Biomedical subjects

A Forge

Publications and source records attributed to A Forge.

At least 19 recordsLinked to original sources

PubMed2006

Relationship of opioid receptors with GABAergic neurons in the rat inferior colliculus.

The inferior colliculus is a critical structure for processing auditory information and receives ascending and descending synaptic auditory projections. In addition to GABAergic and glutamatergic innervations, other neurotransmitter systems are also reported in the inferior colliculus, including opioid peptides. In the present study, the relative distribution of each type of opioid receptor, mu (MOR), delta (DOR) and kappa (KOR) within GABAergic neurons in the inferior colliculus was examined. GABA immunoreactivity was expressed by small, medium and large neurons and distributed in the central nucleus and the pericentral nucleus of the inferior colliculus. Immunostaining for MOR, DOR and KOR receptors was found in both disc-shaped cells and stellate cells. Punctiform beta-endorphin immunolabelling was observed in the proximity of GABA-positive neurons. Co-localization of GABA and MOR receptors was observed in neurons and nerve terminals in the central nucleus, dorsal cortex and external cortex of the inferior colliculus. Quantification of the co-localization patterns determined that a higher proportion of GABA neurons was associated with MOR receptors compared with KOR or DOR receptors.

Animals↗

PubMed2006

Arp2/3 complex-mediated actin polymerisation occurs on specific pre-existing networks in cells and requires spatial restriction to sustain functional lamellipod extension.

The classical Arp2/3-mediated dendritic network defines the cytoskeleton at the leading edge of crawling cells, and it is generally assumed that Arp2/3-mediated actin polymerization generates the force necessary to extend lamellipods. Our previous work suggested that successful lamellipod extension required not only free barbed ends for actin polymerization but also a proper ultrastructural organization of the cytoskeleton. To further explore the structural role of the Arp2/3 complex-mediated networks in lamellipod morphology and function, we performed a detailed analysis of the ultrastructure of the Arp2/3-mediated networks, using the WA domains of Scar and WASp to generate mislocalised Arp2/3 networks in vivo, and to reconstruct de novo Arp2/3-mediated actin nucleation and polymerization on extracted cytoskeletons. We present here evidence that spatially unrestricted Arp2/3-mediated networks are intrinsically three-dimensional and multilayered by nature and, as such, cannot sustain significant polarized extension. Furthermore, such networks polymerize only at preferred locations in extracted cells, corresponding to pre-existing Arp2/3 networks, suggesting that the specific molecular organization of the actin cytoskeleton, in terms of structure and/or biochemical composition, dictates the location of Arp2/3 complex-mediated actin polymerization. We propose that successful lamellipod extension depends not only on localized actin polymerization mediated through local signalling, but also on spatial restriction of the Arp2/3 complex-mediated nucleation of actin polymerization, both in terms of location within the cell and ultrastructural organization of the resulting network.

Actin-Related Protein 2-3 Complex↗

PubMed2006

Caspase-independent pathways of hair cell death induced by kanamycin in vivo.

Cochlear and vestibular sensory cells undergo apoptosis when exposed to aminoglycoside antibiotics in organ culture, but mechanisms of chronic drug-induced hair cell loss in vivo are unclear. We investigated cell death pathways in a mouse model of progressive kanamycin-induced hair cell loss. Hair cell nuclei showed both apoptotic- and necrotic-like appearances but markers for classic apoptotic pathways (cytochrome c, caspase-9, caspase-3, JNK, TUNEL) were absent. In contrast, drug treatment caused EndoG translocation, activation of mu-calpain, and both the synthesis and activation of cathepsin D. Poly (ADP-ribose) polymerase 1 (PARP1) was decreased, but a caspase-derived 89 kDa PARP1 fragment was not present. The mRNA level of PARP1 remained unchanged. Thus, chronic administration of aminoglycosides causes multiple forms of cell death, without a major contribution by classic apoptosis. These results provide a better understanding of the toxic effects of aminoglycosides and are relevant to design protection from aminoglycoside-induced hearing loss.

Animals↗

PubMed2005

Specific loss of connexin 26 expression in ductal sweat gland epithelium associated with the deletion mutation del(GJB6-D13S1830).

