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Date of Award

9-2013

Document Type

Campus Access

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Neuroscience and Behavior

First Advisor

Gerald B. Downes

Second Advisor

Abigail M. Jensen

Third Advisor

James J. Chambers

Subject Categories

Molecular Biology | Neuroscience and Neurobiology | Psychology

Abstract

Excitation and inhibition in the nervous system must be carefully balanced. I utilized the zebrafish (Danio rerio) in order to study this balance in the vertebrate motor system. Zebrafish (Danio rerio) can be used effectively for both forward and reverse genetics. My primary interest lies in genes involved in neural network development and function. Forward genetic mutagenesis screens are particularly powerful as they are unbiased. Using this approach we characterized a mutant based on its abnormal motor behavior. However, mutants identified with abnormal motor behavior may have mutations in muscle proteins. We identified a semi-dominant mutation in the atp2a1 gene, which encodes a protein vital for normal muscle function, from a previously completed mutagenesis screen. In order to investigate genes directly involved in neuronal signaling I used a reverse genetics approach to study GABAA receptors. γ-Aminobutyric acid (GABA) is a major source of inhibition in the motor system. In order to test the effect of blockade of GABAA receptors, I injected gabazine, a GABAA receptor antagonist, into zebrafish embryos. Injected embryos display an abnormal escape response later in development. To determine the GABAA receptor subunits responsible for the observed phenotype, I utilized RNA in situ hybridization to examine the expression of the α class of GABAA receptor subunits. I found that the gabra5 gene, which encodes the GABAA α5 subunit, was expressed in the hindbrain of larvae. To examine the role of the α5 subunit I designed anti-sense morpholinos to target the start codon of the gabra5 gene. Knockdown of the gabra5 gene caused abnormal behavior in larvae similar to that seen in gabazine injected larvae. I have characterized a novel semi-dominant atp2a1 mutant in zebrafish. This mutant completes a zebrafish model system of the human disease Brody's disease. Turning toward a reverse genetic approach I investigated the expression of several GABAA receptor α subunits. I have characterized the behavior of GABAA α5 subunit knockdown embryos and larvae and begun work to generate a stable knockout line. This line will be useful in exploring the function of the α5 subunit and compounds that interact with it.

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