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Abstract
The nervous system requires a fine balance of excitatory and inhibitory signals for proper development and functioning. -Aminobutyric Acid (GABA) is the major inhibitory neurotransmitter in the central nervous system of all vertebrates. GABA is made by the glutamic acid decarboxylase (GAD) enzyme, which exists in two isoforms, GAD67 and GAD65, each of which is encoded by an independent gene, GAD1 and GAD2, respectively. Disruptions in the GAD genes, namely GAD1, have been implicated in several neurological disorders, however little is known about the mechanism(s) that cause these disorders. Like mammals, zebrafish have both known gad genes; however, we recently found molecular evidence for a gad1 paralog in zebrafish. This discovery could allow for studies addressing the mechanistic and functional questions of gad1 gene function in a way that has not been previously possible in mammalian models. We used standard reverse transcription polymerase chain reaction (RT-PCR) and mRNA in situ hybridization to sequence and localize expression of this gad1a gene during zebrafish neural development. We used double fluorescent in situ hybridization (F.I.S.H.) to assess the gad expression pattern both between zebrafish gad genes and to known markers of spinal cord interneurons during development. We also used CRISPR-Cas9 to create different gad1 mutant zebrafish to address questions of gad gene function. We have found that there is differential temporal and spatial expression between the gad1 paralogs during development, while gad1b and gad2 seem to be co-expressed. The presumably distinct expression pattern among this family of genes suggests a complex regulation of the zebrafish gad genes. Analyses of gad1a -/- and gad1b -/- mutant zebrafish show that gad1b -/- zebrafish have significantly reduced levels of GABA as measured by high-performance liquid chromatography (HPLC). The gad1b -/- larvae are hypersensitive to seizure-like behavior and exhibit increased and abnormal brain activity as measured by electrophysiology. Comparatively, the gad1a -/- larvae are somewhat hypersensitive and show less abnormal neurological activity. Taken together, these findings have uncovered a previously unknown genome duplication that has separated gad1 into two genes in zebrafish that both contribute, though in different ways, to proper nervous system development and function.