A whole array of cutaneous syndromes is associated with distinct dominant mutations in GJB2 encoding the gap junction protein connexin 26 (C x 26), including Vohwinkel's syndrome and keratitis-ichthyosis-deafness syndrome. In contrast, recessive GJB2 mutations occur in a large proportion of individuals with hearing loss but no obvious dermatological phenotype. Recently, a large deletion of approximately 342 kb, encompassing the coding region of GJB6 encoding C x 30, but not affecting GJB2, was shown to be associated with hearing loss. From analysis of patient skin, we provide immunohistochemical and bioinformatic data to show that the expression of C x 26 is affected by del(GJB6-D13S1830) in a cell-type-specific manner within the sweat gland. This putative regulatory element of C x 26 expression may be a key factor related to the severe or profound deafness associated with del(GJB6-D13S1830).

Chromosome Deletion↗

PubMed2001

Differential vulnerability of basal and apical hair cells is based on intrinsic susceptibility to free radicals.

The base of the cochlea is more vulnerable to trauma than the apex as seen in the pattern of hair cell damage by cisplatin or aminoglycosides. The differential vulnerability is maintained in organotypic cultures exposed directly to these drugs, suggesting there may be an intrinsic difference in sensitivity to damage along the cochlear spiral. We therefore investigated the survival capacity of isolated outer hair cells and strips dissected from different turns of the guinea pig organ of Corti in short-term culture. Cells were stained with fluorescent indicators of viable or dead cells, calcein-AM and ethidium homodimer. After 5 h at room temperature, up to 90% of outer hair cells from the apex survived, but less than 30% from the base. In contrast, basal inner hair cells remained viable, and supporting cells survived for at least 20 h. The difference in survival capacity between basal and apical outer hair cells coincided with a significantly lower level of the antioxidant glutathione in basal outer hair cells compared with apical outer hair cells. This suggested that basal outer hair cells may be more vulnerable to free-radical damage than apical outer hair cells. The survival of basal outer hair cells was significantly improved by addition of the radical scavengers n-acetyl cysteine, p-phenylenediamine, glutathione, mannitol or salicylate. The protection by antioxidants implies that the accelerated death of basal outer hair cells is due to free-radical damage. The results support an intrinsic susceptibility to free radicals that differs among cochlear cell populations. This differential provides a rational explanation for base-to-apex gradients observed in various forms of cochlear pathology.

Animals↗

PubMed2000

Apoptotic death of hair cells in mammalian vestibular sensory epithelia.

Hair cell death was examined in cultured explants of vestibular organs from mature guinea pigs and gerbils. The effects of gentamicin were compared with those of staurosporine, a membrane-permeable kinase inhibitor that induces programmed cell death in almost all cell types. Under the conditions used staurosporine killed hair cells but supporting cells appeared unaffected, and a topographic pattern of differential sensitivity to staurosporine amongst hair cells, similar to that described for aminoglycoside antibiotics, was revealed. This suggests such differential sensitivity is an inherent property of the hair cell population. Thin sectioning, and examination of whole mount preparations after application of the TUNEL procedure or after double fluorescent labelling with phalloidin and with propidium iodide, which labels nuclei, revealed that hair cells after exposure to gentamicin show features identical to those of apoptotic cells after exposure to staurosporine. Furthermore, cells showing features of apoptosis constitute a major proportion of the hair cells that are ultimately lost following exposure to gentamicin. Incubation of cultures with gentamicin in the presence of broad-spectrum inhibitors of caspases, proteases involved specifically in the cell death pathway, prevented almost all of the hair cell deaths normally triggered by gentamicin. This confirms that apoptosis is the predominant mode of hair cell death after gentamicin exposure. Hair cells exposed to gentamicin in the presence of caspase inhibitors appeared to be preserved intact. This, and the thin section observations, suggests that apoptotic death is the fate of the majority of hair cells affected by that drug and that any sub-lethal damage to hair cells exposed to gentamicin does not result in significant morphological alterations. Hair cell death was also prevented by deferoxamine which has been shown to protect cochlear hair cells in vivo from the effects of gentamicin. Explant cultures of mature vestibular organs may be, therefore, a useful model system for examining putative hair cell protecting agents.

