Publisher: Springer Science and Business Media LLC
Date: 30-01-2016
DOI: 10.1038/GT.2016.8
Publisher: Frontiers Media SA
Date: 20-03-2018
Publisher: Frontiers Media SA
Date: 12-05-2023
DOI: 10.3389/FNINS.2023.1182845
Abstract: Aminoacyl-tRNA synthetases (ARSs) play an essential role in protein synthesis, being responsible for ligating tRNA molecules to their corresponding amino acids in a reaction known as ‘tRNA aminoacylation’. Separate ARSs carry out the aminoacylation reaction in the cytosol and in mitochondria, and mutations in almost all ARS genes cause pathophysiology most evident in the nervous system. Dominant mutations in multiple cytosolic ARSs have been linked to forms of peripheral neuropathy including Charcot-Marie-Tooth disease, distal hereditary motor neuropathy, and spinal muscular atrophy. This review provides an overview of approaches that have been employed to model each of these diseases in vivo , followed by a discussion of the existing animal models of dominant ARS disorders and key mechanistic insights that they have provided. In summary, ARS disease models have demonstrated that loss of canonical ARS function alone cannot fully account for the observed disease phenotypes, and that pathogenic ARS variants cause developmental defects within the peripheral nervous system, despite a typically later onset of disease in humans. In addition, aberrant interactions between mutant ARSs and other proteins have been shown to contribute to the disease phenotypes. These findings provide a strong foundation for future research into this group of diseases, providing methodological guidance for studies on ARS disorders that currently lack in vivo models, as well as identifying candidate therapeutic targets.
Publisher: The Company of Biologists
Date: 15-08-2007
DOI: 10.1242/DEV.001925
Abstract: The adult mammalian cochlea receives dual afferent innervation: the inner sensory hair cells are innervated exclusively by type I spiral ganglion neurons (SGN), whereas the sensory outer hair cells are innervated by type II SGN. We have characterized the spatiotemporal reorganization of the dual afferent innervation pattern as it is established in the developing mouse cochlea. This reorganization occurs during the first postnatal week just before the onset of hearing. Our data reveal three distinct phases in the development of the afferent innervation of the organ of Corti: (1) neurite growth and extension of both classes of afferents to all hair cells (E18-P0) (2) neurite refinement, with formation of the outer spiral bundles innervating outer hair cells (P0-P3) (3) neurite retraction and synaptic pruning to eliminate type I SGN innervation of outer hair cells, while retaining their innervation of inner hair cells (P3-P6). The characterization of this developmental innervation pattern was made possible by the finding that tetramethylrhodamine-conjugated dextran (TMRD) specifically labeled type I SGN. Peripherin and choline-acetyltransferase immunofluorescence confirmed the type II and efferent innervation patterns, respectively, and verified the specificity of the type I SGN neurites labeled by TMRD. These findings define the precise spatiotemporal neurite reorganization of the two afferent nerve fiber populations in the cochlea, which is crucial for auditory neurotransmission. This reorganization also establishes the cochlea as a model system for studying CNS synapse development, plasticity and elimination.
Publisher: Elsevier BV
Date: 07-1987
DOI: 10.1016/0306-4522(87)90214-4
Abstract: Transganglionic transport of horseradish peroxidase or lectin-conjugated horseradish peroxidase from an application site in the cervical trunk of the glossopharyngeal (IXth cranial) nerve of the rat produced extraperikaryal reaction product characteristic of axon terminal processes in three regions of the brain stem: (1) the nucleus of the tractus solitarius, from approximately 2.5 mm rostral to the obex to approximately 3 mm caudal to the obex (2) the spinal trigeminal nucleus at the level of obex (3) the cuneate fasciculus, approximately 3 mm caudal to the obex. In contrast, labelling of the carotid sinus nerve, a branch of the glossopharyngeal nerve which conveys chemoreceptor and baroreceptor afferent fibers from the carotid bifurcation, revealed a restricted central projection to within 1 mm of the obex and corresponding to the intermediate region of the glossopharyngeal nerve projection to the nucleus of the tractus solitarius. Two distinct aggregations of label were observed: (1) rostral to the obex, within the lateral and dorsomedial subnuclei of the nucleus of the tractus solitarius (2) caudal to the obex, within the commissural and ventrolateral subnuclei of the nucleus of the tractus solitarius. Between these two sites the density of labelling was reduced. Retrogradely labelled neurons were demonstrated in the inferior salivatory nucleus and in the nucleus ambiguus after application of lectin-conjugated horseradish peroxidase to the glossopharyngeal nerve. Of the labelled neurons in the nucleus ambiguus (approximately 100), 25% contributed fibers to the carotid sinus nerve. The concentration of extraperikaryal reaction product located rostral to the obex after labelling of the carotid sinus nerve closely matches descriptions of the region of afferent terminations from carotid and aortic baroreceptors in the cat. The concentration of label caudal to the obex may therefore correspond to the region of afferent terminations from carotid chemoreceptors. This study may therefore provide some basis for a separation of the central synapses of primary afferent fibers from the carotid baroreceptors and chemoreceptors in the rat. The labelled neurons of the nucleus ambiguus provide the anatomical substrate for centrifugal control of carotid chemoreceptor activity.
Publisher: Springer International Publishing
Date: 2017
Publisher: Elsevier BV
Date: 03-2009
DOI: 10.1016/J.TINS.2009.01.001
Abstract: We consider the impact of purinergic signaling on the physiology of the special senses of vision, smell, taste and hearing. Purines (particularly ATP and adenosine) act as neurotransmitters, gliotransmitters and paracrine factors in the sensory retina, nasal olfactory epithelium, taste buds and cochlea. The associated purinergic receptor signaling underpins the sensory transduction and information coding in these sense organs. The P2 and P1 receptors mediate fast transmission of sensory signals and have modulatory roles in the regulation of synaptic transmitter release, for ex le in the adaptation to sensory overstimulation. Purinergic signaling regulates bidirectional neuron-glia interactions and is involved in the control of blood supply, extracellular ion homeostasis and the turnover of sensory epithelia by modulating apoptosis and progenitor proliferation. Purinergic signaling is an important player in pathophysiological processes in sensory tissues, and has both detrimental (pro-apoptotic) and supportive (e.g. initiation of cytoprotective stress-signaling cascades) effects.
Publisher: Frontiers Media SA
Date: 20-01-2021
DOI: 10.3389/FNCEL.2020.625879
Abstract: Hypomyelination with brain stem and spinal cord involvement and leg spasticity (HBSL) is a leukodystrophy caused by missense mutations of the aspartyl-tRNA synthetase-encoding gene DARS1 . The clinical picture includes the regression of acquired motor milestones, spasticity, ataxia, seizures, nystagmus, and intellectual disabilities. Morphologically, HBSL is characterized by a distinct pattern of hypomyelination in the central nervous system including the anterior brainstem, the cerebellar peduncles and the supratentorial white matter as well as the dorsal columns and the lateral corticospinal tracts of the spinal cord. Adequate HBSL animal models are lacking. Dars1 knockout mice are embryonic lethal precluding examination of the etiology. To address this, we introduced the HBSL-causing Dars1 D 367 Y point mutation into the mouse genome. Surprisingly, mice carrying this mutation homozygously were phenotypically normal. As hypomorphic mutations are more severe in trans to a deletion, we crossed Dars1 D 367 Y / D 367 Y mice with Dars1-null carriers. The resulting Dars1 D 367 Y /− offspring displayed a strong developmental delay compared to control Dars1 D 367 Y /+ littermates, starting during embryogenesis. Only a small fraction of Dars1 D 367 Y /− mice were born, and half of these mice died with hydrocephalus during the first 3 weeks of life. Of the few Dars1 D 367 Y /− mice that were born at term, 25% displayed microphthalmia. Throughout postnatal life, Dars1 D 367 Y /− mice remained smaller and lighter than their Dars1 D 367 Y /+ littermates. Despite this early developmental deficit, once they made it through early adolescence Dars1 D 367 Y /− mice were phenotypically inconspicuous for most of their adult life, until they developed late onset motor deficits as well as vacuolization and demyelination of the spinal cord white matter. Expression levels of the major myelin proteins were reduced in Dars1 D 367 Y /− mice compared to controls. Taken together, Dars1 D 367 Y /− mice model aspects of the clinical picture of the corresponding missense mutation in HBSL. This model will enable studies of late onset deficits, which is precluded in Dars1 knockout mice, and can be leveraged to test potential HBSL therapeutics including DARS1 gene replacement therapy.
Publisher: Elsevier BV
Date: 09-1996
DOI: 10.1016/S0378-5955(96)00079-2
Abstract: It has been clearly demonstrated that extracellular adenosine 5'-triphosphate (ATP) exerts a potent modulatory activity in the cochlea through its interaction with P2 purinoceptors. However, little is known regarding the metabolism of extracellular ATP in cochlear tissues via ectonucleotidases. This study provides evidence for the presence of ectonucleotidases in the perilymphatic compartment of the guinea pig cochlea. Using microperfusion, ATP (500 microM) was introduced into the cochlear perilymph through the basal turn scala tympani, and effluent was collected from the basal turn scala vestibuli. S les were subsequently analysed for the presence of adenine metabolites using high performance liquid chromatography (HPLC). Cell viability was evaluated by the activity of the intracellular enzyme lactate dehydrogenase (LDH) in the perfusate. ATP was degraded to 122.8 +/- 9.9 microM (25.0 +/- 5.8%) during the passage through the cochlear perilymphatic compartment. Breakdown of ATP resulted in the formation of adenosine 5'-diphosphate (41.5 +/- 9.0 microM), adenosine 5'-monophosphate (201.3 +/- 15.5 microM), adenosine (108.6 +/- 8.3) and inosine (15.0 +/- 1.5 microM). The degradation of ATP was significantly (P < 0.001, Student's t-test) inhibited in the absence of alent cations, Ca2+ and Mg2+ in the perfusate. In control experiments, no spontaneous degradation of ATP was observed in vitro. LDH activity was similar during ATP perfusions (2.9 +/- 0.9%) to control perfusions with artificial perilymph (4.2 +/- 1.0%) indicating well preserved cell integrity in the cochlear perilymphatic compartment. The degradation of extracellular ATP in the presence of intact tissues and its inhibition in the absence of alent cations, a cofactor for ectonucleotidases, provides evidence for ectonucleotidase activity in the perilymphatic fluid space of the cochlea.
Publisher: Springer Science and Business Media LLC
Date: 06-2010
Publisher: S. Karger AG
Date: 2002
DOI: 10.1159/000058297
Publisher: Mary Ann Liebert Inc
Date: 02-2019
DOI: 10.1089/HUM.2018.062
Abstract: Naked plasmid DNA electrotransfer offers advantages over viral-based gene delivery, including being regulatory permissive, but factors influencing expression efficiency and cell fate impact on translational utility. This study compared co-expression of red and green fluorescence reporter plasmids with differing promoters in HEK293 cells and in vivo in guinea pig cochlear mesenchymal cells using Bionic array-Directed Gene Electrotransfer (BaDGE
Publisher: Frontiers Media SA
Date: 08-12-2022
DOI: 10.3389/FNMOL.2022.1061257
Abstract: The leukodystrophy Canavan disease is a fatal white matter disorder caused by loss-of-function mutations of the aspartoacylase-encoding ASPA gene. There are no effective treatments available and experimental gene therapy trials have failed to provide sufficient amelioration from Canavan disease symptoms. Preclinical studies suggest that Canavan disease-like pathology can be addressed by either ASPA gene replacement therapy or by lowering the expression of the N-acetyl-L-aspartate synthesizing enzyme NAT8L. Both approaches in idually prevent or even reverse pathological aspects in Canavan disease mice. Here, we combined both strategies and assessed whether intracranial adeno-associated virus-mediated gene delivery to a Canavan disease mouse model at 12 weeks allows for reversal of existing pathology. This was enabled by a single vector dual-function approach. In vitro and in vivo biopotency assessment revealed significant knockdown of neuronal Nat8l paired with robust ectopic aspartoacylase expression. Following nomination of the most efficient cassette designs, we performed proof-of-concept studies in post-symptomatic Aspa -null mice. Late-stage gene therapy resulted in a decrease of brain vacuoles and long-term reversal of all pathological hallmarks, including loss of body weight, locomotor impairments, elevated N-acetyl-L-aspartate levels, astrogliosis, and demyelination. These data suggest feasibility of a dual-function vector combination therapy, directed at replacing aspartoacylase with concomitantly suppressing N-acetyl-L-aspartate production, which holds potential to permanently alleviate Canavan disease symptoms and expands the therapeutic window towards a treatment option for adult subjects.
