Vesicle traffic in the outer hair cell.

The plasma-membrane marker FM1-43 was employed to reveal the relative significance of different types of endocytic and transcytic mechanisms in outer hair cells (OHCs) of the guinea-pig cochlea. A double-barrel local perfusion system was used to label independently the apical or synaptic pole of the isolated OHC to study mechanisms of vesicle uptake at the poles and of vesicle trafficking along and across the cell. Treatment with an inhibitor of macropino- and phagocytosis, phenylarsine oxide, or of clathrin-mediated endocytic activity, concanavalin A, significantly reduced the dye uptake at both the apical and the synaptic poles, indicating the presence of both clathrin-independent and clathrin-mediated processes at both poles.

MET currents and otoacoustic emissions from mice with a detached tectorial membrane indicate the extracellular matrix regulates Ca near stereocilia.

Key Points: The aim was to determine whether detachment of the tectorial membrane (TM) from the organ of Corti in Tecta/Tectb mice affects the biophysical properties of cochlear outer hair cells (OHCs). Tecta/Tectb mice have highly elevated hearing thresholds, but OHCs mature normally. Mechanoelectrical transducer (MET) channel resting open probability (P ) in mature OHC is ∼50% in endolymphatic [Ca ], resulting in a large standing depolarizing MET current that would allow OHCs to act optimally as electromotile cochlear amplifiers.

Differential deletion of GDNF in the auditory system leads to altered sound responsiveness.

Glial-derived neurotrophic factor (GDNF) has been proposed as a potent neurotrophic factor with the potential to cure neurodegenerative diseases. In the cochlea, GDNF has been detected in auditory neurons and sensory receptor cells and its expression is upregulated upon trauma. Moreover, the application of GDNF in different animal models of deafness has shown its capacity to prevent hearing loss and promoted its future use in therapeutic trials in humans.

Visualizing BDNF Transcript Usage During Sound-Induced Memory Linked Plasticity.

Activity-dependent BDNF (brain-derived neurotrophic factor) expression is hypothesized to be a cue for the context-specificity of memory formation. So far, activity-dependent BDNF cannot be explicitly monitored independently of basal BDNF levels. We used the BLEV ( DNF- - ) reporter mouse to specifically detect activity-dependent usage of exon-IV and -VI promoters through bi-cistronic co-expression of CFP and YFP, respectively.

Gαi Proteins are Indispensable for Hearing.

Background/aims: From invertebrates to mammals, Gαi proteins act together with their common binding partner Gpsm2 to govern cell polarization and planar organization in virtually any polarized cell. Recently, we demonstrated that Gαi3-deficiency in pre-hearing murine cochleae pointed to a role of Gαi3 for asymmetric migration of the kinocilium as well as the orientation and shape of the stereociliary ("hair") bundle, a requirement for the progression of mature hearing. We found that the lack of Gαi3 impairs stereociliary elongation and hair bundle shape in high-frequency cochlear regions, linked to elevated hearing thresholds for high-frequency sound.

The glucocorticoid antagonist mifepristone attenuates sound-induced long-term deficits in auditory nerve response and central auditory processing in female rats.

Systemic corticosteroids have been the mainstay of treatment for various hearing disorders for more than 30 yr. Accordingly, numerous studies have described glucocorticoids (GCs) and stressors to be protective in the auditory organ against damage associated with a variety of health conditions, including noise exposure. Conversely, stressors are also predictive risk factors for hearing disorders.

Double fluorescent labelling of a bipolar epithelial cell in vitro: The outer hair cell.

Background: Fluorescence membrane markers are efficient tools for visualizing the dynamics of membrane recycling processes in living cells. The outer hair cell (OHC) - a bipolar epithelial cell in the cochlea - possesses endocytic activity at both its apical and basal poles. The best visual overview of transcytosis in the OHC is achieved when the cell is isolated, so that both the apical and the basal poles are in the same focal plane to allow confocal imaging.

The chloride-channel blocker 9-anthracenecarboxylic acid reduces the nonlinear capacitance of prestin-associated charge movement.

The basis of the extraordinary sensitivity and frequency selectivity of the cochlea is a chloride-sensitive protein called prestin which can produce an electromechanical response and which resides in the basolateral plasma membrane of outer hair cells (OHCs). The compound 9-anthracenecarboxylic acid (9-AC), an inhibitor of chloride channels, has been found to reduce the electromechanical response of the cochlea and the OHC mechanical impedance. To elucidate these 9-AC effects, the functional electromechanical status of prestin was assayed by measuring the nonlinear capacitance of OHCs from the guinea-pig cochlea and of prestin-transfected human embryonic kidney 293 (HEK 293) cells.