Animals↗

PubMed1999

A missense mutation in myosin VIIA prevents aminoglycoside accumulation in early postnatal cochlear hair cells.

Myosin VIIA is expressed by sensory hair cells in the inner ear and proximal tubule cells in the kidney, the two primary targets of aminoglycoside antibiotics. Using cochlear cultures prepared from early postnatal Myo7a6J mice with a missense mutation in the head region of the myosin VIIA molecule we show that this myosin is required for aminoglycoside accumulation in cochlear hair cells. Hair cells in homozygous mutant Myo7a6J cochlear cultures have disorganized hair bundles, but are otherwise morphologically normal and transduce. However, and in contrast to hair cells from heterozygous Myo7a6J cultures, the homozygous Myo7a6J hair cells do not accumulate [3H]gentamicin and do not exhibit an ototoxic response on exposure to aminoglycoside. Possible roles for myosin VIIA in the process of aminoglycoside accumulation are discussed.

Animals↗

PubMed1999

Gap junctions and connexin expression in the inner ear.

Several different recessive mutations in the connexin26 (Cx26; beta 2) gene have been associated with non-syndromic hereditary deafness. This suggests gap junctions are important to cochlear function. Numerous large gap junctions are present between adjacent supporting cells in both the vestibular and auditory sensory epithelia of the mature inner ear. In vestibular organs, Cx26 is highly expressed, but antibodies of Cx32 (beta 1) also label the supporting cells. In the organ of Corti of the cochlea, Cx26 is the predominant connexin isoform; neither Cx32 nor Cx43 (alpha 1) can be detected by immunohistochemistry. One role for gap junctions between supporting cells may be to provide a pathway for the rapid removal of ions away from the region of the sensory cells during transduction in order to maintain sensitivity. In the cochlea gap junctions are also associated with the basal cells of the stria vascularis, an ion-transporting epithelium that maintains a positive electrical potential in the potassium-rich endolymph fluid which bathes the apical surfaces of the sensory 'hair' cells and which is crucial for auditory transduction. Gap junctions are present between fibrocytes in the spiral ligament that underlies the stria vascularis, and between these fibrocytes and strial basal cells. During cochlear development, the initial formation and subsequent increase in size and number of gap junctions in the stria vascularis coincides with the initial generation and rise of the endocochlear potential. This and other evidence suggests that one role of gap junctions in the cochlea is to provide a pathway for passage of ions to maintain endolymph and, thus, auditory acuity. Mutations to Cx26 could, therefore, disrupt this ion circulation, resulting in deafness.

Amino Acid Sequence↗

PubMed1999

A sugar transporter as a candidate for the outer hair cell motor.

Forces developed by cochlear outer hair cells (OHCs) are responsible for the sharp tuning that underlies sensitivity and frequency selectivity in the ear. OHCs exhibit a voltage-dependent motility involving a 'motor' protein embedded in the basolateral membrane. The motor has so far resisted molecular identification. Here we provide evidence that it may be related to a fructose transporter. We show that OHCs are able to transport this sugar selectively and that the sugar alters electrical properties of the OHC motor. These data can be combined into an integrated model of a sugar carrier, that makes the novel prediction, demonstrated here, that such 'neutral' transporters can be voltage dependent.

Animals↗

PubMed1999

Properties of connexin26 gap junctional proteins derived from mutations associated with non-syndromal heriditary deafness.

Three point mutations of the connexin26 (GJB2) gene associated with hereditary deafness were studied using in vitro expression systems. Mutation M34T results in an amino acid substitution in the first transmembrane domain of the connexin protein, W77R is located in the second transmembrane domain and W44C is in the first extracellular loop. Wild-type and mutated connexin vectors were constructed and transfected into communication-deficient HeLa cells to obtain transient expression of the connexin proteins. Intercellular coupling was subsequently assessed by examining transfer of Lucifer yellow between cells. All three mutations resulted in impaired intercellular coupling. The mechanistic reasons for the functional inadequacies of the mutated proteins were investigated. First, intracellular trafficking and targeting of the expressed connexins were determined by immunohistochemistry. Mutation W77R was inefficiently targeted to the plasma membrane and retained in intracellular stores whereas the other two were targeted to the plasma membrane. Oligomerization assays showed that connexins M34T and W77R failed to assemble efficiently into hexameric gap junction hemichannels, but the W44C mutation did so. A cell-free translation system showed that the mutated proteins were inserted into microsomal membranes but the mutations have different effects on the post-translational properties of the expressed proteins. The results point to the conclusion that mutations in the transmembrane domains of connexin proteins influence gap junction assembly.