Publisher: Public Library of Science (PLoS)
Date: 14-06-2013
Publisher: Elsevier BV
Date: 02-2010
DOI: 10.1016/J.HEARES.2009.12.004
Abstract: Adenosine is a constitutive cell metabolite with a putative role in protection and regeneration in many tissues. This study was undertaken to determine if adenosine signalling pathways are involved in protection against noise injury. A(1) adenosine receptor expression levels were altered in the cochlea exposed to loud sound, suggesting their involvement in the development of noise injury. Adenosine and selective adenosine receptor agonists (CCPA, CGS-21680 and Cl-IB-MECA) were applied to the round window membrane of the cochlea 6h after noise exposure. Auditory brainstem responses measured 48h after drug administration demonstrated partial recovery of hearing thresholds (up to 20dB) in the cochleae treated with adenosine (non-selective adenosine receptor agonist) or CCPA (selective A(1) adenosine receptor agonist). In contrast, the selective A(2A) adenosine receptor agonist CGS-21680 and A(3) adenosine receptor agonist Cl-IB-MECA did not protect the cochlea from hearing loss. Sound-evoked cochlear potentials in control rats exposed to ambient noise were minimally altered by local administration of the adenosine receptor agonists used in the noise study. Free radical generation in the cochlea exposed to noise was reduced by administration of adenosine and CCPA. This study pinpoints A(1) adenosine receptors as attractive targets for pharmacological interventions to reduce noise-induced cochlear injury after exposure.
Publisher: Elsevier BV
Date: 08-2004
Publisher: Springer Berlin Heidelberg
Date: 1991
Publisher: Elsevier BV
Date: 08-2006
DOI: 10.1016/J.BRAINRES.2006.05.094
Abstract: Stimuli such as noise or hypoxia can induce a release of ATP into the cochlear fluid spaces. At nanomolar concentrations, ATP affects neurotransmission and electrochemical regulation of sound transduction. At higher concentrations, ATP may exert cytotoxicity acting on specific P2X(7) receptor subunits, thus contributing to the pathophysiology of noise-induced cochlear injury. Ectonucleoside triphosphate diphosphohydrolases (E-NTPDases) are pivotal to regulation of extracellular nucleotide concentrations and therefore P2 receptor signaling in the cochlea. Here, we characterize the distribution of NTPDase3 ectonucleotidase (preferentially hydrolyzes ATP over ADP) in cochlear tissues and investigate the effect of noise exposure on NTPDase3 expression. Marked NTPDase3 immunoreactivity in the primary afferent neurones of the spiral ganglion, extending in the distal neurite processes to the synapses beneath the inner and outer hair cells, suggests involvement in auditory neurotransmission. Immunolabeling in the lateral wall and epithelial cells lining the cochlear partition was also evident. Semi-quantitative immunohistochemistry revealed increased NTPDase3 immunolabeling in the synaptic regions of the inner and outer hair cells at sound intensities that induce temporary threshold shift. The results suggest a role for NTPDase3 in regulating ATP signaling associated primarily with auditory neurotransmission, and the potential neuroprotective nature of noise-induced up-regulation of this ectonucleotidase in the cochlea.
Publisher: Wiley
Date: 28-04-1999
DOI: 10.1002/(SICI)1096-9861(19990428)407:1<11::AID-CNE2>3.0.CO;2-R
Publisher: Society for Neuroscience
Date: 10-1999
DOI: 10.1523/JNEUROSCI.19-19-08377.1999
Abstract: Extracellular ATP has multimodal actions in the cochlea affecting hearing sensitivity. ATP-gated ion channels involved in this process were characterized in the guinea pig cochlea. Voltage-cl ed hair cells exhibited a P2 receptor pharmacology compatible with the assembly of ATP-gated ion channels from P2X 2 receptor subunits. Reverse transcription-PCR experiments confirmed expression of the P2X 2–1 receptor subunit mRNA isoform in the sensory epithelium (organ of Corti) a splice variant that confers desensitization, P2X 2–2 , was the predominant subunit isoform expressed by primary auditory neurons. Expression of the ATP-gated ion channel protein was localized using a P2X 2 receptor subunit-specific antiserum. The highest density of P2X 2 subunit-like immunoreactivity in the cochlea occurred on the hair cell stereocilia, which faces the endolymph. Tissues lining this compartment exhibited significant P2X 2 receptor subunit expression, with the exception of the stria vascularis. Expression of ATP-gated ion channels at these sites provides a pathway for the observed ATP-induced reduction in endocochlear potential and likely serves a protective role, decoupling the “cochlear lifier” in response to stressors, such as noise and ischemia. Within the perilymphatic compartment, immunolabeling on Deiters’ cells is compatible with purinergic modulation of cochlear micromechanics. P2X 2 receptor subunit expression was also detected in spiral ganglion primary afferent neurons, and immunoelectron microscopy localized these subunits to postsynaptic junctions at both inner and outer hair cells. The former supports a cotransmitter role for ATP in a subset of type I spiral ganglion neurons, and latter represents the first characterization of a receptor for a fast neurotransmitter associated with the type II spiral ganglion neurons.
Publisher: Elsevier BV
Date: 10-2011
DOI: 10.1016/J.AUTNEU.2011.08.002
Abstract: This mini review highlights recent research on the control of breathing that places gliotransmission and purinergic signaling as core drivers to the respiratory circuits in the brainstem. These elements underpin transduction of hypercapnia, hypoxia and acid sensing at central and peripheral chemoreceptors. The processes involve propagation of an extracellular ATP signal and associated P2 receptor activation, where ATP acts on both the glial cells and the associated output cells in the sensor complex - the respiratory rhythm generator neurons and the ventral inspiratory pre-motor neurons. At the peripheral carotid chemoreceptor, the hypoxia sensor likely involves the gasotransmitter H(2)S, complemented by purinergic neurotransmission.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 08-1999
DOI: 10.1097/00001756-199908200-00026
Abstract: Extracellular ATP acts via ionotropic P2X receptors to mediate fast neurotransmission in the central and autonomic nervous systems. Recent data, including identification of P2X2 receptor mRNA expression by spiral ganglion neurones, suggests that purinergic signalling may influence auditory neurotransmission via ATP-gated ion channels assembled from these subunits. Expression of the P2X2 receptor was localized to the region of the spiral ganglion neurone synapses with the inner hair cells using a P2X2 receptor specific antiserum. Whole-cell patch cl ing of neurones cultured from post-natal day 3-5 spiral ganglia demonstrated a heterogeneity of ATP-activated conductances, consistent with the functional expression of P2X2 receptor subunit isoforms along with possible co-expression of additional P2X receptor subunits. These data provide substantive support for a purinergic transmission element at the peripheral auditory synapse.
Publisher: Springer Science and Business Media LLC
Date: 12-05-2015
DOI: 10.1038/NCOMMS8115
Abstract: The dynamic adjustment of hearing sensitivity and frequency selectivity is mediated by the medial olivocochlear efferent reflex, which suppresses the gain of the ‘cochlear lifier’ in each ear. Such efferent feedback is important for promoting discrimination of sounds in background noise, sound localization and protecting the cochleae from acoustic overstimulation. However, the sensory driver for the olivocochlear reflex is unknown. Here, we resolve this longstanding question using a mouse model null for the gene encoding the type III intermediate filament peripherin ( Prph) . Prph (−/−) mice lacked type II spiral ganglion neuron innervation of the outer hair cells, whereas innervation of the inner hair cells by type I spiral ganglion neurons was normal. Compared with Prph (+/+) controls, both contralateral and ipsilateral olivocochlear efferent-mediated suppression of the cochlear lifier were absent in Prph (−/−) mice, demonstrating that outer hair cells and their type II afferents constitute the sensory drive for the olivocochlear efferent reflex.
Publisher: Wiley
Date: 2010
DOI: 10.1002/JNR.22421
Publisher: OAE Publishing Inc.
Date: 2023
Abstract: Aminoacyl-tRNA synthetases are essential, non-redundant enzymes that catalyze the charging of tRNAs with their cognate amino acids. This reaction is a prerequisite for protein translation in all cells. Mutations in human aminoacyl-tRNA synthetases are often associated with defects of the peripheral and central nervous system and are the underlying cause of many rare diseases including neuropathies and leukodystrophies. A comprehensive understanding of aminoacyl-tRNA synthetase expression domains is key to understanding these disorders and developing novel targeted treatment strategies. Here, we describe histological and biochemical methods to analyze the expression pattern of the aspartyl-tRNA synthetase AspRS in human post-mortem brain tissue. The same methods can readily be applied to other members of the aminoacyl-tRNA synthetase superfamily or, more generally, to other cytosolic proteins in the human brain.
Publisher: Elsevier BV
Date: 1990
DOI: 10.1016/0378-5955(90)90221-A
Abstract: Two cholinergically-induced modulations of membrane conductances have been identified in hair cells isolated from the crista ullaris of the leopard frog (Rana pipiens), using the whole cell recording configuration of the patch cl technique. Of 56 crista hair cells tested, 28 showed drug-induced changes in membrane current or membrane potential which were repeatable and could be reversed with washout of drug. The predominant effect (observed in 20 hair cells) of acetylcholine (Ach, 100 microM) to 1mM) or carbachol (1 microM to 50 microM) applied to these hair cells was the reduction of an outward current corresponding to a change in conductance of approximately -0.22 nS. This action by Ach on hair cells has been inferred from previous studies of afferent fiber discharge which reported an increase in firing rate with stimulation of efferent fibers or exogenous application of cholinomimetics (Bernard et al., 1985 Valli et al., 1986 Guth et al., 1986 Norris et al., 1988a). The Ach-induced reduction in outward current was associated with a depolarization of the zero-current membrane potential by approximately +2.5 mV. In a total of 8 hair cells, an Ach-induced reversible increase in outward current was recorded. Changes in conductance were approximately +0.13 nS and were associated with a hyperpolarization of the zero-current membrane potential by approximately -2.2 mV. This current increase is likely to be responsible for the inhibitory post-synaptic potentials (IPSPs) which have previously been recorded intracellularly from acoustico-lateralis hair cells during stimulation of the efferent innervation (Flock and Russell, 1976 Ashmore and Russell, 1982 Art et al., 1984, 1985). Of the remaining 28 hair cells, six cells failed to exhibit any change in membrane conductance or membrane potential in the presence of cholinomimetics while an additional 15 cells exhibited decreases, and 7 cells exhibited increases in outward conductance, during application of Ach or carbachol, which were neither reversible with washout nor repeatable. The Ach-induced decrease in outward current could be reversible blocked by removal of Ca2+ from the external solution. The antagonism of the Ach-induced decrease in outward current by atropine (10(-5) M) suggests that this current may correspond to a facilitatory, 'atropine-preferring' Ach receptor mediated response previously identified in the isolated semicircular canal (Norris et al., 1988a).(ABSTRACT TRUNCATED AT 400 WORDS)
Publisher: Springer Science and Business Media LLC
Date: 10-03-2010
DOI: 10.1007/S00418-010-0682-1
Abstract: Ectonucleoside triphosphate diphosphohydrolases (E-NTPDases) regulate complex extracellular P2 receptor signalling pathways in mammalian tissues by hydrolysing extracellular nucleotides to the respective nucleosides. All enzymes from this family (NTPDase1-8) are expressed in the adult rat cochlea. This study reports the changes in expression of NTPDase5 and NTPDase6 in the developing rat cochlea. These two intracellular members of the E-NTPDase family can be released in a soluble form and show preference for nucleoside 5'-diphosphates, such as UDP and GDP. Here, we demonstrate differential spatial and temporal patterns for NTPDase5 and NTPDase6 expression during cochlear development, which are indicative of both cytosolic and extracellular action via pyrimidines. NTPDase5 is noted during the early postnatal period in developing sensory hair cells and supporting Deiters' cells of the organ of Corti, and primary auditory neurons located in the spiral ganglion. In contrast, NTPDase6 is confined to the embryonic and early postnatal hair cell bundles. NTPDase6 immunolocalisation in the developing cochlea underpins its putative role in hair cell bundle development, probably via cytosolic action, whilst NTPDase5 may have a broader extracellular role in the development of sensory and neural tissues in the rat cochlea. Both NTPDase5 and NTPDase6 colocalize with UDP-preferring P2Y(4), P2Y(6) and P2Y(14) receptors during cochlear development, but this strong association was lost in the adult cochlea. Spatiotemporal topographic expression of NTPDase5 and NTPDase6 and P2Y receptors in adult and developing cochlear tissues provide strong support for the role of pyrimidinergic signalling in cochlear development.
