William F. Sewell

The effects of furosemide on the endocochlear potential and auditory-nerve fiber tuning curves in cats

Furosemide was injected intravenously in anesthetized cats while the endocochlear potential (EP) and tuning curves from fibers of the auditory nerve were simultaneously monitored in the same ear. The characteristic frequencies (CFs) of the fibers studied ranged from 0.25 to 28 kHz. Furosemide administration produced reversible, dose-related decreases in the EP which were accompanied by threshold elevations and alterations in the tuning curves of auditory nerve fibers. There was approximately 1 dB of threshold elevation for every millivolt decrease in the EP. For fibers with CFs above 3 kHz, threshold elevation at CF (in the tip of the tuning curve) was three times that in the tail of the tuning curve. Threshold elevation in the tip was always accompanied by threshold elevation in the tuning curve tail. Threshold elevation was dependent upon the CF of the fiber, with higher CF fibers showing larger threshold shifts than lower CF fibers. Threshold elevation was accompanied by a systematic shift in the CF of the fiber. Fibers with CF above 1 kHz exhibited downward shifts in CF, while those with CF less than 1 kHz generally exhibited upward shifts in CF.

Inner ear drug delivery for auditory applications

Many inner ear disorders cannot be adequately treated by systemic drug delivery. A blood-cochlear barrier exists, similar physiologically to the blood-brain barrier, which limits the concentration and size of molecules able to leave the circulation and gain access to the cells of the inner ear. However, research in novel therapeutics and delivery systems has led to significant progress in the development of local methods of drug delivery to the inner ear. Intratympanic approaches, which deliver therapeutics to the middle ear, rely on permeation through tissue for access to the structures of the inner ear, whereas intracochlear methods are able to directly insert drugs into the inner ear. Innovative drug delivery systems to treat various inner ear ailments such as ototoxicity, sudden sensorineural hearing loss, autoimmune inner ear disease, and for preserving neurons and regenerating sensory cells are being explored.

Single unit clues to cochlear mechanisms

In recent years studies on isolated hair cells have suggested that there is an inherent tuning of hair cells determined by their mechanical and electrical properties. However, tuning for mammalian cochleas appears to be much more complicated since there are typically two types of receptor cells (inner and outer hair cells) imbedded in a highly organized framework of supporting cells, membranes and fluids. The major neural output of the cochlea can be monitored by recording the activity of myelinated axons of spiral ganglion cells, not only under normal conditions, but also when the discharge patterns are altered by ototoxic drugs, acoustic trauma or olivocochlear bundle stimulation. A model system with two excitatory influences, one sharply tuned and highly sensitive, and a second, broadly tuned and relatively insensitive, can account for much of the existing data. Results from single-neuron marking studies support the notion that these two influences probably involve interactions between inner and outer hair cells. More global influences such as the endocochlear potential also can act on auditory-nerve fibers through the hair-cell systems. Thus, the inherent frequency selectivity of the receptor cell is only one of many factors that determine the tuning of mammalian auditory-nerve fibers.

Drug delivery for treatment of inner ear disease: current state of knowledge.

Delivery of medications to the inner ear has been an area of considerable growth in both the research and clinical realms during the past several decades. Systemic delivery of medication destined for treatment of the inner ear is the foundation on which newer delivery techniques have been developed. Because of systemic side effects, investigators and clinicians have begun developing and using techniques to deliver therapeutic agents locally. Alongside the now commonplace use of intratympanic gentamicin for Menieres disease and the emerging use of intratympanic steroids for sudden sensorineural hearing loss, novel technologies, such as hydrogels and nanoparticles, are being explored. At the horizon of inner ear drug-delivery techniques, intracochlear devices that leverage recent advances in microsystems technology are being developed to apply medications directly into the inner ear. Potential uses for such devices include neurotrophic factor and steroid delivery with cochlear implantation, RNA interference technologies, and stem-cell therapy. The historical, current, and future delivery techniques and uses of drug delivery for treatment of inner ear disease serve as the basis for this review.