Animals↗

PubMed1998

Hair cell recovery in the vestibular sensory epithelia of mature guinea pigs.

The progression of recovery of the vestibular sensory epithelia of guinea pigs after gentamicin-induced hair cell injury was assessed quantitatively and qualitatively. Evaluations were made of the number of cells bearing hair bundles by using scanning electron microscopy (SEM) and of identifiable hair cells in thin sections. Both assessment procedures showed that an initial loss of hair cells in utricular maculae is followed by significant recovery in the number of hair cells present. SEM also showed recovery in saccules comparable to that in utricles. During the recovery, progressive maturation of hair bundles, which exhibited features similar to those seen during normal ontogenetic development of hair cells, could be identified. The pattern and extent of hair cell loss and subsequent reappearance revealed by SEM corresponded with that derived from analysis of thin sections. This suggests that repair of nonlethally damaged hair cells is unlikely but, rather, that new hair cells are produced. An apparent decrease in supporting cell numbers was observed coincident with the increase in hair cell numbers. This complements previous morphological observations, which have suggested new hair cells arise from direct, nonmitotic transdifferentiation of supporting cells. The quantitative analyses indicate that more than half of the hair cells that are lost are replaced, but the recovery process does not result in complete restoration of the epithelium. Eight months after the end of drug treatment, the number of hair cells present was still significantly less than normal, and several other abnormalities persisted. There was also no evidence of any hair cell recovery in the organ of Corti. Thus, there appear to be limitations on the capacity for spontaneous replacement of lost hair cells in the mammalian inner ear.

Animals↗

PubMed1998

Intercellular junctional maturation in the stria vascularis: possible association with onset and rise of endocochlear potential.

The postnatal maturation of intercellular junctions of marginal and basal cells of the stria vascularis was examined in the gerbil using thin sections and freeze fracture techniques. Immunohistochemical methods were used to determine the presence of Na,K-ATPase postnatally. The onset and growth of endocochlear potential (EP) was also measured. In marginal cells, the apical surface and junctional region around the apical pole of the cell was found to have adult-like characteristics by the time of onset of EP, whilst the increase in staining for Na.K-ATPase temporally coincided with an increasing density of intra-membrane protein particles on the infoldings of marginal cell lateral membranes. Maturation of the junctional specialisations of the basal cells was found to correspond temporally with the period of onset and rise of EP. Tight junctions between basal cells first appeared as small, broken strands composed of widely spaced particles at 6 days after birth (DAB). These junctional strands increased in number and in particle density until adult-like at 16 DAB when they covered large areas of the basal cell lateral membrane. Gap junctions on the apical membrane of basal cells first appeared as small patches of loosely packed junctional elements at 6 DAB. Between 8 and 16 DAB the area of membrane occupied by the gap junctions increased, reaching a mature conformation by 18 DAB. The results suggest that EP maturation is dependent upon the development of sealing between the basal cells by tight junctions and also the establishment and development of gap junctions in the apical plasma membrane of basal cells, associated with intermediate cells.

Age Factors↗

PubMed1997

Myosin VIIA is required for aminoglycoside accumulation in cochlear hair cells.