Publisher: The Royal Society
Date: 22-09-1992
Abstract: Acetylcholine (ACh) and adenosine 5'-triphosphate (ATP) are shown to act in opposing fashion on guinea-pig cochlear outer hair cells (OHCS) via receptors localized within different fluid compartments of the organ of Corti. The cholinergic (efferent) receptors localized at the basal (perilymphatic) region of these cells activated a rapidly desensitizing hyperpolarizing K+ current. In contrast, purinergic (ATP) receptors were localized at the apical (endolymphatic) surface of OHCS and activated a depolarizing nonselective cation current which exhibited inward rectification and lacked desensitization. Localization of the receptors was determined by using whole-cell patch-cl , by recording onset latencies and response litudes to pulses of either ACh or ATP pressure-applied at selected sites along the length of isolated OHCS. Under voltage-cl at -60 mV, the largest ACh-induced (outward) currents were recorded when ACh was directed at the basal region of the cells. Conversely, the maximum (inward) ATP currents were obtained when ATP was directed toward the apical surface of these cells. Onset latencies increased rapidly from a minimum of approximately 10 ms for either ACh or ATP as the drug pipette was moved away from these optimal sites. The ATP response was antagonized by amiloride in a dose-dependent manner with a KD of approximately 400 microM. The localization of P2-type purinoceptors to the endolymphatic surface of OHCS suggests that ATP mediates a humoral modulation of the mechano-electrical transduction process.
Publisher: Elsevier BV
Date: 02-1988
DOI: 10.1016/0378-5955(88)90092-5
Abstract: We present preliminary evidence that acetylcholine has three distinct modes of action on the discharge rate of afferent fibers from the frog isolated semicircular canal. These are: (I) a facilitatory effect which is of mixed muscarinic-nicotinic type and is mediated by a receptor which we call "atropine-preferring' (II) a suppressive effect unmasked by atropine and antagonized by strychnine whose action is mediated by a "strychnine-preferring" receptor (III) a suppressive effect produced by the cholinesterase inhibitors eserine and echothiophate, which being antagonized by curare, is mediated by a "curare-preferring" receptor.
Publisher: Elsevier BV
Date: 02-2001
DOI: 10.1016/S0165-3806(00)00149-8
Abstract: The expression pattern of the ATP-gated ion channel P2X(1) receptor subunit was studied in the developing rat cochlea by riboprobe in situ hybridisation and immunohistochemistry. Embryonic (E12, E14, E16 and E18) and postnatal (P0, P2, P4, P6, P10 and adult) rat cochleae were examined. Both mRNA and protein localisation techniques demonstrated comparable P2X(1) receptor expression from E16 until P6 but this expression was absent at later developmental stages. P2X(1) receptor mRNA expression was localised within the otic capsule and associated mesenchyme (from E16 to P6), spiral limbus (from P0 to P6) and within the spiral ligament adjacent to the insertion of Reissner's membrane (from P2 to P6). P2X(1) receptor protein had a similar distribution based upon immunoperoxidase localisation. P2X(1) receptor-like immunoreactivity was detected in the otic capsule and the surrounding mesenchyme (from E16 to P6), spiral limbus (from P0) and epithelial cells of Reissner's membrane (from P2 to P6). The spiral ganglion neurones showed the earliest P2X(1) receptor expression (from E16 to P6). This became associated with immunolabelling of their afferent neurite projections to the base of the developing inner and outer hair cells (observed from E18 and peaking at P2). Immunolabelling of the efferent nerve fibres of the intraganglionic spiral bundle (from E18 to P6) within the spiral ganglion was also observed. The results suggest that ATP-gated ion channels assembled from P2X(1) receptor subunits provide a signal transduction pathway for development of afferent and efferent innervation of the sensory hair cells and purinergic influence on cochlear morphogenesis.
Publisher: Elsevier BV
Date: 04-1989
DOI: 10.1016/0378-5955(89)90070-1
Abstract: Hair cells isolated from the crista ullaris of the frog (Rana pipiens) remained viable for up to 5 h and were studied using whole cell voltage- and current cl recordings. Morphological characteristics of isolated crista hair cells were compared with hair cells studied in situ using light- and electron microscopy. While other labyrinthine hair cells such as mammalian inner and outer hair cells of the cochlea, saccular hair cells of the frog, and cochlear hair cells of the turtle typically have a cylindrical shape, the crista hair cells in the frog are predominantly bulbous, having a thin elongated trunk projecting from a spherical base just large enough to enclose the nucleus. This shape correlates well with the compressed packing configuration of hair cells of the crista ullaris observed in situ in the histological material. The support cells often failed to separate adjacent hair cells, particularly the apical ends of the hair cells. Maximal cell density on the sensory epithelial ridge appears to be achieved by this arrangement. The mean resting membrane potential (Vz) of isolated crista hair cells was -44.8 mV. Cells with smooth surfaces and apparent opacity had the most negative Vz potentials. As the cells appeared to deteriorate, there was development of transparency and cell surface granulation. Such cells had more positive initial Vz values. Cells with Vz values more positive than -15 mV exhibited a distinct, contoured nucleus. Cells lacking these indicators of deterioration were characterized by input resistances of 1.9 +/- 0.31 G omega and membrane time constants of 13 +/- 2.5 ms. A large complex outwardly rectifying current was identified which was abolished by substituting Cs+ for K+ in the internal solution. The outward K+ current had two major components: a fast tetraethylammonium (TEA)-insensitive, voltage dependent I(A)-type current which showed voltage dependent inactivation and a TEA sensitive current which had characteristics of a calcium dependent IK(Ca)-type current. Transient changes (20 ms duration) in membrane potential mimicking that which could be produced by the transduction current during cilial displacement potently modulated the I(A) current. Depolarizing current pulses of greater than 63800 pA were required to elicit membrane voltage oscillations. The resulting membrane potential offset of at least 40 mV is well beyond the magnitude of hair cell receptor potentials making it unlikely that these oscillations would play a role in enhancing frequency selectivity.(ABSTRACT TRUNCATED AT 400 WORDS)
Publisher: Springer US
Date: 1990
Publisher: Elsevier BV
Date: 11-1999
DOI: 10.1016/S0169-328X(99)00244-2
Abstract: Ectonucleotidases provide the signal termination mechanism for purinergic transmission, including fast excitatory neurotransmission by ATP in the CNS. This study provides evidence for ectonucleotidase expression in the rat cochlea, brain and other tissues. In addition to detection of rat ecto-ATPase and ecto-ATPDase in these tissues, we identify a novel ecto-ATPase splice variant arising from the loss of a putative exon (193 bp) in the C-terminal coding region. This is the first evidence of alternative splicing in the ecto-ATPase gene family. Splicing of the 193-bp putative exon containing a stop codon extends the open reading frame and provides translation of an additional 50 amino acids compared with the isoform isolated earlier from the rat brain (rEATPase(A) GenBank accession #Y11835). The splice variant (rEATPase(B) GenBank accession #AF129103) encodes 545 amino acids with a predicted protein molecular mass of 60 kDa. rEATPase(B) contains a long cytoplasmic tail (62 amino acids) with three potential protein kinase CK2 phosphorylation sites not present in rEATPase(A). Co-expression of two ecto-ATPase isoforms with different regulatory sites suggests that the extracellular ATP signal levels may be differently influenced by intracellular feedback pathways.
Publisher: Frontiers Media SA
Date: 24-05-2016
Publisher: Elsevier BV
Date: 1986
DOI: 10.1016/0300-9629(86)90484-6
Abstract: Rats reared from birth at 30 degrees C show a permanent deficit in body temperature regulation. To test the ability of heat-reared rats to respond to an adrenergic drive, carbon dioxide production was measured at five doses of noradrenaline in rats reared at either 20 or 30 degrees C. Noradrenaline-induced carbon dioxide production was greater at all doses in 20 degrees C rats, but sensitivity to noradrenaline was the same in heat-reared and control animals. These findings support the conclusion that the thermoregulatory deficit induced by heat rearing is due to a change induced in the nervous system.
Publisher: Springer Science and Business Media LLC
Date: 10-2015
Publisher: Wiley
Date: 26-02-2013
DOI: 10.1111/EJN.12158
Abstract: The physiological significance of canonical transient receptor potential (TRPC) ion channels in sensory systems is rapidly emerging. Heterologous expression studies show that TRPC3 is a significant Ca(2+) entry pathway, with dual activation via G protein-coupled receptor (GPCR)-phospholipase C-diacylglycerol second messenger signaling, and through negative feedback, whereby a fall in cytosolic Ca(2+) releases Ca(2+) -calmodulin channel block. We hypothesised that the latter process contributes to cochlear hair cell cytosolic Ca(2+) homeostasis. Confocal microfluorimetry with the Ca(2+) indicator Fluo-4 acetoxymethylester showed that, when cytosolic Ca(2+) was depleted, Ca(2+) re-entry was significantly impaired in mature TRPC3(-/-) inner and outer hair cells. The impact of this disrupted Ca(2+) homeostasis on sound transduction was assessed with the use of distortion product otoacoustic emissions (DPOAEs), which constitute a direct measure of the outer hair cell transduction that underlies hearing sensitivity and frequency selectivity. TRPC3(-/-) mice showed significantly stronger DPOAE (2f1 - f2 ) growth functions than wild-type (WT) littermates within the frequency range of best hearing acuity. This translated to hyperacusis (decreased threshold) measured by the auditory brainstem response (ABR). TRPC3(-/-) and WT mice did not differ in the levels of temporary and permanent threshold shift arising from noise exposure, indicating that potential GPCR signaling via TRPC3 is not pronounced. Overall, these data suggest that the Ca(2+) set-point in the hair cell, and hence membrane conductance, is modulated by TRPC3s through their function as a negative feedback-regulated Ca(2+) entry pathway. This TPRC3-regulated Ca(2+) homeostasis shapes the sound transduction input-output function and auditory neurotransmission.
Publisher: American Physiological Society
Date: 11-1992
DOI: 10.1152/JN.1992.68.5.1642
Abstract: 1. A-type outward currents were studied in sensory hair cells isolated from the semicircular canals (SCC) of the leopard frog (Rana pipiens) with whole-cell voltage- and current-cl ing techniques. 2. There appear to be two classes of A-type outward-conducting potassium channels based on steady-state, kinetic, pharmacological parameters, and reversal potential. 3. The two classes of A-type currents differ in their steady-state inactivation properties as well as in the kinetics of inactivation. The steady-state inactivation properties are such that a significant portion of the fast channels are available from near the resting potential. 4. The inactivating channels studied do not appear to be calcium dependent. 5. The A-channels in hair cells appear to subserve functions that are analogous to IA functions in neurons, that is, modulating spike latency and Q (the oscillatory d ing function). The A-currents appear to temporally limit the hair cell voltage response to a current injection.