High-throughput behavioral screening method for detecting auditory response defects in zebrafish

We have developed an automated, high-throughput behavioral screening method for detecting hearing defects in zebrafish. Our assay monitors a rapid escape reflex in response to a loud sound. With this approach, 36 adult zebrafish, restrained in visually isolated compartments, can be simultaneously assessed for responsiveness to near-field 400 Hz sinusoidal tone bursts. Automated, objective determinations of responses are achieved with a computer program that obtains images at precise times relative to the acoustic stimulus. Images taken with a CCD video camera before and after stimulus presentation are subtracted to reveal a response to the sound. Up to 108 fish can be screened per hour. Over 6500 fish were tested to validate the reliability of the assay. We found that 1% of these animals displayed hearing deficits. The phenotypes of non-responders were further assessed with radiological analysis for defects in the gross morphology of the auditory system. Nearly all of those showed abnormalities in conductive elements of the auditory system: the swim bladder or Weberian ossicles.

A possible neurotransmitter role for CGRP in a hair-cell sensory organ

We report that calcitonin gene-related peptide (CGRP) increases the discharge rate of afferent fibers innervating hair cells in the lateral line organ of Xenopus laevis. We have localized CGRP-like immunoreactivity in small, presumably efferent, fibers innervating the lateral line organ. In addition to providing evidence for a neurotransmitter role for CGRP in a sensory system, these results may help explain the non-cholinergic excitatory effect seen with efferent stimulation in this and other hair cell organs such as the inner ear.

Morphology and cell type heterogeneities of the inner ear epithelia in adult and juvenile zebrafish (Danio rerio).

Although the zebrafish has become an important model for genetic analysis of the vertebrate auditory system, a comprehensive description of the zebrafish ear has been provided for embryonic and larval development only (Haddon and Lewis [ 1996 ] J. Comp. Neurol. 365:113). Here we describe the development of sensory maculae in juvenile fish and the morphology of the adult zebrafish ear. This description was obtained via three‐dimensional reconstruction of serial sections and confocal microscopy of immunolabeled preparations and includes the Weberian ossicles and fluid spaces. Phalloidin staining, which labels actin filaments of stereocilia, was used to delineate the sensory epithelia, to visualize the distribution of hair cells, to estimate their density in different areas of the maculae, and to perform hair cell counts. Morphology of ciliary bundles in different regions of the lagena, saccule, utricle, macula neglecta, and cristae was characterized with an anti‐acetylated tubulin antibody and by phalloidin staining. We have identified two antibodies characterized by region‐specific staining patterns in the inner ear epithelia. Zn‐1 antibody staining largely correlates with the presence of short‐bundle hair cells in the peripheral regions of sensory epithelia. Zn‐4 antibody, on the other hand, labels a zone of epithelial cells surrounding the sensory maculae. These analyses extend previous observations of cell‐type heterogeneity in both sensory and nonsensory epithelia of the fish ear. J. Comp. Neurol. 438:173–190, 2001.

Furosemide selectively reduces one component in rate-level functions from auditory-nerve fibers

Furosemide, an ototoxic diuretic, was administered intravenously while rate- and phase-level functions of auditory nerve fibers were measured in the cat. Normal level functions can demonstrate two components distinguished by an abrupt shift in the phase of the response as the sound level is increased. Furosemide, administered at doses that decrease the endocochlear potential, selectively reduces the discharge rate in response to tones at sound levels below that of the abrupt phase shift.

Auditory sensitivity regulation via rapid changes in expression of surface AMPA receptors

We report a robust regulation of surface AMPA receptors in mouse auditory neurons, both with application of glutamate receptor agonists in cultured neurons and in response to acoustic stimulation in vivo. The reversible reduction of surface AMPA receptors following acoustic stimulation correlated with changes in acoustic sensitivity. Thus we show that AMPA receptor cycling is important for optimizing synaptic transfer at one of the most exacting synapses in the body.

Pharmacological alterations of the activity of afferent fibers innervating hair cells

To determine whether some of the substances that may be present in hair-cell sensory organs could affect neural activity in afferent fibers, we examined 56 compounds for the ability to alter the discharge rate of afferent fibers innervating hair cells in the lateral line organ of Xenopus laevis, the African clawed frog. These compounds included amino acids, glutamyl dipeptides, standard neurotransmitter candidates, and other constituents of tissues and body fluids. Substances found to be excitatory included some neutral amino acids (alanine, serine, threonine, asparagine, glutamine, and proline), ATP, carnosine, histidine, and barium chloride. Compounds that suppressed discharge included the aromatic amino acids (phenylalanine, tryptophan, and tyrosine), serotonin, and gamma-glutamyl dipeptides. GABA and acidic amino acids (glutamate, aspartate, and cysteine sulfinate) produced a brief excitation followed by a suppression of discharge rate. Several of these substances were active at sufficiently low concentrations that their presence in body fluids may affect afferent fiber discharge rate under normal or pathological conditions.