Myosin VIIA is expressed by sensory hair cells and has a primary structure predicting a role in membrane trafficking and turnover, processes that may underlie the susceptibility of hair cells to aminoglycoside antibiotics. [3H]Gentamicin accumulation and the effects of aminoglycosides were therefore examined in cochlear cultures of mice with different missense mutations in the myosin VIIA gene, Myo7a, to see whether myosin VIIA plays a role in aminoglycoside ototoxicity. Hair cells from homozygous mutant Myo7ash1 mice, with a mutation in a nonconserved region of the myosin VIIA head, respond rapidly to aminoglycoside treatment and accumulate high levels of gentamicin. Hair cells from homozygous mutant Myo7a6J mice, with a mutation at a highly conserved residue close to the ATP binding site of the myosin VIIA head, do not accumulate [3H]gentamicin and are protected from aminoglycoside ototoxicity. Hair cells from heterozygotes of both alleles accumulate [3H]gentamicin and respond to aminoglycosides. Although aminoglycoside uptake is thought to be via apical surface-associated endocytosis, coated pit numbers on the apical membrane of heterozygous and homozygous Myo7a6J hair cells are similar. Pulse-chase experiments with cationic ferritin confirm that the apical endocytotic pathway is functional in homozygous Myo7a6J hair cells. Transduction currents can be recorded from both heterozygous and homozygous Myo7a6J hair cells, suggesting it is unlikely that the drug enters via diffusion through the mechanotransducer channel. The results show that myosin VIIA is required for aminoglycoside accumulation in hair cells. Myosin VIIA may transport a putative aminoglycoside receptor to the hair cell surface, indirectly translocate it to sites of membrane retrieval, or retain it in the endocytotic pathway.

Adenosine Triphosphate↗

PubMed1997

Postnatal maturation of the organ of Corti in gerbils: morphology and physiological responses.

The organ of Corti, the sensory epithelium of hearing in mammals, matures postnatally in the gerbil. Quantitative analyses of the postnatal development of the organ of Corti, including supporting cells and the basilar membrane, were carried out. The morphological study confirmed that maturation of the sensory cells proceeds with a base-to-apex gradient, with the outer hair cells appearing to mature before the inner hair cells. Maturation of the supporting cells and the basilar membrane commenced first in the middle turn. Expansion of the second row of Deiters' cells began at 6 days after birth in the middle turn, before enlargement of the pillar cell heads at 8 days postnatally. Pillar cell head enlargement continued until 20 days postnatally in the middle turn. The tunnel of Corti and spaces of Nuel appeared first in the middle turn between 8 and 10 days postnatally. The maturation of the basilar membrane involved the thickening of the central hyaline layer and a reduction in the epithelial cells on the tympanic aspect. This process continued until about 20 days after birth. The cochlear microphonic potential, whole nerve action potential, and stimulus frequency otoacoustic emissions were recorded from 12 days after birth onward and related to changes in organ of Corti morphology. The results show that changes in the accessory structures continue throughout the period of onset and development of cochlear responses between 12 and 20 days after birth, and may therefore influence the micromechanical responses of the organ of Corti to acoustic stimuli during this period.

Animals↗

PubMed1997

Structural development of sensory cells in the ear.

The hair cells of the auditory and balance systems of the inner ear have precise structures and orientations related to function. Hair cells differentiate from a homogenous cell population with the initiation of afferent synaptogenesis and appearance of the apical hair bundle being the first definitive structural sign of hair cell development. The cytoskeletal network then develops and the intercellular membrane junctions become more complex. As auditory function is established in mammalian cochlear hair cells, the lateral membrane acquires certain specializations. Accompanying this there is a change from afferent to efferent innervation of outer hair cells.

Journal Article↗

PubMed1997

Morphological evidence for supporting cell to hair cell conversion in the mammalian utricular macula.