Publisher: SAGE Publications
Date: 1990
DOI: 10.1177/000348949009900113
Abstract: Amino-oxyacetic acid (AOAA) was evaluated as a palliative in tinnitus. Sixty-six patients with tinnitus presumed to be of cochlear origin were given either a placebo or 75 mg of AOAA four times a day for 1 week. Response was evaluated by both audiometric measurement of tinnitus loudness and subjective rating by patients of change or no change in tinnitus severity. Because loudness measurements and self-rating have not been shown to be independent, and since the aim of clinical treatment of tinnitus is the alleviation of subjective distress, greater weight was given to the patient's self-rating. A total of 21 % of all patients reported a subjective decrease in tinnitus severity, usually within 3 to 4 days after the start of AOAA use. Patients with tinnitus caused by presbycusis or Meniere's disease were the most likely to respond to AOAA treatment with a reduction in tinnitus severity, whereas those with drug-induced tinnitus were the least likely to respond. Nausea and dysequilibrium were the most common side effects of AOAA use. Of the 21 % of patients who responded to AOAA, 71 % developed some type of side effect. Amino-oxyacetic acid produces a reduction in the severity of tinnitus in about 20% of patients however, the incidence of side effects makes the drug unacceptable for clinical use.
Publisher: Elsevier BV
Date: 03-1998
DOI: 10.1016/S0378-5955(98)00004-5
Abstract: This study investigated the characteristics of ecto-nucleotidases in tissues lining the perilymphatic cavity of the cochlea. The perilymphatic space of the isolated guinea-pig cochlea was maintained with oxygenated artificial perilymph (AP) perfused at a rate of 100 microl/min. Following AP perfusion, either adenosine triphosphate (ATP), adenosine diphosphate (ADP) or adenosine monophosphate (AMP) was introduced into scala tympani, and perfusion arrested for 2 min for substrate incubation with cochlear tissues. Effluent collected from the cochlea was assayed for adenine nucleotide metabolites by reverse-phase high-performance liquid chromatography (RP-HPLC). Extracellular ATP and ADP were rapidly and sequentially hydrolysed to adenosine by Ca2+/Mg2+-dependent and Ca2+/Mg2+-independent enzymatic mechanisms. The degradation of extracellular ATP, ADP and AMP occurred in the presence of intact tissues, as demonstrated by the limited lactate dehydrogenase (LDH) activity (0-2.2%). ATPase activity was not affected by inhibitors of intracellular ATPases (oligomycin, ouabain, N-ethylmaleimide, 100 microM NaN3) and non-specific alkaline phosphatase (beta-glycerophosphate). The hydrolysis of ATP was inhibited by 5 mM NaN3, suramin, ATPgammaS, La3+ and CTP, the hydrolysis of ADP by beta,gamma-imidoATP, and AMP degradation by alpha,beta-methyleneADP. Ecto-ATPase, ecto-ADPase and ecto-5'-nucleotidase followed Michaelis-Menten hyperbolic kinetics, with estimated Km values of 2282 microM, 6619 microM and 881 microM, respectively. Our results indicate the presence of considerable ecto-nucleotidase activity within scala tympani of the cochlea, and support its role as the terminating mechanism for P2 receptor signalling known to occur in the cochlea. A competition plot is consistent with ATP and ADP degradation mediated by the same enzyme (ecto-ADP diphosphohydrolase) with two different catalytic sites.
Publisher: Elsevier BV
Date: 12-2020
Publisher: Elsevier BV
Date: 07-2010
Publisher: Elsevier BV
Date: 2017
DOI: 10.1016/J.NBD.2016.10.008
Abstract: The recently diagnosed leukodystrophy Hypomyelination with Brain stem and Spinal cord involvement and Leg spasticity (HBSL) is caused by mutations of the cytoplasmic aspartyl-tRNA synthetase geneDARS. The physiological role of DARS in translation is to accurately pair aspartate with its cognate tRNA. Clinically, HBSL subjects show a distinct pattern of hypomyelination and develop progressive leg spasticity, variable cognitive impairment and epilepsy. To elucidate the underlying pathomechanism, we comprehensively assessed endogenous DARS expression in mice. Additionally, aiming at creating the first mammalian HBSL model, we genetically engineered and phenotyped mutant mice with a targetedDarslocus. DARS, although expressed in all organs, shows a distinct expression pattern in the adult brain with little immunoreactivity in macroglia but enrichment in neuronal subpopulations of the hippoc us, cerebellum, and cortex. Within neurons, DARS is mainly located in the cell soma where it co-localizes with other components of the translation machinery. Intriguingly, DARS is also present along neurites and at synapses, where it potentially contributes to local protein synthesis.Dars-null mice are not viable and die before embryonic day 11. Heterozygous mice with only one functionalDarsallele display substantially reduced DARS levels in the brain yet these mutants show no gross abnormalities, including unchanged motor performance. However, we detected reduced pre-pulse inhibition of the acoustic startle response indicating dysfunction of attentional processing inDars Our results, for the first time, show an in-depth characterization of the DARS tissue distribution in mice, revealing surprisingly little uniformity across brain regions or between the major neural cell types. The complete loss of DARS function is not tolerated in mice suggesting that the identified HBSL mutations in humans retain some residual enzyme activity. The mild phenotype of heterozygousDars-null carriers indicates that even partial restoration of DARS levels would be therapeutically relevant. Despite the fact that they do not resemble the full spectrum of clinical symptoms, the robust pre-pulse inhibition phenotype ofDars
Publisher: Springer Science and Business Media LLC
Date: 04-06-2018
DOI: 10.1007/S00418-018-1683-8
Abstract: Aminoglycoside ototoxicity results in permanent loss of the sensory hair cells in the mammalian cochlea. It usually begins at the basal turn causing high-frequency hearing loss. Here we describe previously unreported resistance of hair cells to neomycin ototoxicity in the extreme basal (hook) region of the developing cochlea of the C57BL/6 mouse. Organ of Corti explants from mice at postnatal day 3 were incubated (37 °C, 5% CO
Publisher: Springer Science and Business Media LLC
Date: 07-07-2020
Publisher: Springer Science and Business Media LLC
Date: 13-06-2013
Publisher: Springer Science and Business Media LLC
Date: 06-2010
Publisher: MDPI AG
Date: 16-04-2020
DOI: 10.3390/BIOM10040617
Abstract: Scorpion venoms are a rich source of bioactive molecules, but characterisation of toxin peptides affecting cytosolic Ca2+, central to cell signalling and cell death, is limited. We undertook a functional screening of the venom of the Australian scorpion Hormurus waigiensis to determine the breadth of Ca2+ mobilisation. A human embryonic kidney (HEK293) cell line stably expressing the genetically encoded Ca2+ reporter GCaMP5G and the rabbit type 1 ryanodine receptor (RyR1) was developed as a biosensor. Size-exclusion Fast Protein Liquid Chromatography separated the venom into 53 fractions, constituting 12 chromatographic peaks. Liquid chromatography mass spectroscopy identified 182 distinct molecules with 3 to 63 components per peak. The molecular weights varied from 258 Da—13.6 kDa, with 53% under 1 kDa. The majority of the venom chromatographic peaks (tested as six venom pools) were found to reversibly modulate cell monolayer bioimpedance, detected using the xCELLigence platform (ACEA Biosciences). Confocal Ca2+ imaging showed 9/14 peak s les, with molecules spanning the molecular size range, increased cytosolic Ca2+ mobilization. H. waigiensis venom Ca2+ activity was correlated with changes in bio-impedance, reflecting multi-modal toxin actions on cell physiology across the venom proteome.
Publisher: The Royal Society
Date: 22-05-1991
Abstract: Acetylcholine has long been thought to be the neurotransmitter of the cochlear efferent system in mammals although the evidence is largely indirect. By using whole-cell recordings from isolated outer hair cells, we show that acetylcholine activates a large rapidly desensitizing outward potassium current. This corresponds to hyperpolarization of the membrane potential from rest. The half maximal dose for acetylcholine was 13.5 microM with a cooperativity of 2. The response was not due to a conventional muscarinic action of acetylcholine for it was not blocked by 0.1 microM atropine and muscarinic antagonists but it could be blocked by 0.1 microM curare, suggesting that it shared many properties of a nicotinic receptor. It was, however, inhibited by 10 microM strychnine. The potassium current activated by acetylcholine required external calcium and was characterized by a significant delay at room temperature. This points to the involvement of a second messenger system, possibly calcium itself.
Publisher: Elsevier BV
Date: 09-1988
DOI: 10.1016/0378-5955(88)90043-3
Abstract: Histamine and other imidazole-containing substances were found to increase ullar nerve afferent firing rate while both H1 and H2 histamine antagonists effectively inhibited ullar nerve activity. A specific inhibitor of histidine decarboxylase, the enzyme which catalyses the synthesis of histamine, reduced ullar nerve firing in a dose-dependent manner. These observations suggest a physiological role for histamine in the inner ear. Maintenance of a response to histamine after de-efferentation of the crista ullaris supports the hypothesis that the site of action is the hair cell antagonism of the histamine response by a cholinergic antagonist, atropine, and antagonism of a cholinergically mediated facilitation by the histaminergic antagonist pyrilamine, indicate that the site of action may involve the acetylcholine receptor complex on the crista ullaris hair cells. The observation that imidazole-containing compounds cause significant effects on semicircular canal neurotransmission provides an important finding with regard to the site of action of antihistamines used for the treatment of vertigo and motion sickness.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 11-1996
DOI: 10.1097/00001756-199611040-00051
Abstract: The distribution of the P2x2 purinoceptor subunit protein, which forms ATP-gated ion channels by homo- and hetero-multimeric assembly, was examined in the adult rat and guinea-pig cerebellum using two novel antisera generated against separate 18 amino acid sequences located in the predicted extracellular domain of this subunit. These antisera, the first available for labelling the P2x2R subunit protein, were validated by selective labelling of a fusion protein containing the target amino acid sequences, and in cerebellum, by peptide specific block of immunoreactivity and by comparison with the distribution of P2x2R mRNA. P2x2R-like immunoreactivity was seen in Purkinje cells, specifically the soma and dendrites, neurons in the granular and molecular layers and deep cerebellar nuclei. The identification of P2x2R-like immunoreactivity within the cerebellar neural circuitry is consistent with a role for extracellular ATP acting as a fast neurotransmitter in motor learning and coordination of movement. Additionally, labelling of neuroglia and fibre tracts supports a erse role for extracellular ATP in CNS homeostasis.
Publisher: Wiley
Date: 02-11-2017
DOI: 10.1002/LARY.26885
Abstract: Biological components of perilymph affect the electrical performance of cochlear implants. Understanding the perilymph composition of common animal models will improve the understanding of this impact and improve the interpretation of results from animal studies and how it relates to humans. Analysis and comparison of the proteomes of human, guinea pig, and cat perilymph. Multiple perilymph s les from both guinea pigs and cats were analysed via liquid chromatography with tandem mass spectrometry. Proteins were identified using the Mascot database. Human data were obtained from a published dataset. Proteins identified were refined to form a proteome for each species. Over 200 different proteins were found per species. There were 81, 39, and 64 proteins in the final human, guinea pig, and cat proteomes, respectively. Twenty-one proteins were common to all three species. Fifty-two percent of the cat proteome was found in the human proteome, and 31% of the guinea pig was common to human. The cat proteome had similar complexity to the human proteome in three protein classes, whereas the guinea pig had a similar complexity in two. The presence of albumin was significantly higher in human perilymph than in the other two species. Immunoglobulins were more abundant in the human than in the cat proteome. Perilymph proteomes were compared across three species. The degree of crossover of proteins of both guinea pig and cat with human indicate that these animals suitable models for the human cochlea, albeit the cat perilymph is a closer match. NA. Laryngoscope, 128:E47-E52, 2018.