Generation of somatic electromechanical force by outer hair cells may be influenced by prestin-CASK interaction at the basal junction with the Deiter's cell.

The motor protein, prestin, situated in the basolateral plasma membrane of cochlear outer hair cells (OHCs), underlies the generation of somatic, voltage-driven mechanical force, the basis for the exquisite sensitivity, frequency selectivity and dynamic range of mammalian hearing. The molecular and structural basis of the ontogenetic development of this electromechanical force has remained elusive. The present study demonstrates that this force is significantly reduced when the immature subcellular distribution of prestin found along the entire plasma membrane persists into maturity, as has been described in previous studies under hypothyroidism.

Impact of heart valve decellularization on 3-D ultrastructure, immunogenicity and thrombogenicity.

Decellularized xenogeneic tissue represents an interesting material for heart valve tissue engineering. The prospect objective is removal of all viable cells while preserving extracellular matrix (ECM) integrity. The major concerns of all decellularization protocols remain ECM disruption, immunogenicity and thrombogenicity.

Facilitated noninvasive visualization of collagen and elastin in blood vessels.

Multiphoton imaging is a powerful tool for three-dimensional visualization of extracellular matrix components such as collagen and elastin in fresh, nonfixed, and nonembedded tissues. We have previously published data on the induction of the second harmonic generation signal of collagen and autofluorescence of elastin using a tunable multiphoton laser system. Without staining, a second harmonic generation signal was detected for collagen when excited at wavelength lambda(ex) = 840 nm.

Membrane traffic in outer hair cells of the adult mammalian cochlea.

Outer hair cells (OHCs), the sensory-motor cells responsible for the extraordinary frequency selectivity and dynamic range of the cochlea, rapidly endocytose membrane and protein at their apical surface. Endocytosis and transcytosis in isolated OHCs from the mature guinea-pig cochlea were investigated using the amphipathic membrane probe FM1-43. We observed membrane transport from the apical surface to both the basolateral wall and the subnuclear pole.

Norfluoxetine and fluoxetine have similar anticonvulsant and Ca2+ channel blocking potencies.

Norfluoxetine is the most important active metabolite of the widely used antidepressant fluoxetine but little is known about its pharmacological actions. In this study the anticonvulsant actions of norfluoxetine and fluoxetine were studied and compared to those of phenytoin and clonazepam in pentylenetetrazol-induced mouse epilepsy models. Pretreatment with fluoxetine or norfluoxetine (20mg/kg s.

Intrinsic laryngeal muscle reinnervation with nerve-muscle pedicle.

Objective: To test the application of the nerve-muscle pedicle (NMP) technique for selective reinnervation of previously denervated posterior cricoarytenoid (PCA) muscle.

Methods: The left recurrent laryngeal nerve (RLN) was severed in 5 mongrel dogs, and an ansa cervicalis-sternohyoid muscle pedicle was sutured to the left PCA muscle. Three dogs underwent a sham operation.

Differential effects of fluoxetine enantiomers in mammalian neural and cardiac tissues.

Racemic fluoxetine is a widely used SSRI antidepressant compound having also anticonvulsant effect. In addition, it was shown that it blocked several types of voltage gated ion channels including neural and cardiac calcium channels. In the present study the effects of enantiomers of fluoxetine (R(-)-fluoxetine and S(+)-fluoxetine) were compared on neuronal and cardiac voltage-gated Ca2+ channels using the whole cell configuration of patch clamp techniques, and the anticonvulsant action of these enantiomers was also evaluated in a mouse epilepsy model.

A detailed procedure and dissection guide for the isolation of spiral ganglion cells of the guinea pig for electrophysiological experiments.

In the present study step-by-step instructions are provided for a preparative technique employed for the removal of the spiral ganglion from the inner ear of the guinea pig. Removal of the temporal bone is followed by opening of the bulla and excision of the modiolus. All major steps of the technique are illustrated with photographs.

Removal of Ca(2+) following depolarization-evoked cytoplasmic Ca(2+) transients in freshly dissociated pyramidal neurones of the rat dorsal cochlear nucleus.

Cytoplasmic [Ca(2+)] ([Ca(2+)](i)) was measured using Fura-2 in pyramidal neurones isolated from the rat dorsal cochlear nucleus (DCN). The kinetic properties of Ca(2+) removal following K(+) depolarization-induced Ca(2+) transients were characterized by fitting exponential functions to the decay phase. The removal after small transients (<82 nM peak [Ca(2+)](i)) had monophasic time course (time constant of 6.