The possible origin of the immature hair cells that appear in the utricular maculae of guinea pigs following gentamicin-induced hair cell death was investigated. Guinea pigs were continuously infused with bromodeoxyuridine, to label proliferating cells and their progeny, for 2 weeks after inducing damage to the inner ear on one side with gentamicin. The opposite ear in each animal served as control. Serial sections were cut through the entire utricular maculae of both ears of each animal and the number of labelled cells in the epithelium and underlying connective tissue was counted. Label was present in cells in the sensory epithelium in the utricles from the drug exposed ears but not in the controls. The nuclei of cells in the underlying connective tissue were also labelled in both ears. Some of the labelled nuclei in the epithelium were at the level normally occupied by hair cells, but most were at the level of supporting cell nuclei. However, the total number of labelled nuclei in the sensory epithelium was small; the maximum was 12 in one animal. The number of labelled nuclei in the connective tissue of the treated ears was significantly greater than the number in the untreated ear. This confirms that cell proliferation is stimulated in the mature mammalian utricular macula after hair cell loss, but the extent to which it occurs appears to be insufficient to explain the recovery in hair cell numbers which is observed. Detailed thin section studies of the utricular maculae of gentamicin-treated animals over a prolonged post-treatment period were also performed. In utricles which had suffered damage, there were cells which, like supporting cells but unlike hair cells, were resting on basement membrane, but which possessed at their apical ends organized bundles of microvilli similar to immature hair cell stereocilia. Other cells with more obvious stereocilia remained in contact with the basement membrane via and a small feet process. In still other cells, where a stereociliary bundle was obvious and almost mature in appearance, there was a foot process extending towards the basement membrane but not quite in contact, suggesting it had just detached. All these cells were contacted by nerve endings and specialization of the membranes were apparent at the site of cell-neurone contact. The morphological characteristics of these cells are consistent with phenotypic conversion of supporting cells into hair cells and this may account for some of the hair cell production in the mature mammalian vestibular sensory epithelia after hair cell death.

Animals↗

PubMed1995

Two modes of hair cell loss from the vestibular sensory epithelia of the guinea pig inner ear.

In the vestibular and auditory neurosensory epithelia of poikilothermic vertebrates and of birds, damaged sensory "hair" cells are often deleted by extrusion from the apical surface. In contrast, in the adult mammalian auditory epithelium (the organ of Corti), the bodies of damaged hair cells degenerate within the epithelium. To determine whether this apparent difference is species related or is associated with the differing structural organisation of the epithelia, hair cell deletion in the mammalian vestibular end-organs was examined. The structural organisation of these tissues is closer to that of the inner ear epithelia of lower vertebrates than to the organ of Corti. Hair cell loss was induced by chronic, systemic treatment of guinea pigs with the ototoxic aminoglycoside antibiotic gentamicin. The vestibular sensory epithelia were examined at various times after treatment via scanning electron microscopy, thin sectioning, and staining f-actin with fluorescently labelled phalloidin. Two distinct modes of hair cell loss were identified: 1) degeneration of hair cells within the epithelium, which often showed morphological features consistent with those described for apoptosis, and 2) extrusion of intact cells from the apical surface. Neither process caused the formation of obvious lesions through the epithelial surfaces. Expansion of adjacent supporting cells during hair cell deletion resulted in repair that appeared to preserve permeability barriers. There was also no evidence of inflammation accompanying hair cell removal. Thus, with both modes of hair cell loss, it appeared that deletion of hair cells was achieved without disruption of tissue architecture or integrity. This may be important for subsequent repair and regeneration processes to operate.

Animals↗

PubMed1995

Postnatal development of membrane specialisations of gerbil outer hair cells.

Using a combination of freeze-fracture and thin sections, this study examines the maturation of the membrane specialisations of the gerbil outer hair cells (OHC) between 2 and 16 days after birth (DAB). The apical membrane, the junctional region around the neck of the cell, and the lateral and basal membranes are described. The results suggest a sequential development of the different components of the lateral wall. Intramembrane protein particles (IMP), the putative OHC motor elements, were found to be present at low density at 2 DAB and increased in density from 2200 IMP/microns 2 at 2 DAB to 4131/ microns 2 at 8 DAB. OHCs have been reported as showing electromotility from 8 DAB onward. IMPs continue to increase in density until mature values are attained at 16 DAB. Sub-surface cisternae did not appear until 8 DAB, with a single layer being complete by 10 DAB. Pillar structures, proposed to be related to the cytoskeletal lattice, first appear at 10 DAB. The apical membrane of the immature hair cell is characterised by the presence of pits related to the endocytosis of vesicles, and tip-links between stereocilia, thought to be associated with sites of ion channel opening, are present at 2 DAB. The junctional region comprises two areas which mature at differing rates: an apical-most region which attains an adult-like appearance by 8 DAB and a basal-ward region which continues to increase in complexity until mature at 16 DAB. The functional significance of the results are discussed in relation to the possible roles of the junctional regions and the proposed sites of the OHC motor elements.

Aging↗