Publisher: Elsevier BV
Date: 2002
DOI: 10.1016/S0306-4522(01)00454-7
Abstract: Spiral ganglion neurones provide the afferent innervation to cochlear hair cells. Little is known of the molecular physiological processes associated with the differentiation of these neurones, which occurs up to and beyond hearing onset. We have identified novel A-type (inactivating) potassium currents in neonatal rat spiral ganglion neurones in situ, which have not previously been reported from the mammalian cochlea, presumably as a consequence of altered protein expression associated with other preparations. Under whole-cell voltage cl , voltage steps activated both A-type and non-inactivating outward currents from around -55 mV. The litude of the A-type currents was dependent on the holding potential, with steady-state inactivation relieved at hyperpolarised potentials. At -60 mV (close to the resting potential in situ) the currents were approximately 30% enabled. The inactivation kinetics and the degree of inactivation varied between cells, suggesting heterogeneous expression of multiple inactivating currents. A-type currents provided around 60% of total conductance activated by depolarising voltage steps from the resting potential, and were very sensitive to bath-applied 4-aminopyridine (0.01-1 mM). Tetraethylammonium (0.1-30 mM) also blocked the majority of the A-type currents, and the non-inactivating outward current, but left residual fast inactivating A-type current. Under current cl , neurones fired single tetrodotoxin-sensitive action potentials. 4-Aminopyridine relieved the A-type current mediated stabilisation of membrane potential, resulting in periodic small litude action potentials. This study provides the first electrophysiological evidence for A-type potassium currents in neonatal spiral ganglion neurones and shows that these currents play an integral role in primary auditory neurone firing.
Publisher: Wiley
Date: 02-1997
DOI: 10.1113/JPHYSIOL.1997.SP021896
Abstract: 1. Whole-cell patch-cl recordings were used to determine the variation in the P2X receptor conductance, activated by extracellular ATP, in outer hair cells (OHCs) isolated from each of the four turns of the guinea-pig cochlea. 2. In standard solution (containing 1.5 mM Ca2+) slope conductances were determined in OHCs of known origin from current-voltage relationships obtained from voltage r s applied between -100 and +50 mV. Membrane conductance throughout this voltage range was greatest in OHCs originating from the basal (high frequency encoding) region of the cochlea. This gradient in OHC conductance from apex to base of the cochlea can be attributed to variation in expression of both a negatively activated K+ conductance and a TEA-sensitive outwardly rectifying K+ conductance. OHC slope conductance measured about a membrane potential of -75 mV increased from a mean of 33.5 nS in the apical region (turn 4) to 96.8 nS in the basal region (turn 1) of the cochlea. 3. Removal of external Ca2+ reduced OHC conductance by an average of 25%, reflecting a Ca2+ dependence of the background conductances in these cells. In zero external Ca2+ the mean slope conductance measured at -75 mV in the apical turn was 25.0 nS compared with 73.8 nS in the basal turn. 4. In Ca(2+)-free solution both 2 mM and 4 microM ATP produced inward currents that were progressively larger in OHCs originating from more basal regions of the cochlea. The steady-state inward current elicited by 2 mM extracellular ATP increased from -1.44 to -3.26 nA for turns 4 and 1, respectively. 5. The P2X receptor conductance was determined between -100 and +50 mV by comparing voltage r s in the presence and absence of extracellular ATP in Ca(2+)-free solution. The conductance was inwardly rectifying with a reversal potential close to 0 mV. Measured close to the resting membrane potential of the cells (-75 mV), 2 mM ATP elicited an average 300% increase in conductance in parallel with the systematic increase in background conductance which occurs in OHCs originating from the more basal regions of the cochlea. The conductance at -75 mV activated by 2 mM ATP increased from a mean of 59.6 nS in turn 4 OHCs to a mean of 166.2 nS in turn 1 OHCs. The conductance activated by 4 microM ATP was also greater in the basal turn OHCs (45.3 nS) than in the apical region OHCs (5.9 nS). 6. The number of ATP-gated ion channels on in idual OHCs, presumed to be localized to the stereocilia, increases from approximately 6000 in turn 4 cells to 16,500 in turn 1 cells, based on estimates of unitary conductance and average maximum ATP-activated OHC conductance (2 mM ATP).
Publisher: S. Karger AG
Date: 2003
DOI: 10.1159/000069478
Abstract: Extracellular adenosine 5′-triphosphate (ATP)-gated ion channels assembled from P2X receptor subunits exhibit subunit-selective allosteric modulation by protons and alent cations. In voltage-cl ed guinea-pig cochlear outer hair cells (OHC) and Deiters’ cells (DC), H sup + /sup and Cu sup + /sup , but not Zn sup + /sup , enhanced the P2X receptor-mediated inward currents. Acid pH (6.5) potentiated OHC ATP-gated currents by 45%. Co-application of Cu sup + /sup (1–40 µ i M /i ) with ATP increased the response by 20%. In DCs, ATP-gated currents were potentiated 85% by acid pH, and 70% by Cu sup + /sup . Alkaline pH inhibited ATP-gated inward currents by 73% in OHCs and 85% in DCs. Zn sup + /sup was either ineffective (1–10 µ i M /i ) or inhibitory (40–400 µ i M /i ). Recombinant rat P2X sub /sub receptor-mediated inward currents in i Xenopus /i oocytes displayed allosteric modulation that was different from the native guinea-pig cochlear P2X receptors. The oocyte ATP-gated inward current was potentiated 450% by shifting from pH 7.5 to pH 6.5, and 130% with 40 µ i M /i Cu sup + /sup . The enhanced response to ATP with acid pH and Cu sup + /sup is a signature of the P2X sub /sub subunit. In contrast to native guinea-pig cochlear cells, extracellular Zn sup + /sup (40 µ i M /i ) increased the recombinant ATP-gated inward current by 200% in oocytes. These results suggest that the positive allosteric modulation of cochlear OHC and DC ATP-gated ion channels by protons and Cu sup + /sup arises in part from the P2X sub /sub receptor subunit, with additional regulatory elements.
Publisher: Springer Science and Business Media LLC
Date: 09-12-2005
DOI: 10.1007/S00418-005-0119-4
Abstract: ATP-gated non-selective cation channels assembled from P2X(3) receptor subunits contribute to transduction and neurotransmitter signaling in peripheral sensory systems and also feature prominently in the development of the central nervous system. In this study, P2X(3) receptor expression was characterized in the mouse cochlea from embryonic day 18 (E18) using confocal immunofluorescence. From E18 to P6, spiral ganglion neuron cell bodies and peripheral neurites projecting to the inner and outer hair cells were labeled. The inner spiral plexus associated with the inner hair cell synapses had a stronger fluorescence signal than outer spiral bundle fibers which provide the afferent innervation to the outer hair cells. Labeling in the cell bodies and peripheral neurites diminished around P6, and was no longer detected after the onset of hearing (P11, P17, adult). In opposition to the axiom that P2X(3) expression is neuron-specific, inner and outer sensory hair cells were labeled in the base and mid turn region at E18, but at P3 only the outer hair cells in the most apical region of the cochlea continued to express the protein. These data suggest a role for P2X(3) receptor-mediated purinergic signaling in cochlear synaptic reorganization, and establishment of neurotransmission, which occurs just prior to the onset of hearing function.
Publisher: Springer Science and Business Media LLC
Date: 1988
DOI: 10.1007/BF00604059
Publisher: Springer Science and Business Media LLC
Date: 18-07-2023
DOI: 10.1007/S12975-023-01173-1
Abstract: Canonical transient receptor potential (TRPC) non-selective cation channels, particularly those assembled with TRPC3, TRPC6, and TRPC7 subunits, are coupled to G αq -type G protein-coupled receptors for the major classes of excitatory neurotransmitters. Sustained activation of this TRPC channel-based pathophysiological signaling hub in neurons and glia likely contributes to prodigious excitotoxicity-driven secondary brain injury expansion. This was investigated in mouse models with selective Trpc gene knockout (KO). In adult cerebellar brain slices, application of glutamate and the class I metabotropic glutamate receptor agonist (S)-3,5-dihydroxyphenylglycine to Purkinje neurons expressing the GCaMP5g Ca 2+ reporter demonstrated that the majority of the Ca 2+ loading in the molecular layer dendritic arbors was attributable to the TRPC3 effector channels ( Trpc3 KO compared with wildtype (WT)). This Ca 2+ dysregulation was associated with glutamate excitotoxicity causing progressive disruption of the Purkinje cell dendrites (significantly abated in a GAD67-GFP - Trpc3 KO reporter brain slice model). Contribution of the G αq -coupled TRPC channels to secondary brain injury was evaluated in a dual photothrombotic focal ischemic injury model targeting cerebellar and cerebral cortex regions, comparing day 4 post-injury in WT mice, Trpc3 KO , and Trpc1/3/6/7 quadruple knockout ( Trpc QKO ), with immediate 2-h (primary) brain injury. Neuroprotection to secondary brain injury was afforded in both brain regions by Trpc3 KO and Trpc QKO models, with the Trpc QKO showing greatest neuroprotection. These findings demonstrate the contribution of the G αq -coupled TRPC effector mechanism to excitotoxicity-based secondary brain injury expansion, which is a primary driver for mortality and morbidity in stroke, traumatic brain injury, and epilepsy.
Publisher: Springer Science and Business Media LLC
Date: 18-05-2020
Publisher: Frontiers Media SA
Date: 06-08-2019
Publisher: Elsevier BV
Date: 2004
Publisher: Elsevier BV
Date: 12-2017
DOI: 10.1016/J.NEUROPHARM.2016.10.004
Abstract: This review categorizes functionally validated actions of defined scorpion toxin (SCTX) neuropeptides across ion channel subclasses, highlighting key trends in this rapidly evolving field. Scorpion envenomation is a common event in many tropical and subtropical countries, with neuropharmacological actions, particularly autonomic nervous system modulation, causing significant mortality. The primary active agents within scorpion venoms are a erse group of small neuropeptides that elicit specific potent actions across a wide range of ion channel classes. The identification and functional characterisation of these SCTX peptides has tremendous potential for development of novel pharmaceuticals that advance knowledge of ion channels and establish lead compounds for treatment of excitable tissue disorders. This review delineates the unique specificities of 320 in idual SCTX peptides that collectively act on 41 ion channel subclasses. Thus the SCTX research field has significant translational implications for pathophysiology spanning neurotransmission, neurohumoral signalling, sensori-motor systems and excitation-contraction coupling. This article is part of the Special Issue entitled 'Venom-derived Peptides as Pharmacological Tools.'
Publisher: Informa UK Limited
Date: 06-2007
DOI: 10.1128/MCB.00379-07
Publisher: Springer Science and Business Media LLC
Date: 06-2010
Publisher: Society for Neuroscience
Date: 15-08-2012
DOI: 10.1523/JNEUROSCI.6446-11.2012
Abstract: Canonical transient receptor potential (TRPC3) nonselective cation channels are effectors of G-protein-coupled receptors (GPCRs), activated via phospholipase C–diacylglycerol signaling. In cerebellar Purkinje cells, TRPC3 channels cause the metabotropic glutamate receptor (mGluR)-mediated slow EPSC (sEPSC). TRPC3 channels also provide negative feedback regulation of cytosolic Ca 2+ , mediated by a C terminus “calmodulin and inositol trisphosphate receptor binding” (CIRB) domain. Here we report the alternative splicing of the TRPC3 mRNA transcript (designated TRPC3c), resulting in omission of exon 9 (approximately half of the CIRB domain) in mice, rats, and guinea pigs. TRPC3c expression is brain region specific, with prevalence in the cerebellum and brainstem. The TRPC3c channels expressed in HEK293 cells exhibit increased basal and GPCR-activated channel currents, and increased Ca 2+ fluorescence responses, compared with the previously characterized (TRPC3b) isoform when activated via either the endogenous M3 muscarinic acetylcholine receptor, or via coexpressed mGluR1. GPCR-induced TRPC3c channel opening rate (cell-attached patch) matched the maximum activation achieved with inside-out patches with zero cytosolic Ca 2+ , whereas the GPCR-induced TRPC3b activation frequency was significantly less. Both TRPC3 channel isoforms were blocked with 2 m m Ca 2+ , attributable to CIRB domain regulation. In addition, genistein blocked Purkinje cell ( S )-2-amino-2-(3,5-dihydroxyphenyl) acetic acid (mGluR1)-activated TPRC3 current as for recombinant TRPC3c current. This novel TRPC3c ion channel therefore has enhanced efficacy as a neuronal GPCR-Ca 2+ signaling effector, and is associated with sensorimotor coordination, neuronal development, and brain injury.
