Biomedical subjects

William F Sewell

Publications and source records attributed to William F Sewell.

6 recordsLinked to original sources

PubMed2005

Inner ear drug delivery via a reciprocating perfusion system in the guinea pig.

Rapid progress in understanding the molecular mechanisms associated with cochlear and auditory nerve degenerative processes offers hope for the development of gene-transfer and molecular approaches to treat these diseases in patients. For therapies based on these discoveries to become clinically useful, it will be necessary to develop safe and reliable mechanisms for the delivery of drugs into the inner ear, bypassing the blood-labyrinthine barrier. Toward the goal of developing an inner ear perfusion device for human use, a reciprocating microfluidic system that allows perfusion of drugs into the cochlear perilymph through a single inlet hole in scala tympani of the basal turn was developed. The performance of a prototype, extracorporeal reciprocating perfusion system in guinea pigs is described. Analysis of the cochlear distribution of compounds after perfusion took advantage of the place-dependent generation of responses to tones along the length of the cochlea. Perfusion with a control artificial perilymph solution had no effect. Two drugs with well-characterized effects on cochlear physiology, salicylate (5 mM) and DNQX (6,7-Dinitroquinoxaline-2,3-dione; 100 and 300 microM), reversibly altered responses. The magnitude of drug effect decreased with distance from the perfusion pipette for up to 10 mm, and increased with dose and length of application.

Animals↗

PubMed2005

Mutations that affect the survival of selected amacrine cell subpopulations define a new class of genetic defects in the vertebrate retina.

Amacrine neurons are among the most diverse cell classes in the vertebrate retina. To gain insight into mechanisms vital to the production and survival of amacrine cell types, we investigated a group of mutations in three zebrafish loci: kleks (kle), chiorny (chy), and bergmann (bgm). Mutants of all three genes display a severe loss of selected amacrine cell subpopulations. The numbers of GABA-expressing amacrine interneurons are sharply reduced in all three mutants, while cell loss in other amacrine cell subpopulations varies and some cells are not affected at all. To investigate how amacrine cell loss affects retinal function, we performed electroretinograms on mutant animals. While the kle mutation mostly influences the function of the inner nuclear layer, unexpectedly the chy mutant phenotype also involves a loss of photoreceptor cell activity. The precise ration and arrangement of amacrine cell subpopulations suggest that cell-cell interactions are involved in the differentiation of this cell class. To test whether defects of such interactions may be, at least in part, responsible for mutant phenotypes, we performed mosaic analysis and demonstrated that the loss of parvalbumin-positive amacrine cells in chy mutants is due to extrinsic (cell-nonautonomous) causes. The phenotype of another amacrine cell subpopulation, the GABA-positive cells, does not display a clear cell-nonautonomy in chy animals. These results indicate that environmental factors, possibly interactions among different subpopulations of amacrine neurons, are involved in the development of the amacrine cell class.

Amacrine Cells↗

PubMed2005

A method for intracochlear drug delivery in the mouse.

The confluence of two rapidly emerging research arenas - development of mouse models of human deafness and inner ear drug therapy for treatment and prevention of hearing loss - provides an opportunity for unprecedented approaches to study and treat deafness. Toward such goals, we have developed a method for intracochlear drug delivery in the mouse. The bulla was exposed using a ventral approach and the stapedial artery cauterized. An opening made into the inferior-medial aspect of the bulla, where the basal cochlear wall fuses with tympanic bulla, provided direct access to the scala tympani without separately opening the bulla or elevating auditory response thresholds. Cochlear responses, assayed by frequency-specific effects on ABRs and DPOAEs, were stable with infusion (1 microl/h) of an artificial perilymph solution (80 min). The glutamate receptor antagonist, CNQX (100 microM; 175 min), reduced ABR responses without affecting DPOAEs. Salicylate (5mM; 165 min) altered both. Both drugs had greatest effects at high frequencies, but distributed throughout the cochlea and were reversible. The safe delivery of drugs into the cochlea by this approach has immediate application in the study and treatment of various forms of human hearing loss that can be modeled in the mouse.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed2004

Pharmacology of acetylcholine-mediated cell signaling in the lateral line organ following efferent stimulation.

Cholinergic efferent fibers modify hair cell responses to mechanical stimulation. It is hypothesized that calcium entering the hair cell through a nicotinic receptor activates a small-conductance (SK), calcium-activated potassium channel to hyperpolarize the hair cell. The calcium signal may be amplified by calcium-induced calcium release from the synaptic cisternae. Pharmacological tests of these ideas in the intact cochlea have been technically difficult because of the complex and fragile structure of the mammalian inner ear. We turned to the Xenopus laevis lateral line organ, whose simplicity and accessibility make it a model for understanding hair cell organ function in a relatively intact system. Drugs were applied to the inner surface of the skin while monitoring the effects of efferent stimulation on afferent fiber discharge rate. Efferent effects were blocked by antagonists of SK channels including apamin (EC50 = 0.5 microM) and dequalinium (EC50 = 12 microM). The effect of apamin was not enhanced by co-administration of phenylmethylsulfonyl fluoride, a proteolysis inhibitor. Efferent effects were attenuated by ryanodine, an agent that can interfere with calcium-induced calcium release, although relatively high (mM) concentrations of ryanodine were required. Fluorescent cationic styryl dyes, 4-di-2-asp and fm 1-43, blocked efferent effects, although it was not possible to observe specific entry of the dye into the base of hair cells. These pharmacological findings in the Xenopus lateral line organ support the hypothesis that effects of efferent stimulation are mediated by calcium entry through the nicotinic receptor via activation of SK channels and suggest the generality of this mechanism in meditating cholinergic efferent effects.

Acetylcholine↗

PubMed2004

Afferent synaptic transmission in a hair cell organ: pharmacological and physiological analysis of the role of the extended refractory period.

One feature of neuronal discharge proposed to play a role in coding temporal information is the relative refractory period that follows each action potential. In neurons innervating hair cells, there is an extended refractory period that can last </=100 ms. We have taken a pharmacological approach to examine the extended refractory period in the Xenopus lateral line organ. We show that each action potential in the afferent fiber, whether generated spontaneously or through an antidromic electrical pulse, decreases the probability of subsequent afferent discharge for a period of </=100 ms. We show that the extended refractory period can be modulated with drugs that alter glutamatergic transmission between the hair cell and the afferent fiber. The extended refractory period can be enhanced by perfusion with agents that reduce synaptic activity. These agents include blockers of voltage-dependent transmitter release, such as cobalt, as well as glutamate receptor antagonists, such as CNQX and kynurenic acid. Conversely, perfusion with agents that increase synaptic activity through activation of the glutamate receptors, such as AMPA or kainate, reduces the magnitude of suppression during the extended refractory period. The extended refractory period is greatly reduced by iberiotoxin and tetraethylammonium (TEA), indicating it may be mediated in large part by a calcium-dependent potassium channel. The ability to modulate the extended refractory period with changes in synaptic input suggests a simple, dynamic mechanism by which strong input (i.e., large or frequent excitatory postsynaptic potentials) can be strengthened and weak inputs weakened.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed2002

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.

Acoustic Stimulation↗