Publisher: SAGE Publications
Date: 11-2002
DOI: 10.1177/002215540205001102
Abstract: Cellular, molecular, and physiological studies have demonstrated an important signaling role for ATP and related nucleotides acting via P2 receptors in the cochlea of the inner ear. Signal modulation is facilitated by ectonucleotidases, a heterologous family of surface-located enzymes involved in extracellular nucleotide hydrolysis. Our previous studies have implicated CD39/NTPDase1 and CD39L1/NTPDase2, members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family, as major ATP-hydrolyzing enzymes in the tissues lining the cochlear endolymphatic and perilymphatic compartments. NTPDase1 hydrolyzes both nucleoside triphosphates and diphosphates. In contrast, NTPDase2 is a preferential nucleoside triphosphatase. This study characterizes expression of these E-NTPDases in the mouse cochlea by immunohistochemistry. NTPDase1 can be immunolocalized to the cochlear vasculature and neural tissues (primary auditory neurons in the spiral ganglion). In contrast, NTPDase2 immunolabeling was principally localized to synaptic regions of the sensory inner and outer hair cells, stereocilia and cuticular plates of the outer hair cells, supporting cells of the organ of Corti (Deiters’ cells and inner border cells), efferent nerve fibers located in the intraganglionic spiral bundle, and in the outer sulcus and root region of the spiral ligament. This differential expression of NTPDase1 and 2 in the cochlea suggests spatial regulation of P2 receptor signaling, potentially involving different nucleotide species and hydrolysis kinetics.
Publisher: Elsevier BV
Date: 07-1995
Abstract: A truncated form of the P2xR1 purinoceptor subunit (which we designate P2xR1-2) was detected in rat pituitary gland and the secretory epithelial tissue (stria vascularis) of the cochlea using RT-PCR of solid-phase cDNA libraries. PCR products corresponding to the P2xR1 purinoceptor subunit (1) were obtained from vas deferens, brain and microdissected cochlear sensory epithelial tissues including organ of Corti, sacculus and crista ullaris. Cloning and sequencing revealed that the P2xR1-2 product included an 85-bp insertion in a region corresponding to a novel C-terminal end of the second membrane spanning domain and continuing as the cytoplasmic domain. A stop codon sequence after the first 51 bp of the insert effectively truncates this subunit, reducing the final cytoplasmic domain by 90% compared with the previously published P2xR1(-1) sequence, thereby reducing the overall peptide by approximately 25%. The region of the receptor lost in the truncated version coded for a number of serine roline rich regions which may act as potential intracellular regulatory sites.
Publisher: S. Karger AG
Date: 1997
DOI: 10.1159/000259233
Abstract: The ability to identify the expression of the protein subunits which assemble to form ionotropic receptors for acetylcholine and extracellular adenosine 5'-triphosphate (ATP) in in idual cells of the inner ear provides ex les of the high resolution and exquisite sensitivity which molecular biology brings to the study of hearing and balance. The data from these studies provide both fine detail with respect to the classification of the elements involved and an overview of the sites of potential interaction of both extracellular and intracellular signalling pathways. The high sensitivity necessitates a molecular physiological approach when using these techniques so that these data on the site and extent of expression can be balanced against functional significance. With the demonstration of expression of the alpha 9 subunit of the nicotinic acetylcholine receptor in cochlear outer hair cells, molecular biology has provided an explanation for the unusual cholinergic receptor pharmacology of the olivocochlear efferent innervation which has confounded investigators for decades. In addition, a role for extracellular ATP as a signalling molecule regulating electrochemical gradients and neurotransmission within the inner ear is supported by the extent of P2 receptor expression in this tissue, data which beg for intense functional study.
Publisher: Springer Science and Business Media LLC
Date: 02-1990
DOI: 10.1007/BF00204814
Publisher: Portland Press Ltd.
Date: 24-01-2005
DOI: 10.1042/BJ20040852
Abstract: The present study provides functional characterization of alternative splicing of the NTPDase2 (ecto-nucleoside triphosphate diphosphohydrolase-2) involved in the regulation of extracellular nucleotide concentrations in a range of organ systems. A novel NTPDase2β isoform produced by alternative splicing of the rat NTPDase2 gene provides an extended intracellular C-terminus and distinguishes itself from NTPDase2α isoform in gaining several intracellular protein kinase CK2 (casein kinase 2) phosphorylation sites and losing the intracellular protein kinase C motif. The plasmids containing NTPDase2α or NTPDase2β cDNA were used to stably transfect Chinese-hamster ovary-S cells. Imaging studies showed that NTPDase2α was predominantly membrane-bound, whereas NTPDase2β had combined cell surface and intracellular localization. α and β isoforms showed variations in alent cation dependence and substrate specificity for nucleoside-5′-triphosphates and nucleoside-5′-diphosphates. NTPDase2β exhibited reduced ATPase activity and no apparent ADPase activity. NTPDase2 isoforms demonstrated similar sensitivity to inhibitors such as suramin and pyridoxal phosphate-6-azophenyl-2′,4′-disulphonic acid, and differential regulation by protein kinases. NTPDase2β was up-regulated by intracellular protein kinase CK2 phosphorylation, whereas NTPDase2α activity was down-regulated by protein kinase C phosphorylation. The results demonstrate that alternative coding of the intracellular C-terminal domain contributes distinctive phenotypic variation with respect to extracellular nucleotide specificity, hydrolysis kinetics, protein kinase-dependent intracellular regulation and protein trafficking. These findings advance the molecular physiology of this enzyme system by characterizing the contribution of the C-terminal domain to many of the enzyme's signature properties.
Publisher: Frontiers Media SA
Date: 26-09-2022
DOI: 10.3389/FNEUR.2022.962227
Abstract: In the post-natal mouse cochlea, type II spiral ganglion neurons (SGNs) innervating the electromotile outer hair cells (OHCs) of the ‘cochlear lifier' selectively express the type III intermediate filament peripherin gene ( Prph) . Immunolabeling showed that Prph knockout (KO) mice exhibited disruption of this (outer spiral bundle) afferent innervation, while the radial fiber (type I SGN) innervation of the inner hair cells (~95% of the SGN population) was retained. Functionality of the medial olivocochlear (MOC) efferent innervation of the OHCs was confirmed in the Prph KO, based on suppression of distortion product otoacoustic emissions (DPOAEs) via direct electrical stimulation. However, “contralateral suppression” of the MOC reflex neural circuit, evident as a rapid reduction in cubic DPOAE when noise is presented to the opposite ear in wildtype mice, was substantially disrupted in the Prph KO. Auditory brainstem response (ABR) measurements demonstrated that hearing sensitivity (thresholds and growth-functions) were indistinguishable between wildtype and Prph KO mice. Despite this comparability in sound transduction and strength of the afferent signal to the central auditory pathways, high-intensity, broadband noise exposure (108 dB SPL, 1 h) produced permanent high frequency hearing loss (24–32 kHz) in Prph KO mice but not the wildtype mice, consistent with the attenuated contralateral suppression of the Prph KO. These data support the postulate that auditory neurons expressing Prph contribute to the sensory arm of the otoprotective MOC feedback circuit.
Publisher: Springer Science and Business Media LLC
Date: 03-08-2019
Publisher: Springer Science and Business Media LLC
Date: 02-1998
DOI: 10.1007/BF02740601
Abstract: Nearly two-thirds of military suicides involve firearms, and safe firearm storage is rare. To examine whether US military service members endorse greater openness to safe firearm storage depending on the content of the visual message they are randomly assigned to view. This comparative effectiveness study used a 3 × 2 × 2 factorial design to randomize US military service members to view 1 of 12 visual messages on safe firearm storage. Willingness to use safe firearm storage practices was assessed immediately before and after exposure to the message. Participants were recruited using the KnowledgePanel Calibration approach. Inclusion criteria included current membership in the US military and current firearm ownership. The KnowledgePanel s le was fielded from December 3 to 27, 2021, with a 76% completion rate and 45 in iduals determined to be qualified (28% qualification rate). The opt-in s le was fielded December 7, 2021, through January 4, 2022, with 699 in iduals (3%) qualified and 674 included in the final data set. Messages shared the same image and text on safe firearm storage but varied in messenger occupation (eg, primary care physician, security forces, or combat controller), the presence of text validating the perspective of firearm owners, and the presence of text validating the drive for home protection. Outcomes included changes in willingness to use 4 at-home (unloaded, separate from ammunition, in a locked location, and with a locking device) and 3 away-from-home (with family or friend, at a firearm retailer, or at a law enforcement agency) firearm storage practices. All analyses, including s le descriptives, are based on weighted data. Of the 719 in iduals in the data set, 367 (median [range] age, 33.64 [18-86] years 80.4% male 71.4% White) who endorsed not currently storing firearms using the methods assessed were included in analyses. In a multivariate analysis of variance, a significant interaction was found among time, messenger profession, gun-friendly text, and home protection text across all outcomes (Wilks' λ F = 2.09 P = .01 pη2 = 0.040) however, in a post hoc repeated-measures analysis of variance, the interaction was statistically significant only for storing firearms away from home with a trusted family member or friend (F = 5.42 P = .005 pη2 = 0.030). The profession of the messenger was more consistently associated with shifts in willingness than was the message content, although this varied across storage options. The findings of this comparative effectiveness study suggest that several combinations of messenger and content may be associated with willingness to endorse safe firearm storing practices, with particularly consistent positive findings for messages featuring security forces. The scalability and dosage potential of this intervention may render visual messaging valuable for promoting safe firearm storage at the population level.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 14-10-2008
DOI: 10.1126/SCISIGNAL.1162329
Abstract: Optics and electrophysiology reveal the dynamics of an ATP-gated ion channel signaling complex.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 03-1997
DOI: 10.1097/00001756-199703240-00004
Abstract: The site of extracellular ATP signalling in the retina was investigated by examining expression of the P2X2 receptor (P2X2R) subunit which assembles to form ATP-gated ion channels. Indirect in situ RT-PCR in situ hybridization localized the presence of mRNA for the P2X2R subunit within the soma of photoreceptors, inner nuclear layer neurones and the retina ganglion cells. Use of an antiserum specific for the P2X2R subunit confirmed the expression of the protein by these cells and demonstrated a particularly dense immunolabelling within the inner plexiform layer containing the dendritic processes of the retina ganglion cells. The outer segment of the photoreceptors also exhibited P2X2R-like immunoreactivity. The extensive expression of ATP-gated ion channel protein within the retina suggests that extracellular ATP plays erse neurohumoral roles in regulation of visual processing and cellular homeostasis.
Publisher: Society for Neuroscience
Date: 15-08-1997
Publisher: Elsevier BV
Date: 06-2017
Publisher: S. Karger AG
Date: 2001
DOI: 10.1159/000046824
Publisher: Springer Science and Business Media LLC
Date: 22-04-2022
Publisher: American Association for the Advancement of Science (AAAS)
Date: 23-04-2014
DOI: 10.1126/SCITRANSLMED.3008177
Abstract: Neurotrophin gene delivery, achieved by “close-field” electroporation of mesenchymal cells adjacent to the cochlear implant, improved hearing in deafened guinea pigs.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 08-1998
DOI: 10.1097/00001756-199808030-00008
Abstract: Reissner's membrane forms a partition between the endolymphatic and perilymphatic cochlear compartments. Expression of the P2X2 receptor subunit which assembles to form ATP-gated ion channels was detected in guinea-pig Reissner's membrane using the reverse transcription polymerase chain reaction (RT-PCR). The P2X2 receptor subunit protein was localized to the epithelial cells which line the endolymphatic surface of Reissner's membrane using confocal immunofluorescence. The P2X receptor expression in Reissner's membrane was functionally confirmed using whole-cell voltage-cl . An inwardly rectifying conductance was activated in Reissner's membrane epithelial cells in the presence of extracellular ATP (100 microM). This conductance had a pharmacological profile compatible with the P2X2 receptor designation, but exhibited substantial desensitisation which may be attributable to additional P2X receptor subunits. This study indicates that extracellular ATP, a humoral factor within scala media, acts via ATP-gated ion channels expressed by Reissner's membrane epithelial cells to decrease the driving force for sound transduction.
Publisher: Elsevier BV
Date: 11-2011
Publisher: S. Karger AG
Date: 2022
DOI: 10.1159/000524312
Abstract: b i Introduction: /i /b This retrospective cohort study of myringoplasty performed at Tauranga Hospital, Bay of Plenty, New Zealand from 2010 to 2020 sought to identify predictive factors for successful myringoplasty with particular consideration given to the known high prevalence of middle ear conditions in New Zealand Māori. b i Methods: /i /b Outcomes were surgical success (perforation closure at 1 month) and hearing improvement, which were correlated against demographic, pathological, and surgical variables. b i Results: /i /b 174 patients underwent 221 procedures (139 in children under 18 years old), with 66.1% of patients being New Zealand Māori and 24.7% New Zealand European ethnicity. Normalized by population demographics, New Zealand Māori were 2.3 times overrepresented, whereas New Zealand Europeans were underrepresented by 0.34 times (a 6.8 times relative treatment differential). The rate of surgical success was 84.6%, independent of patient age, gender, and ethnicity. A postauricular approach and the use of temporalis fascia grafts were both correlated with optimal success rates, whereas early postoperative infection (& #x3c month) was correlated with ∼3 times increased failure. Myringoplasty improved hearing in 83.1% of patients (average air-bone gap reduction of 10.7 dB). New Zealand Māori patients had ∼4 times greater preoperative conductive hearing loss compared to New Zealand Europeans, but benefited the most from myringoplasty. b i Discussion/Conclusion: /i /b New Zealand Māori and pediatric populations required greater access to myringoplasty, achieving good surgical and audiological outcomes. Myringoplasty is highly effective and significantly improves hearing, particularly for New Zealand Māori. Pediatric success rates were equivalent to adults, supporting timely myringoplasty to minimize morbidity from untreated perforations.
Publisher: Wiley
Date: 29-02-2008
Publisher: Springer International Publishing
Date: 2016
Publisher: Springer Science and Business Media LLC
Date: 17-02-2018
DOI: 10.1007/S12975-018-0615-1
Abstract: It is generally accepted that the cerebellum is particularly vulnerable to ischaemic injury, and this may contribute to the high mortality arising from posterior circulation strokes. However, this has not been systematically examined in an animal model. This study compared the development and resolution of matched photothrombotic microvascular infarcts in the cerebellar and cerebral cortices in adult 129/SvEv mice of both sexes. The photothrombotic lesions were made using tail vein injection of Rose Bengal with a 532 nm laser projected onto a 2 mm diameter aperture over the target region of the brain (with skull thinning). Infarct size was then imaged histologically following 2 h to 30-day survival using serial reconstruction of haematoxylin and eosin stained cryosections. This was complemented with immunohistochemistry for neuron and glial markers. At 2 h post-injury, the cerebellar infarct volume averaged ~ 2.7 times that of the cerebral cortex infarcts. Infarct volume reached maximum in the cerebellum in a quarter of the time (24 h) taken in the cerebral cortex (4 days). Remodelling resolved the infarcts within a month, leaving significantly larger residual injury volume in the cerebellum. The death of neurons in the core lesion at 2 h was confirmed by NeuN and Calbindin immunofluorescence, alongside activation of astrocytes and microglia. The latter persisted in the region within and surrounding the residual infarct at 30 days. This comparison of acute focal ischaemic injuries in cerebellar and cerebral cortices provides direct confirmation of exacerbation of neuropathology and faster kinetics in the cerebellum.
Publisher: Wiley
Date: 20-04-1998
DOI: 10.1002/(SICI)1096-9861(19980420)393:4<403::AID-CNE1>3.0.CO;2-4
Publisher: Frontiers Media SA
Date: 22-06-2021
DOI: 10.3389/FNCEL.2021.661857
Abstract: Central Nervous System (CNS) homeostasis and function rely on intercellular synchronization of metabolic pathways. Developmental and neurochemical imbalances arising from mutations are frequently associated with devastating and often intractable neurological dysfunction. In the absence of pharmacological treatment options, but with knowledge of the genetic cause underlying the pathophysiology, gene therapy holds promise for disease control. Consideration of leukodystrophies provide a case in point we review cell type – specific expression pattern of the disease – causing genes and reflect on genetic and cellular treatment approaches including ex vivo hematopoietic stem cell gene therapies and in vivo approaches using adeno-associated virus (AAV) vectors. We link recent advances in vectorology to glial targeting directed towards gene therapies for specific leukodystrophies and related developmental or neurometabolic disorders affecting the CNS white matter and frame strategies for therapy development in future.
Publisher: Elsevier BV
Date: 12-2000
DOI: 10.1016/S0165-0270(00)00322-8
Abstract: The cochlea presents a considerable challenge to the study of sound transduction and auditory neurotransmission. This arises from the location of the sensory, supporting and secretory epithelia, and primary auditory neurons within a complex ossified spiral structure comprised of three separate fluid-filled chambers. We have developed a novel cochlear slice preparation, which provides access to the highly differentiated tissues while retaining structural integrity and cell viability. Our technique for slicing the cochlea and imaging tissue structure facilitates the study of peripheral auditory signaling in situ. The preparation was developed in the neonatal rat (postnatal days 4-14) and is based on the use of vibrating blade microtome slicing after perfusing the perilymphatic compartments with chilled Pluronic F127 NF, a block copolymer gel. This material is liquid when cold, and sets when warmed to room temperature, stabilizing the cochlear fluid-filled compartments and thereby supporting the cochlear partition during slicing. Slices (150-300 microm) of neonatal rat cochlea, imaged using infrared videomicroscopy, allow tight-seal voltage cl recordings from a variety of cell types. Recordings obtained from primary auditory neurons, hair cells, supporting cells, and Reissner's membrane epithelial cells verify the viability of the tissues in the preparation. Data includes novel evidence for glutamatergic and purinergic co-transmission by primary auditory neurons. The preparation has considerable potential in a range of molecular physiological applications requiring cell-specific targeting with retention of cell connectivity.
Publisher: Elsevier BV
Date: 07-2000
DOI: 10.1016/S0165-1838(00)00116-8
Abstract: Investigation of the multiple roles of extracellular nucleotides in the cochlea has developed from analysis of ATP-activated conductances in single sensory hair cells. Molecular probes such as radiolabelled ATP analogues and radiolabelled mRNA for ATP-gated ion channel subunits (P2X receptors) rapidly revealed the extensive nature of ATP signalling in this sensory organ. This has provided a foundation for physiological investigations which put extracellular nucleotides at the centre of homeostatic regulation of the driving force for sound transduction, modulation of mechanical tuning, control of cochlear blood flow and auditory neurotransmission. The purinergic signal transduction pathways associated with these processes have several novel features of significance to the broader field of purinergic neuroscience. In turn, these studies have benefited from the recent experimental advances in the field of purinergic signalling, a significant component of which is associated with the work of Professor Geoffrey Burnstock.
Publisher: Elsevier BV
Date: 05-1994
DOI: 10.1016/0378-5955(94)90054-X
Abstract: Poly(A)+ RNA was extracted from rat cochleae using guanidinium thiocyanate and oligo(dT)-cellulose, and converted into cDNA by reverse transcriptase using an oligo(dT) primer. Oligonucleotides complementary to conserved 5' and 3' regions of alpha and beta subunits of the neuronal nicotinic acetylcholine receptor subunit (nAChR) family were then used as primers to screen the cochlear cDNA via the polymerase chain reaction (PCR) procedure. PCR products of approximately 900 bp length, purified by agarose gel electrophoresis, were nick translated to produce [32P]-dCTP labelled probes for Southern Blot screening of nAChR cDNAs. Of the four alpha and three beta subunits screened, only alpha 5 and beta 4 nAChR cDNAs hybridized. The alpha 5 PCR product was cloned and sequenced and proved to be identical to published sequence for alpha 5. The detection of alpha 5 and beta 4 nAChR subunit expression in cochlear tissue supports previous electrophysiological and immunocytochemical evidence for nAChR-mediated centrifugal control of hearing function.
Publisher: Frontiers Media SA
Date: 15-05-2019
Publisher: Elsevier BV
Date: 2007
DOI: 10.1016/J.BIOCEL.2007.01.003
Abstract: Membrane-bound NTPDase2 is a member of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) enzyme family involved in the regulation of P2 receptor signaling. NTPDase2 has broad substrate specificity for extracellular nucleotides, but hydrolyses nucleoside 5'-triphosphates with high preference over nucleoside 5'-diphosphates. In this study, we have sought to determine how enzyme substrates acting on P2 receptors affect intracellular NTPDase2 trafficking. To achieve this, Chinese hamster ovary (CHO) cells were transiently transfected with rat-specific NTPDase2 cDNA tagged with green fluorescent protein (GFP), to allow direct visualisation of subcellular localisation and trafficking of NTPDase2. Cells were superfused with NTPDase2 substrates (ATP and UTP) and synthetic nucleotide analogues (ATPgammaS and ADPbetaS), and confocal image stacks were acquired at regular time intervals. NTPDase2 incorporation into the plasma membrane was determined by comparative analysis of fluorescence intensity in the cytosolic and membrane compartments. GFP-tagged NTPDase2 was fully functional and ATP and ATPgammaS induced membrane incorporation of GFP-NTPDase2 from putative intracellular stores, whilst UTP and ADPbetaS were ineffective. The increased ATP hydrolysis rate correlated with increased NTPDase2 trafficking to the plasma membrane. ATP-induced NTPDase2 trafficking was mediated by activation of endogenous P2X receptors involving Ca2+ entry rather than by P2Y receptor-induced release of Ca2+ from intracellular stores. Our results suggest that P2X receptor activation stimulates insertion of latent NTPDase2 into the plasma membrane. The increase in surface-located NTPDase2 may reflect a regulatory mechanism counteracting excessive stimulation and desensitisation of P2 receptors.
Publisher: Wiley
Date: 10-2003
Publisher: Wiley
Date: 12-1988
DOI: 10.1113/JPHYSIOL.1988.SP017371
Abstract: 1. An attempt has been made to test the hypothesis that in the nucleus of the tractus solitarius (NTS) in the rat, the most caudal region of synaptic terminals of the carotid sinus nerve, just caudal to the obex, represents mainly the site of synapse of chemoreceptor fibres from the carotid body. 2. Under halothane anaesthesia, the neurotoxin kainic acid was used to lesion this region and a second region, immediately rostral to obex, where terminals are thought to arise mainly from baroreceptor fibres of the carotid sinus nerve. 3. Measurements based on the distribution of fluorescent dye co-injected with the kainic acid showed that the two groups of 100 nl microinjections were centered 0.82 mm apart and that the injectate spread through mean distances of 0.57 mm (caudal microinjections) and 0.52 mm (rostral microinjections). Nissl staining was used to determine cellular degeneration. The caudal lesions mostly involved ventrolateral and commissural subnuclei of NTS and the rostral lesions involved lateral and dorsolateral subnuclei. 4. Ventilatory sensitivity to hypoxia was tested under light halothane anaesthesia, 1 day after lesioning. To enhance the responses, the contralateral carotid sinus nerve was sectioned prior to experiments. Caudal lesions reduced the ventilatory response to inspired oxygen (20.9-9.6% O2) by a mean of 67% and rostral lesions by 18% of the effect produced by carotid sinus nerve section on that side. Subsequent section of the carotid sinus nerve on the side of the NTS lesion confirmed that caudal lesions produced effects comparable to those of carotid body denervation rostral lesions did not. 5. These results strongly support the hypothesis that chemoreceptor and baroreceptor afferent fibres in the carotid sinus nerve synapse at substantially separable sites in the nucleus of the tractus solitarius. The identification of the site in NTS caudal to the obex as the principal site of carotid chemoreceptor synapses places them close to but not upon respiratory premotor neurones of the same nucleus.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 1998
DOI: 10.1097/00001756-199801260-00019
Abstract: Spiral ganglion neurones in rat cochlea express three different isoforms of the P2X2 receptor subunit which assemble into ATP-gated ion channels. Two of these P2X2R subunit isoforms have previously been detected in other auditory tissues. The third isoform (designated P2X2-3R) has not been described. This isoform lacks 39 bp immediately prior to the stop codon, corresponding to a 13 amino acid deletion of the extreme C-terminus domain. Using direct in situ RT-PCR, expression of P2X2R mRNA was confined to a subpopulation of type I spiral ganglion neurones. This study supports a role for extracellular ATP as a neurotransmitter for a discrete population of auditory neurones where variation in P2X2R isoform assembly may confer functional heterogeneity, including enhanced desensitization.
Publisher: Springer Science and Business Media LLC
Date: 06-2010
Publisher: Oxford University Press (OUP)
Date: 06-05-2020
Abstract: Hyperphosphorylation and deposition of tau in the brain characterizes frontotemporal dementia and Alzheimer’s disease. Disease-associated mutations in the tau-encoding MAPT gene have enabled the generation of transgenic mouse models that recapitulate aspects of human neurodegenerative diseases, including tau hyperphosphorylation and neurofibrillary tangle formation. Here, we characterized the effects of transgenic P301S mutant human tau expression on neuronal network function in the murine hippoc us. Onset of progressive spatial learning deficits in P301S tau transgenic TAU58/2 mice were paralleled by long-term potentiation deficits and neuronal network aberrations during electrophysiological and EEG recordings. Gene-expression profiling just prior to onset of apparent deficits in TAU58/2 mice revealed a signature of immediate early genes that is consistent with neuronal network hypersynchronicity. We found that the increased immediate early gene activity was confined to neurons harbouring tau pathology, providing a cellular link between aberrant tau and network dysfunction. Taken together, our data suggest that tau pathology drives neuronal network dysfunction through hyperexcitation of in idual, pathology-harbouring neurons, thereby contributing to memory deficits.
Publisher: Elsevier BV
Date: 10-1995
DOI: 10.1016/0378-5955(95)00151-1
Abstract: There is accumulating evidence for a purinergic humoral system involved in the control of cochlear function. Evidence of specific P2 purinoceptors on cochlear tissues implies a role for extracellular adenosine triphosphate (ATP) in the cochlea. To further this hypothesis a study was undertaken to determine if there was any specific source of purine compounds in cochlear tissues. Cochlear tissues (the sensory epithelium and lateral wall) from the guinea pig were incubated with the acridine derivative quinacrine dihydrochloride (5 x 10(-6) M in phosphate-buffered saline for 30 min at room temperature) which fluoresces on binding to high concentrations of ATP. Most cochlear tissues showed a diffuse green fluorescence slightly above the background level. However, a region of the marginal cells of the stria vascularis showed a specific punctate fluorescence. Optical sectioning of these cells by confocal microscopy revealed that the fluorescent structures in these marginal cells was confined to a region up to 10 microns from their endolymphatic surface. Similar cells studied by transmission electron microscopy showed membrane-bound vesicles located in the same region of the cell. These data imply that purine compounds are localized in discrete structures, perhaps vesicles, within the marginal cells which could serve as a source of extracellular ATP in the cochlea.
Publisher: Springer Science and Business Media LLC
Date: 24-09-2014
Publisher: Wiley
Date: 12-1983
Abstract: This study utilizes anterograde axonal transport of cobaltous-lysine and conventional silver-staining techniques to study the central projections of the horizontal semicircular canal branch of the VIII nerve within the vestibular nuclei of the carpet shark Cephaloscyllium isabella. Two major terminating axon fields were observed, one caudal and one rostral to the entrance of the VIII nerve, corresponding to the ventral vestibular nucleus and superior vestibular nucleus, respectively. Both fields appear to be located within the ventral portion of the nuclei indicating an apparent sub ision of the VIII nerve projections within the brainstem. The resolution of the sensitive cobalt tracer indicates the presence of both dendritic and pericellular termination of these primary afferent fibres. In the area immediately caudal to the entrance of the VIII nerve a number of labelled primary afferent fibres project to the ventral region of the intermediate nucleus. Other fibres follow the visceral sensory root VII and terminate proximal to the sulcus limitans of His within the dendritic field of the neurons of the nucleus magnocellularis. Some fibres turn ventromedially from the main group of the ascending fibres and terminate in the area of the inferior reticular formation.
Publisher: Frontiers Media SA
Date: 09-05-2023
DOI: 10.3389/FNINS.2023.1182874
Abstract: Protein synthesis is a fundamental process that underpins almost every aspect of cellular functioning. Intriguingly, despite their common function, recessive mutations in aminoacyl-tRNA synthetases (ARSs), the family of enzymes that pair tRNA molecules with amino acids prior to translation on the ribosome, cause a erse range of multi-system disorders that affect specific groups of tissues. Neurological development is impaired in most ARS-associated disorders. In addition to central nervous system defects, diseases caused by recessive mutations in cytosolic ARSs commonly affect the liver and lungs. Patients with biallelic mutations in mitochondrial ARSs often present with encephalopathies, with variable involvement of peripheral systems. Many of these disorders cause severe disability, and as understanding of their pathogenesis is currently limited, there are no effective treatments available. To address this, accurate in vivo models for most of the recessive ARS diseases are urgently needed. Here, we discuss approaches that have been taken to model recessive ARS diseases in vivo , highlighting some of the challenges that have arisen in this process, as well as key results obtained from these models. Further development and refinement of animal models is essential to facilitate a better understanding of the pathophysiology underlying recessive ARS diseases, and ultimately to enable development and testing of effective therapies.
Publisher: Wiley
Date: 12-1983
Abstract: This study utilizes retrograde axonal transport of cobaltous-lysine, and conventional silver and Golgi staining techniques to study the abducens motor nucleus innervating the external rectus muscle of the carpet shark. The nucleus consists of 300-400 motoneurons located immediately ventrolateral to the medial longitudinal fasciculus (MLF), distributed over about 1.25 mm in a rostrocaudal direction at the level of exit of the VI nerve. The axons of the motoneurons form seven or eight discrete ventrally directed fascicles which, having exited from the brainstem, group together to form the abducens (VI) nerve. The motoneurons are on average about 16 micron in diameter, are bipolar, and their dendrites have a transverse orientation. Typically one set of dendrites penetrates the MLF and the other set extends ventrally into the reticular formation.
Publisher: Elsevier BV
Date: 08-2002
DOI: 10.1016/S0378-5955(02)00460-4
Abstract: Extracellular ATP and other extracellular nucleotides acting via P2 receptors in the inner ear initiate a wide variety of signalling pathways important for regulation of hearing and balance. Ectonucleotidases are extracellular nucleotide-metabolising enzymes that modulate purinergic signalling in most tissues. Major ectonucleotidases in the cochlea are likely members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family. In this study, we provide a detailed description of NTPDase1 and NTPDase2 distribution in cochlear tissues using immunocytochemistry. E-NTPDase immunoreactivity was not equally distributed in the tissues bordering scala media. It was observed in the organ of Corti, including sensory and supporting cells, but was notably absent from Reissner's membrane and most of the marginal cells of the stria vascularis. NTPDase1 expression was most prominent in the cochlear vasculature and cell bodies of the spiral ganglion neurones, whereas considerable NTPDase2 immunoreactivity was detected in the stria vascularis. Both E-NTPDases were expressed in the cuticular plates of the sensory hair cells and nerve fibres projecting from the synaptic area underneath the inner and outer hair cells. E-NTPDase localisation corresponds to the reported distribution of some P2X receptor subunits (P2X(2) in particular) in sensory, supporting and neural cells and also P2Y receptor distribution in the vasculature and secretory tissues of the lateral wall. The role for E-NTPDases in purinergic signalling is most likely to regulate extracellular nucleoside triphosphate and diphosphate levels and thus provide termination for extracellular ATP signalling that has been linked to control of cochlear blood flow, electrochemical regulation of sound transduction and to neurotransmission in the cochlea.
Publisher: Elsevier BV
Date: 03-2002
DOI: 10.1016/S0028-3908(01)00184-8
Abstract: Spiral ganglion neurones provide the primary afferent innervation to sensory hair cells within the mammalian cochlea. Recent evidence suggests that their function may be modulated by purinergic signalling mechanisms, associated with release of adenosine 5'-triphosphate (ATP). Utilising a newly developed slice preparation of the neonatal rat cochlea, we have investigated the response of neurones in situ, to purinergic agonists and antagonists using whole-cell voltage cl recordings. In cells identified as type I spiral ganglion neurones on the basis of morphology and voltage-dependent conductances, pressure-applied ATP, alpha,beta-methyleneATP (alpha,beta-meATP), 2-methylthioATP (2-MeSATP) and adenosine 5'-diphosphate (ADP) elicited a consistent phenotype of desensitising, inwardly rectifying current. The ATP-activated currents were reversibly blocked by the P2X receptor antagonists pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 10 microM), and 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP IC(50) 407 nM). Neurones were more sensitive to ATP at low pH. The EC(50) value for ATP shifted from 18 microM at pH 7.3, to 1 microM at pH 6.3, with Hill coefficients of approximately 1. The results indicate that ATP-gated ion channels in spiral ganglion neurones arise from a specific heteromultimeric assembly of P2X receptor subunits which has no correspondence with present recombinant P2X receptor models.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 12-02-2007
Publisher: Society for Neuroscience
Date: 11-1994
DOI: 10.1523/JNEUROSCI.14-11-06992.1994
Abstract: Fluorescence imaging of extracellular adenosine-5′-triphosphate (ATP) binding sites on inner and outer hair cells isolated from the guinea pig organ of Corti was achieved using the fluorescent analog of ATP, 2′- (or-3′)-O-(trinitrophenyl)adenosine-5′- triphosphate (TNP-ATP 30–75 microM). This analog, which fluoresces on binding to these sites, was pressure applied by micropipette while hair cells were viewed by fluorescence microscopy. Fluorescence imaging revealed a widespread distribution of extracellular binding sites, including the stereocilia, cuticular plate, and the basolateral margins of the cells, but particularly in infracuticular and infranuclear regions. In support of extracellular binding, simultaneous electrophysiological recordings demonstrated that rapid washout of TNP-ATP-induced fluorescence was dependent upon cell integrity. Suramin, a nonselective P2 purinoceptor antagonist, coapplied with TNP-ATP, reduced the fluorescence observed on the stereocilia and apical surface of the cuticular plate only. This implies that binding sites on the apical surface of hair cells are P2 receptors, consistent with previous electrophysiological evidence for localization of P2 receptors to the apical surface of cochlear hair cells (Housley et al., 1992). Binding of TNP-ATP to P2 purinoceptors was confirmed by its antagonism of the inward current elicited by ATP (10 microM) in voltage-cl ed hair cells. Fluorescence from the basolateral margin was significantly quenched when TNP-ATP was applied in alent cation-free solution. Because alent cations are required for ATPase activity, this finding provides evidence for the presence of ecto-ATPases on the basolateral membrane of hair cells. The alent cation-free condition had no significant effect on the ATP-gated P2 purinoceptor conductance. We propose that there are two classes of ATP binding sites on cochlear hair cells: apically located P2 purinoceptors gating nonselective cation channels and basolaterally located ecto- ATPases that may be involved in purine turnover.
Start Date: 06-2015
End Date: 12-2018
Amount: $350,000.00
Funder: Australian Research Council
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End Date: 04-2015
Amount: $354,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 02-2015
End Date: 12-2018
Amount: $384,700.00
Funder: Australian Research Council
View Funded ActivityStart Date: 2014
End Date: 12-2014
Amount: $250,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 2018
End Date: 12-2018
Amount: $443,311.00
Funder: Australian Research Council
View Funded ActivityStart Date: 2010
End Date: 12-2015
Amount: $280,000.00
Funder: Australian Research Council
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